Current Protocols in Mouse Biology (Volume 1)

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CURRENT PROTOCOLS in Mouse Biology

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You have full text access to this content

Current Protocols in Mouse Biology

Online ISBN: 9780470942390 DOI: 10.1002/9780470942390 Editors & Contributors

EDITORIAL BOARD Johan Auwerx Ecole Polytechnique Fédérale de Lausanne Stephen D. Brown Harwell Science and Innovation Campus, UK Monica Justice Baylor College of Medicine David D. Moore Baylor College of Medicine Susan L. Ackerman The Jackson Laboratory Joseph Nadeau Institute for Systems Biology CONTRIBUTORS Cristina Antal Mouse Clinical Institute Illkirch, France and Institut d’Histologie, Faculté de Médecine Strasbourg, France Johan Auwerx Institut Clinique de la Souris (ICS) Illkirch, France and Ecole Polytechnique Fédérale de Lausanne Lausanne, Switzerland Abdelkader Ayadi Institut Clinique de la Souris (ICS) Illkirch, France Bernard Baertschi Institute for Biomedical Ethics University of Geneva Geneva, Switzerland Gareth T. Banks Neurobehavioural Genetics MRC Harwell

Harwell Science and Innovation Campus Oxfordshire, United Kingdom Isabelle Barde School of Life Sciences and “Frontiers in Genetics” National Program Ecole Polytechnique Fédérale de Lausanne (EPFL) Lausanne, Switzerland Fernando J. Benavides The University of Texas M.D. Anderson Cancer Center Science Park-Research Division Smithville, Texas Marie-Christine Birling Institut Clinique de la Souris (ICS) Illkirch, France Gemma Brufau Department of Pediatrics Center for Liver, Digestive, and Metabolic Diseases University Medical Center Groningen University of Groningen Groningen, The Netherlands Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire Université de Strasbourg, and Collége de France Illkirch, France Nathalie Chartoire Institut Clinique de la Souris (ICS) Illkirch, France Luis E. Donate Spanish National Cancer Research Centre (CNIO) Madrid, Spain Pascal Escher IRO-Institute for Research in Ophthalmology Sion, Switzerland and Department of Ophthalmology University of Lausanne Lausanne, Switzerland Jérôme N. Feige MusculoSkeletal Diseases Novartis Institute for Biomedical Research Basel, Switzerland Giséle Ferrand Ecole Polytechnique Fédérale de Lausanne Lausanne, Switzerland Shumin Gao The University of Medicine & Dentistry of New Jersey New Jersey Medical School Newark, New Jersey Françoise Gofflot Institut Clinique de la Souris (ICS) Illkirch, France and Université Catholique de Louvain Life Science Institute Louvain-la-Neuve, Belgium Isabelle Goncalves da Cruz Institut Clinique de la Souris (ICS) Illkirch, France

Jacob B. Griffin Roche Madison Inc. Madison, Wisconsin Albert K. Groen Department of Pediatrics Center for Liver, Digestive, and Metabolic Diseases University Medical Center Groningen University of Groningen Groningen, The Netherlands and Department of Laboratory Medicine Center for Liver, Digestive, and Metabolic Diseases University Medical Center Groningen University of Groningen Groningen, The Netherlands Jean-Louis Guénet Département de Biologie du Développement Institut Pasteur Paris, France Marcel Gyger EPFL—Center of Phenogenomics Lausanne, Switzerland Yann Hérault Mouse Clinical Institute Illkirch, France and Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) CNRS/INSERM/Université Louis Pasteur Illkirch, France David Ho The University of Medicine & Dentistry of New Jersey New Jersey Medical School Newark, New Jersey Chull Hong The University of Medicine & Dentistry of New Jersey New Jersey Medical School Newark, New Jersey Neil J. Ingham Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton, Cambridge, United Kingdom Ralf Kühn German Research Center for Environmental Health Munich, Germany and Technical University Munich Munich, Germany David A. Largaespada Department of Genetics, Cell Biology, and Development University of Minnesota Minneapolis, Minnesota, Center for Genome Engineering University of Minnesota Minneapolis, Minnesota, and Masonic Cancer Center University of Minnesota Minneapolis, Minnesota Pontus Lundberg Department of Biomedicine Experimental Hematology University Hospital Basel

Basel, Switzerland Stefan Marcaletti MusculoSkeletal Diseases Novartis Institute for Biomedical Research Basel, Switzerland Manuel Mark Mouse Clinical Institute Illkirch, France, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) CNRS/INSERM/Université Louis Pasteur Illkirch, France and Hôpital Universitaire de Strasbourg Strasbourg, France Daniel Metzger Institut de Génétique et de Biologie Moléculaire et Cellulaire Université de Strasbourg, and Collége de France Illkirch, France Michael S. Minett Molecular Nociception Group Wolfson Institute for Biomedical Research University College London London, United Kingdom and London Pain Consortium Kings College London London, United Kingdom Salvatore Modica Institute of Food, Nutrition, and Health ETH Zurich Schwerzenbach, Switzerland Branden Moriarty Department of Genetics, Cell Biology, and Development University of Minnesota Minneapolis, Minnesota, Center for Genome Engineering University of Minnesota Minneapolis, Minnesota, and Masonic Cancer Center University of Minnesota Minneapolis, Minnesota Antonio Moschetta Laboratory of Lipid Metabolism and Cancer Department of Translational Pharmacology Consorzio Mario Negri Sud Santa Maria Imbaro, Italy and Clinica Medica “A. Murri” Department of Internal and Public Medicine University Aldo Moro of Bari Bari, Italy Francisca Mulero Spanish National Cancer Research Centre (CNIO) Madrid, Spain Stéphanie Muller Mouse Clinical Institute Illkirch, France Stefania Murzilli Laboratory of Lipid Metabolism and Cancer Department of Translational Pharmacology Consorzio Mario Negri Sud

Santa Maria Imbaro, Italy Patrick M. Nolan Neurobehavioural Genetics MRC Harwell Harwell Science and Innovation Campus Oxfordshire, United Kingdom Sandra Offner School of Life Sciences and “Frontiers in Genetics” National Program Ecole Polytechnique Fédérale de Lausanne (EPFL) Lausanne, Switzerland Selina Pearson Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton, Cambridge, United Kingdom Kathryn Quick Molecular Nociception Group Wolfson Institute for Biomedical Research University College London London, United Kingdom and London Pain Consortium Kings College London London, United Kingdom Hannah G. Radley-Crabb School of Anatomy and Human Biology The University of Western Australia Crawley, Australia Richard R. Ribchester Euan MacDonald Centre for Motor Neurone Disease Research University of Edinburgh Edinburgh, Scotland, United Kingdom Daniel F. Schorderet IRO-Institute for Research in Ophthalmology Sion, Switzerland, Department of Ophthalmology University of Lausanne Lausanne, Switzerland, and EPFL-Ecole Polytechnique Fédérale Lausanne, Switzerland Manuel Serrano Spanish National Cancer Research Centre (CNIO) Madrid, Spain Radek Skoda Department of Biomedicine Experimental Hematology University Hospital Basel Basel, Switzerland Bart Staels Université Lille Nord de France Lille, France Inserm, U1011 Lille, France UDSL Lille, France Institut Pasteur de Lille Lille, France Karen P. Steel Wellcome Trust Sanger Institute

Wellcome Trust Genome Campus Hinxton, Cambridge, United Kingdom Anne Tailleux Université Lille Nord de France Lille, France Inserm, U1011 Lille, France UDSL Lille, France Institut Pasteur de Lille Lille, France Charles Thomas Center of Phenogenomics (CPG) Ecole Polytechnique Fédérale de Lausanne Lausanne, Switzerland Didier Trono School of Life Sciences and “Frontiers in Genetics” National Program Ecole Polytechnique Fédérale de Lausanne (EPFL) Lausanne, Switzerland Dorothy E. Vatner The University of Medicine & Dentistry of New Jersey New Jersey Medical School Newark, New Jersey Stephen F. Vatner The University of Medicine & Dentistry of New Jersey New Jersey Medical School Newark, New Jersey Sonia Verp School of Life Sciences and “Frontiers in Genetics” National Program Ecole Polytechnique Fédérale de Lausanne (EPFL) Lausanne, Switzerland Xavier Warot Institut Clinique de la Souris (ICS) Illkirch, France and Ecole Polytechnique Fédérale de Lausanne Lausanne, Switzerland Benedikt Wefers German Research Center for Environmental Health Munich, Germany Olivia Wendling Institut Clinique de la Souris (ICS) Illkirch, France and Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/Université Louis Pasteur Illkirch, France John N. Wood Molecular Nociception Group Wolfson Institute for Biomedical Research University College London London, United Kingdom and London Pain Consortium Kings College London London, United Kingdom Christine I. Wooddell Roche Madison Inc. Madison, Wisconsin

Wolfgang Wurst German Research Center for Environmental Health Munich, Germany, Technical University Munich Munich, Germany, Max-Planck-Institute of Psychiatry Munich, Germany, and Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE) Munich, Germany Guofeng Zhang School of Anatomy and Human Biology The University of Western Australia Crawley, Australia Xin Zhao The University of Medicine & Dentistry of New Jersey New Jersey Medical School Newark, New Jersey

Characterization and Validation of Cre-Driver Mouse Lines Françoise Gofflot,1,3 Olivia Wendling,1,2 Nathalie Chartoire,1 Marie-Christine Birling,1 Xavier Warot,1,4 and Johan Auwerx1,4 1

Institut Clinique de la Souris (ICS), Illkirch, France Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, Illkirch, France 3 Université Catholique de Louvain, Life Science Institute, Louvain-la-Neuve, Belgium 4 Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 2

ABSTRACT Conditional gene manipulations in mice are increasingly popular strategies in biomedical research. These approaches rely on the production of conditional genetically engineered mutant mouse (GEMM) lines with mutations in protein-encoding genes. These conditional GEMMs are then bred with one or several transgenic mouse lines expressing a site-specific recombinase, most often the Cre recombinase, in a tissue-specific manner. Conditional GEMMs can only be exploited if Cre transgenic mouse lines are available to generate somatic mutations, and thus the number of Cre transgenic lines has significantly increased over the last 15 years. Once produced, these transgenic lines must be validated for reliable, efficient, and specific Cre expression and Cre-mediated recombination. In this overview, the minimum level of information that is ideally required to validate a Cre-driver transgenic line is first discussed. The vagaries associated with validation procedures are considered next, and some solutions are proposed to assess the expression and activity of constitutive or inducible Cre recombinase before undertaking extensive breeding C 2011 by John experiments and exhaustive phenotyping. Curr. Protoc. Mouse Biol. 1:1-15 Wiley & Sons, Inc. Keywords: site-specific recombination r conditional mutagenesis r inducible Cre r functional genomic

INTRODUCTION Much of the recent progress in mammalian functional genomics has been driven by the use of genetically engineered mutant mouse (GEMM) lines. Informative mutations can now be generated in almost any mouse gene, either through classic gene targeting (conventional germline knockouts) or, increasingly, through conditional gene targeting, a strategy that allows temporal and spatial control of the onset of gene ablation/modification (Lewandoski, 2001; Metzger and Chambon, 2001; Branda and Dymecki, 2004; Argmann et al., 2005). The most successful approach for conditional gene targeting is based on the Cre-loxP system (Sauer and Henderson, 1989; Lakso et al., 1992; Rajewsky et al., 1996; Nagy, 2000; Collins et al., 2007; Birling et al., 2009), in which the allele of interest is flanked by recognition sites for the Cre DNA recombinase, the loxP sites. When such “floxed” mice are bred with transgenic mice expressing the Cre recombinase in a tissue-specific fash-

ion, the gene of interest is knocked out/altered only in this particular tissue. An added sophistication is the inclusion of temporal control, which can be achieved using ligand-activated chimeric recombinases composed, for instance, of the fusion of the Cre recombinase with the ligand-binding domain of a mutated form of the estrogen receptor (ER), which can be activated only by synthetic ER ligands (e.g., tamoxifen), but not by natural estrogen-like compounds (Feil et al., 1996; Danielian et al., 1998; Schwenk et al., 1998; Vasioukhin et al., 1999; Metzger and Chambon, 2001; Hayashi and McMahon, 2002). This strategy avoids problems with early lethality, developmental effects, and compensatory mechanisms, which are often apparent in classical germline or somatic knockout models. In 2006 large collaborative research efforts were launched by the European Commission, the U.S. National Institutes of Health (NIH), and Genome Canada to establish libraries of mutant mouse ES cell lines, each of which

Current Protocols in Mouse Biology 1: 1-15, March 2011 Published online March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/9780470942390.mo100103 C 2011 John Wiley & Sons, Inc. Copyright

Cre-Driver Mouse Lines

1 Volume 1

establishment

carries an altered or “floxed” allele of a single gene (Austin et al., 2004; Auwerx et al., 2004; Collins et al., 2007). These mutant ES cell mutations can be readily transformed into mice using blastocyst injection, and the mutation activated by crossing the mouse bearing the floxed allele with a Cre-driver strain to induce the mutation in spatially and temporally determined patterns. The full power of conditional GEMMs, however, can only be exploited if transgenic mouse lines expressing the Cre recombinase in a tissue-, organ-, and cell-type-specific manner are available to allow the creation of somatic mutations. When searching the literature, investigators will find many Cre-driver transgenic lines that have been used successfully. However, for multiple reasons, data available for mouse Cre lines are often incomplete. Ideally, a minimum level of information should be available to users to allow selection of appropriate Cre transgenic lines for genedeletion experiments–specifically: (1) specificity and efficiency of Cre expression and Cre-mediated deletion; (2) reproducibility of the deletion from animal to animal for the same floxed allele; (3) reproducibility of

transgenic founder transgenic line step 1: Cre expression

validation

the deletion with different floxed alleles; (4) timing of Cre expression and Cre-mediated deletion for noninducible Cre mouse lines; (5) kinetics and efficiency of Cre induction and absence of leakage for inducible Cre mouse lines; and (6) phenotypes caused by either integration-mediated mutagenesis, by Cre “toxicity,” or by passenger genes in the construct. Although community efforts are underway to fully characterize newly produced Credriver mice (e.g., CREATE European Project, http://dev.creline.org/home; Mouse Clinical Institute, http://www.ics-mci.fr/crezoo.html), this ideal level of information is typically not known for currently available mice. To ensure the correct interpretation of resulting phenotypes, it is thus up to investigators to carefully verify the most critical parameters before setting up their experiment. In that context, this overview discusses critical parameters associated with production, validation, and use of Cre-driver transgenic lines, and presents some simple assays that can be used for characterization of these mouse lines. These assays can be combined to characterize newly produced strains, ultimately streamlining the establishment of

no

RT-qPCR

characterization

breeding

yes

G2 - tg

step 2 x Reporter line

no

qPCR

discard

yes

G2 - tg

step 3

step 3: Cre activity anatomical level

x colorimetric reporter line

step 4: phenotyping

clinical phenotyping SHIRPA/biochemistry

histology (␤-gal –AP)

upload information in database


G1 - tg

analysis

discard step 2: Cre activity DNA level

G0 - tg

G2 - tg

ready to be used

Figure 1 Flow chart combining simple assays allowing one to characterize Cre recombinase expression and activity in transgenic Cre lines from the genomic to the cellular level. Each step can also be applied to complete or confirm available data on Cre-driver lines. G, generation; tg, transgenic; β–gal, β-galactosidase; AP, alkaline phosphatase; RT-qPCR, quantitative real-time reverse transcriptase PCR; qPCR, quantitative PCR.

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homogeneously validated Cre lines, or individual assays can be applied to existing lines for which available information is incomplete and/or requires confirmation. A flow chart summarizing the appropriate application of the strategies described in this article is shown in Figure 1.

SPECIFICITY AND EFFICIENCY OF Cre EXPRESSION Because the key feature of conditional gene targeting is its spatial or temporal restriction, the first parameter to control is the fidelity of Cre recombinase expression. Indeed, all systems used to generate tissue-specific Creexpressing mice rely on appropriate promoters and/or enhancers to control the expression

of Cre in a specific cell lineage. However, recombination could occur in cells outside of the desired target tissue due to unexpected or inappropriate expression of the transgene. This can be due to incomplete information about the gene whose promoter is used to drive recombinase expression, a transgene-insertion effect, or, in the case of inducible Cre, a “leakage effect” of the construct in other tissues. A broad, rapid, and cost-effective screen to verify the full expression pattern of the Cre recombinase in the selected transgenic lines can be performed by quantitative real-time reverse transcriptase PCR (RT-qPCR). By testing a large range of organs, information on ectopic or unexpected expression can be obtained rapidly before starting crosses with the floxed-allele transgenic mice.

Table 1 List of 25 Samples for Analysis of Cre Expression by RT-qPCR

System

Organ

Vascular

Aorta Heart

Digestive

Jejunum Colon Liver Pancreas Stomach

Skeletal

Bone

Metabolism

BATa WATb Muscle

Respiratory

Lung

Hematopoietic

Spleen Skin

Nervous

Olfactory bulbs Cortical and subcortical area Hypothalamus Hippocampus and thalamus Cerebellum Brainstem Spinal Cord

Urogenital

Ovary/Testis Kidney

Sensory a BAT, brown adipose tissue. b WAT, white adipose tissue.

Eye Cre-Driver Mouse Lines

3 Current Protocols in Mouse Biology

Volume 1

A pX

ERT2

Cre

RT-qPCR Primers

pA

␤-globin

Ex Ex

In

Ex

B E10

E9

E9 Cre excision

*

Marker loxP

E10

WT allele

RXR␣⌬AF2(LNL)

LNL allele

RXR␣⌬AF2(L)

L allele

loxP

*

E9

RXR␣

E10 loxP

Figure 2 (A) Schematic representation of the CreERT2 transgene used for pronuculei injection (Feil et al., 1997; Indra et al., 1999). The pair of primers used for RT-qPCR amplification of Cre transcripts is located in the β-globin region between a tissue-specific promoter X (pX) and the CreERT2 gene. (B) RXRαAF2(LNL) mouse line. This line is used as a floxed reporter line for determination of Cre recombinase activity by qPCR. A floxed Neo cassette is inserted between exon 9 and 10 of the RXRα gene, and a mutation (*) is present in exon 10 (Mascrez et al., 1998). Three pairs of primers have been designed that allow specific amplification of the wild-type (WT, green), floxed (LNL, blue), and excised (L, pink) alleles. Ex, exon; In, Intron; pA, polyadenylation site; pX, promoter of the gene X.

Experiments can be performed on one male and one female per line, with a minimum number of 25 samples, as suggested in Table 1, in order to cover the major body systems. However, depending on the expected tissue specificity of Cre recombinase expression, additional samples can be added. According to the Cre transgene used to generate the selected Cre-driver line, a set of primers is designed in a common part of the Cre cassette. In the case of the CreERT2 cassette, which has been largely used to produce tamoxifen-inducible Cre expression (Feil et al., 1997; Indra et al., 1999), specific primers can be designed in the β-globin intron (Fig. 2A). As a control of sensitivity, the expression of the endogenous gene whose promoter is used to drive Cre expression should also be measured. Standard validated procedures for RT-qPCR can be applied, as described elsewhere (Bookout et al., 2006; Gofflot et al., 2007; http://empress.har. mrc.ac.uk/), and in accordance with Minimum


Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009; http://www.genequantification.de/miqe.html). As an example of the predictive value of this assay, analysis in the Ppm1a-CreERT2 line is described here, in which CreERT2 expression is driven by the promoter of the ubiquitous “protein phosphatase 1A, magnesiumdependent, alpha isoform” gene (LifschitzMercer et al., 2001). Also described is the Vil1-CreERT2 mouse line, in which the recombinase is targeted to the epithelial cells of the intestinal crypts (Meseguer and Catterall, 1987; Pinto et al., 1999; Robine et al., 1997). Both lines are produced and available at the Mouse Clinical Institute (http://www.icsmci.fr/crezoo.html). Several lines generated after microinjection of the same construct were analyzed, and both the expression pattern and expression level were compared. In the Ppm1a-CreERT2 line, Cre transcripts were

Figure 3 (figure appears on next page) Characterization of Cre expression in the ubiquitous Ppm1a-CreERT2 (A, B) and digestive tract-specific Vil1-CreERT2 (C) mouse lines. (A, C) Comparison of relative Cre expression between different transgenic lines (N is at least 2 for each line) determined by RT-qPCR in 25 tissue samples. (B) Comparison of relative expression of Cre versus the endogenous Ppm1a mRNAs (N = 2).

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0

Figure 3 Line A

Current Protocols in Mouse Biology Muscle Cortex-subcortex

BAT WAT Muscle Cortex-subcortex

WAT

Muscle

Cortex-subcortex

Cerebellum Olfactory bulbs Spinal cord Hypothalamus Hippocampus-thalamus Brainstem Ovary Testis Lung Eye Kidney

Cerebellum Olfactory bulbs Spinal cord Hypothalamus Hippocampus-thalamus Brainstem Ovary Testis Lung Eye Kidney

Cerebellum

Olfactory bulbs

Spinal cord

Hypothalamus

Hippocampus-thalamus

Brainstem Ovary Testis Lung Eye

Kidney

WAT

BAT

Skin

Spleen

Line C

Bones

Stomach

Line B

Pancreas

Liver

Colon

Skin

20

BAT

40

Skin

60 Spleen

80

Spleen

Line B Bones

100

Bones

20

Stomach

40

Stomach

60

Liver

80

Pancreas

Ppm1a

Liver

100

Pancreas

Colon

Duodenum

Heart

Relative expression (%)

Line A

Colon

Duodenum

Cre

Duodenum

C Heart

B

Heart

0 Aorta

0

Aorta


100

Aorta


A Line D

80

60

40

20

(legend appears on previous page)


5

Volume 1

amplified in 25 RNA samples obtained from two animals per line. Relative Cre expression was easily detected in most of the 25 samples analyzed, confirming the ubiquitous nature of the promoter selected (Fig. 3A). One transgenic line (line C) expressed the transgene at a higher level, with little variation between individuals and organs. In these lines, the expression level of Cre mRNA was close to that of the endogenous Ppm1a mRNA, as illustrated in Figure 3B. Notably the testis, an organ expressing high levels of the Ppm1a mRNA, expressed the highest amount of CreERT2 mRNA. Cre, and Ppm1a mRNAs were detected at very low levels in the pancreas, a tissue notorious for its high content of RNase, making RNA extraction challenging (Chirgwin et al., 1979). In contrast, amplification of Cre transcripts in 24 organs from two different Vil1-CreERT2 mouse lines revealed significant Cre mRNA expression only in the jejunum, colon, and testis (Fig. 3C), with relative levels of Cre mRNA being higher in line B. For both lines, the level of Cre expression was in the same range as the level of the endogenous Vil1 mRNA (data not shown). Importantly, this analysis revealed the presence of Cre transcripts in the testis, a site of expression that was not expected for the Vil1 promoter. Infidelity of Cre expression and recombination in the germline has previously been reported by others (Schmidt-Supprian and Rajewsky, 2007). This example emphasizes the importance of verifying ectopic/unexpected expression to avoid misinterpretation of phenotype due to recombination in other cells than the desired target.

SPECIFICITY AND EFFICIENCY OF Cre-MEDIATED DELETION


Although RT-qPCR can provide easy and sensitive detection of Cre expression, the information most needed by investigators is about Cre-mediated recombination. As opposed to Cre expression analysis, Cre activity can only be tested in animals that have been crossed with mouse lines harboring a floxed allele and, for inducible Cre lines, that have been injected either with the inducer or vehicle. Although for most published Cre lines recombination properties have been validated by reporter gene studies, users should be aware that the efficiency of recombination can be locus dependent, and, therefore, the recombination pattern obtained with a particular reporter gene does not necessarily predict that of other floxed genes (see Vooijs et al., 2001; J. Becker and B. Kieffer, pers. comm.). Indeed,

the chromatin structure at the locus of interest, the state of DNA methylation, and the transcriptional activity seem to affect the efficiency of recombination. In addition, it has been reported that the ability of a floxed target gene to be recombined could also vary between cell types (Kellendonk et al., 1999). This was potentially explained by differential accessibility of Cre to loxP sites due to cell type- and development-specific chromatin conformations. Before starting an extensive phenotypic analysis, it is thus mandatory to monitor recombination at the target locus. The most common procedures used to examine the pattern of Cre-mediated recombination in various tissues are Southern blot analysis and simple PCR. Although these procedures are robust, they do not provide a quantitative evaluation of the recombination and are of restricted sensitivity, especially when limited samples are available. An alternative to score for both the efficiency and specificity of Cre recombinase deletion at the DNA level is quantitative PCR. This procedure can (i) provide information on Cre excision efficiency with high sensitivity and reproducibility, (ii) evaluate the reproducibility of deletion from animal to animal on the same floxed alleles, (iii) evaluate the reproducibility of deletion between different floxed lines and, (iv) for inducible Cre lines, verify the efficiency of the tamoxifen induction on the CreERT2 transgene activity. This analysis of Cre recombination activity is performed in animals that have been crossed with any mouse lines harboring a floxed allele, either a so-called “reporter line” or the transgenic line with the floxed allele of interest. As an example, at the Mouse Clinical Institute, the reporter line used to test the recombinase excision activity by qPCR is a floxed RXRα transgenic line, the RXRαAF2 (LNL) (Mascrez et al., 1998; Fig. 2B). Crosses between Cre lines and this reporter line are set up to obtain double transgenic mice, and three pairs of primers are used to specifically amplify the wild-type (WT), floxed (LNL), and excised (L) alleles (Figure 2B). In the case of CreERT2 transgenic animals, bigenic mice are injected either with tamoxifen or vehicle before analysis. For this step, the selection of samples can be made on the basis of the RT-qPCR data, if available—i.e., only positive Cre expression samples are analyzed. Additional samples of one positive system may also be analyzed indepth, e.g., more segments of the digestive tract can be analyzed as illustrated below in the case of the Vil1-CreERT2 .

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A 100% 80% 60% 20% 0% % WT

% LNL

%L

100%

Kidney

Ovary/testis

Spinal cord

Celebellum

Brain

Muscle

Skin

Pancreas

Colon

40% 20% 0%

Liver

80% 60%

Aorta

Proportion of alleles

40%

B 100% 80%

Proportion of alleles

60% 40% 20% 0% % WT

% LNL

%L

100% 80% 60% 40%

Kidney

Ovary/testis

Spinal cord

Celebellum

Cortical and subcortical

Muscle

Tail skin

Pancreas

Liver

Colon

lleum

Jejunum

Duodenum

0%

Aorta

20%

Figure 4 Determination of Cre-mediated excision by qPCR in the ubiquitous Ppm1a-CreERT2 (A) and digestive tract-specific Vil1-CreERT2 (B) mouse lines. Comparison of the percentage of excised allele (L) versus floxed (LNL) and wild type (WT) allele in double transgenic animals Ppm1a-CreERT2 /RXRαAF2(LNL) and Vil1-CreERT2 /RXRαAF2(LNL) mice injected with vehicle (top) or with tamoxifen (bottom). For tamoxifen injections, tamoxifen (Sigma, cat. no. T56648) was prepared at 10 mg/ml in sunflower seed oil (Sigma, cat. no. S5007). Intraperitoneal injection of 100 μl of this solution was performed for 5 consecutive days (1 mg/mouse/day) with mice aged 10 weeks old, and whose weight was >20 g. Identical amounts of sunflower seed oil (vehicle) were administered following the same protocol to control mice.

To illustrate the predictive value and sensitivity of this test, Figure 4 shows the analysis of the Ppm1a-CreERT2 and Vil1-CreERT2 mouse lines. In samples dissected from Ppm1aCreERT2 /RXRαAF2 (LNL) mice injected with vehicle, only the WT and floxed alleles were present, while the excised allele was detected in all the 11 tissues analyzed from tamoxifeninjected double transgenic animals (Fig. 4A). The proportion of excision ranged from 5% to

43%, the WT allele being 50%. The highest level of excision was observed in the skin and liver, and the lowest levels in the three samples from the central nervous system (CNS). The relatively low level of excision in the brain of the Ppm1a-CreERT2 mice, with regard to Cre mRNA expression levels, is most likely due to insufficient entrance of tamoxifen into brain cells. Indeed, induction of Cre activity in the brain seems to be slower than in other organs,



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perhaps due to the blood-brain barrier and/or to the slower renewal of cells. For brain targeting, it is thus recommended to perform analysis 1 month, instead of 1 week, after the last injection of tamoxifen (Metzger and Chambon, 2001; Weber et al., 2001). In comparison, in bigenic Vil1-CreERT2 / RXRαAF2 (LNL) mice injected with tamoxifen, the excised allele was detected only in the digestive tract, at a relative proportion of 15% to 30% (Fig. 4B). No excised allele was detected in the reproductive organs of the selected line, despite the fact that Cre expression was measured by RT-qPCR in the testis. This could be due either to the sensitivity of this test or to lower tamoxifen access to this tissue.

ANATOMICAL PATTERN OF Cre-MEDIATED DELETION As most transgenic Cre lines are driven by cell-specific promoters, the required level of information for validation is the location

of Cre excision activity within specific functional or cellular compartments of an organ. The classic way to characterize Cre activity at the cellular level is to cross Cre-driver lines with colorimetric reporter lines, such as the ROSA26, ACZL, and ZAP reporter mouse lines (Fig. 5). In the ROSA26 reporter line, the ROSA26 allele is targeted with a Cre excision– conditional lacZ reporter (Soriano, 1999). In the ACZL reporter line, a floxed CAT transcription unit prevents lacZ expression in absence of Cre-mediated recombination (Akagi et al., 1997). The Z/AP reporter line (Lobe et al., 1999) utilizes two reporters: the lacZ reporter marks cells before excision occurs, while the heat-resistant human placental alkaline phosphatase (hAP) marks cells after Cre-mediated DNA excision. As for the qPCR test, the analysis is performed on samples dissected from double transgenic animals. However, samples need first to be embedded, sectioned, and stained for either Xgal and/or hAP.

No Cre

ROSA26

pROS A26

PGK Neo pA

loxP

LacZ

Cre

no staining

pA

Ref

Soriano (1999)

loxP

pROS A26

LacZ

-Gal

pA

loxP

p Actin

CAT

pA

ACZL

loxP

p Actin

LacZ

no staining

pA

Akagi et al. (1997)

loxP

LacZ

pA

LacZ

pA

-Gal

loxP

eCMV

p Actin

Z/AP

loxP

eCMV

p Actin

AP

pA

-Gal

Lobe et al. (1999)

loxP

AP

pA

AP

loxP

Figure 5 Schematic representation of the construction and activity of the three most popular colorimetric reporter lines used to test for Cre activity in Cre-driver transgenic lines. β-gal, β-galactosidase; AP, alkaline phosphatase; CMV, cytomegalovirus; PGK, phosphoglycerate kinase; CAT, chloramphenicol acetyltransferase; pA, polyadenylation site. Cre-Driver Mouse Lines

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aorta

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Figure 6 Characterization of Cre activity at histological level in the Ppm1a-CreERT2 mice line. (A) Cre-mediated expression of the reporter gene β-galactosidase in nine organs dissected from double-transgenic Ppm1a-CreERT2 /ROSA26 mice injected with tamoxifen. (B) XGal (ROSA, ACZL) and hAP (Z/AP) staining of muscle sections revealing (i) the reporter expression pattern in three different colorimetric reporter lines crossed with a CMV-Cre deleter mice (bottom row) (Dupe et al., 1997), and (ii) the localization of Cre activity in Ppm1a-CreERT2 mice crossed with each of these reporter lines (top row).

In the case of CreERT2 transgenic animals, bigenic mice are also injected either with tamoxifen or with vehicle 1 week or 1 month before sample collection. The selection of samples could be made on the basis of the RT-qPCR and/or qPCR analysis; in that case, only tissues in which positive Cre expression and/or recombination activity have been ob-

served are further analyzed, limiting the number of samples to be processed. However, for mouse lines in which Cre is targeted not only to a particular tissue but to a specific cell type that may represent only a very small proportion of the organ cellular population, the sensitivity of RT-qPCR or qPCR may be limited, and it is advisable to confirm negative results at the



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histological level to avoid discarding potentially valuable lines. A matter of concern with this procedure is the potential lack of ubiquitous expression of the reporter in existing reporter lines, especially at adult stages. In addition, tissue-specific Cre lines generated by pronuclear injection often exhibit mosaic expression of the recombinase (Schwenk et al., 1998), meaning that only sub-regions of the tissue/organ show active recombination. To illustrate these points, Cre activity was characterized in detail at the cellular level in Ppm1aCreERT2 mice crossed with the ROSA26 reporter line. Bigenic Ppm1a-CreERT2 /ROSA26 mice were injected with either tamoxifen or vehicle. Xgal staining of sections revealed the activity of the Cre recombinase in all but one organ, the muscle, analyzed in tamoxifeninjected double transgenic mice (Fig. 6A). In all organs positively labeled, the area of excision within an organ was mosaic and the cell excision within a tissue was not complete. The liver and skin displayed the highest level of excision, while the lowest level was observed in the brain, in line with the qPCR analysis. No significant Cre activity was detected in mice injected with vehicle (data not shown). The absence of staining observed in the muscle was unexpected in light of the Cre expression and activity data. To confirm this observation, Cre recombination activity was evaluated, and detected, in the muscle after crosses with the ACZL and ZAP reporter mouse lines (Fig. 6B, bottom row). This analysis revealed that absence of Xgal staining in the muscle of Ppm1a-CreERT2 /ROSA26 mice was associated with the ROSA26 reporter line and not with absence of Cre recombination activity. This could be due to the absence or weak expression of the ROSA26 reporter in this tissue, a hypothesis supported by the higher Cre excision level observed in the ACZL and ZAP reporter mouse lines, which express higher levels of the reporter gene in skeletal muscle, as evaluated after crossing with a ubiquitous deleter, the CMV-Cre mouse line (Dupe et al., 1997; Fig. 6B, top row). The cellular characterization of Cre activity in the Ppm1a-CreERT2 underscores the value of using different reporter mouse lines according to the targeted organ/tissue. Among the colorimetric reporter lines classically used in the literature, a preliminary comparison indicated that the ROSA26 line seems the most adequate for the majority of promoters, as it showed reporter activity in the larger number

of organs evaluated after crossing with a ubiquitous Cre-deleter mouse line, while the ACZL and Z/AP displayed expression in a more restricted number of tissues (O. Wendling and D. Metzger, unpub. observ.). Our study, however, revealed the usefulness of these two lines for tissue-specific analysis, e.g., in the skeletal muscle. To address the problem of the lack of ubiquitousness of reporter lines, detection of Cre mRNA or Cre protein at the anatomical level by in situ hybridization (ISH) or immunohistochemistry (IHC) can be used. These two procedures are discussed below.

ADDITIONAL OR ALTERNATIVE PROCEDURES TO DETECT Cre EXPRESSION To map Cre expression at the anatomical level, nonradioactive in situ hybridization (ISH) using digoxigenin-labeled probes can be performed in a standard 5-step procedure: hybridization of the probe to pretreated tissue at 65◦ C; stringent post-hybridization washes; blocking steps to prepare for the immunodetection; primary antibody anti-DIGAP incubation; and colorimetric alkaline phosphatase detection (Chotteau-Lelievre et al., 2006; Gofflot et al., 2007; Knoll et al., 2007). Although this procedure may require a higher level of expertise, it also has several advantages. It can be used in parallel with, or in place of, the RT-qPCR, as it scores for Cre expression and does not require crosses with a transgenic floxed line. As such, it is thus totally independent of any reporter activity, and also independent with respect to cellular access of inducer such as tamoxifen. It can be combined with other detection procedures, either by double in situ hybridization or immunohistochemistry, allowing precise characterization of the cellular population targeted by Cre expression. For those less familiar with ISH, immunodetection procedures for Cre protein have been previously described (Kaelin et al., 2006; Knoll et al., 2007). To illustrate these two procedures, two inducible tissue-specific Cre lines were used: the Tph2-CreERT2 (gift of P. Chambon and D. Metzger) and the Ins1-CreERT2 , in which Cre expression is targeted to highly restricted cell populations, the raphe serotonergic neurons and the β-cells of pancreatic islets, respectively. As illustrated in Figure 7, ISH was successfully used to detect Cre mRNA in the raphe nuclei of the Tph2-CreERT2 mice (Fig. 7A). Cre expression was similar to lacZ staining on brain sections from tamoxifen-injected

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Tph2-CreERT2/ROSA26

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Ins1-CreERT2/ROSA26

Figure 7 Characterization of Cre activity at histological level in the Tph2-CreERT2 and Ins1CreERT2 mouse lines. (A) Sections through the pons, midbrain, and medulla revealing Cre activity in the raphe nuclei through Cre-mediated expression of the reporter gene β-galactosidase (Tph2CreERT2 /ROSA26 mice injected with tamoxifen, top row) and Cre expression through ISH with a specific Cre probe (Tph2-CreERT2 mice, bottom row). (B) IHC detection of Cre protein and Xgal staining of pancreas sections of tamoxifen or vehicle-injected Ins1-CreERT2 /ROSA26 mice revealed specific Cre protein content and Cre activity restricted to the islets of Langerhans. ISH procedures have been described elsewhere (Chotteau-Lelievre et al., 2006; Gofflot et al., 2007). For IHC, primary rabbit anti-Cre (1:8000 dilution, VWR, cat. no. 69050-3) was used with goat antirabbit antibody coupled to horseradish peroxidase (1:100 dilution; Invitrogen, cat. no. G-21234) as secondary antibody. After washing, Cre was visualized by FITC-tyramide amplification (1/50, 30-min incubation) (PerkinElmer, cat. no. SAT701B).

Tph2-CreERT2 /ROSA26 mice, revealing that all 9 nuclei of the raphe in the midbrain and medulla were specifically labeled. As demonstrated by the analysis of the Ppm1a-CreERT2 line, the pancreas is an organ for which the isolation of RNA is particularly challenging (Chirgwin et al., 1979) and Cre mRNA could not be detected reliably in pancreas samples of Ins1CreERT2 mice. In that particular case, IHC was used and allowed the detection of Cre protein in the islets of Langerhans of the

Ins1-CreERT2 mice (Fig. 7B). This result was further confirmed at the histological level in Ins1-CreERT2 /ROSA26 mice, in which specific Cre-mediated excision was detected by lacZ staining in the islets of Langerhans of the pancreas.

PHENOTYPIC CHARACTERIZATION OF Cre LINES Transgenic lines produced by conventional transgenesis can develop unexpected



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phenotypes due to integration-mediated mutagenesis or passenger genes in the construct (Lusis et al., 2007). In addition, a high level of Cre protein expression can result in cellular toxicity (Forni et al., 2006; Schmidt-Supprian and Rajewsky, 2007). In that context, successfully characterized lines should also be subjected to a final functional test. Indeed, functional abnormalities in Cre mice could be a confounding factor for the interpretation of the phenotype observed when that Cre line is used to delete a gene of interest. When producing a new Cre-driver transgenic line, a standard and simple behavioral, biochemical, and metabolic phenotyping procedure would allow one to discard Cre mouse lines with interfering phenotypes before distribution and archiving. First, to evaluate the general health and basic neurological status, the modified SHIRPA protocol (http://empress.har.mrc.ac.uk/browser/) can be used, as it is a rapid, high-throughput non-invasive and non-stressful test suited for a global evaluation of the phenotype (Mandillo et al., 2008). Second, clinical and

basal metabolic parameters in G2 mice maintained under basal chow–fed conditions should be monitored (Champy et al., 2004). Finally, some specific tests could be used to evaluate the functions of the organ(s) to which Cre expression is specifically targeted. For example, blood pressure, heart rate, heart weight, and histology could be specifically evaluated in mice with Cre targeted to cardiac muscle, while rotarod test, grip strength, endurance running, and muscle histology could be investigated in mice with Cre targeted to skeletal muscle (see http://empress.har.mrc.ac.uk/browser/ for detailed procedures). Instructive of the importance of such a phenotypic characterization, the Vil1-CreERT2 mice have a severe functional abnormality which is due to the presence of a passenger gene, the G-protein coupled receptor Tgr5 (Thomas et al., 2008) in the BAC construct, precluding its use for metabolic studies (Fig. 8A). Tgr5 mRNA is 5-fold overexpressed in the Vil1-CreERT2 mouse line

A BAC RP23-278N11 Chromosome 1 :74,302,024-74,495,075

193.05 kb

Pnkd ENSMUSG00000026179 Arpc2 ENSMUSG00000006304

Vil 1 ENSMUSG00000026175 Ctdsp1 ENSMUSG00000026176

Tgr5 (Gpbar1) ENSMUSG00000064272 Gm216 ENSMUSG00000073650 Aamp ENSMUSG00000006299 Slc11a1 ENSMUSG00000026177 Tmbim1 ENSMUSG00000006301 Usp37 ENSMUSG00000033364

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Figure 8 Phenotypic analysis of the Vil1-CreERT2 transgenic line. (A) Schematic representation of the mouse BAC used for the construction of the Vil1-CreERT2 transgene. (B) Expression of mRNA levels of the GPCR Tgr5 in the ileum of control and Vil1-CreERT2 transgenic mice. (C) Mean ± SD of the area under the curve during an oral glucose tolerance test in control (N = 8) and Vil1-CreERT2 (N = 8) mice fed a high-fat, high-sucrose diet for 12 weeks. (D-E) Insulin and GLP-1 levels measured in the serum 15 min after the administration of the oral glucose load in the control and Vil1-CreERT2 transgenic mice of panel (B). Cre-Driver Mouse Lines

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(Fig. 8B). As Tgr5 controls the expression of GLP-1 in entero-endocrine cells (Thomas et al., 2009), which in turn improves insulin secretion, Vil1-CreERT2 mice have improved glucose tolerance with a reduced area under the curve (AUC) in oral glucose tolerance tests (Fig. 8C). The higher insulin release as a consequence of the increase of GLP-1 levels after a glucose challenge explains the improved glucose tolerance (Fig. 8D-E). In other cases, the phenotype could be the consequence of insertional mutagenesis. In such conditions, it can be worth testing Cre lines derived from another founder.

SUMMARY Advances in the sophisticated manipulation of the mouse genome now allow the generation of mutant mice for disease modeling and functional analysis by conditional mutagenesis. In the coming years, site-specific recombination transgenic mice will become necessary tools for most scientists to generate conditional mutations. Although a few initiatives attempt to establish and/or index a range of transgenic mouse lines that express Cre recombinase in different tissues (e.g., Gensat, http://www.gensat.org/index. html; CreXMice, http://nagy.mshri.on.ca/cre/; MUGEN Mutant Mice database, http://bioit. fleming.gr/mugen/mde.jsp; CREATE, http:// dev.creline.org/home), most of the current data are disparate, heterogenous, and incomplete. It will thus be up to Cre users to thoroughly characterize and/or validate the appropriate Cre-driver mouse lines before undertaking extensive breeding experiments and exhaustive mutant analyses that could otherwise lead to inconclusive or incorrect conclusions. As already mentioned, investigators will have to test their selected Cre lines on their own conditional mutants, as efficiency of Cre-mediated excision varies from one allele to another. When bigenic mice are generated and phenotyped, it is mandatory to check in parallel that Cre-mediated excision has indeed occurred in the organ of interest. In this overview, a set of basic assays is described that can be used either to confirm existing data or as a streamlined and standardized characterization scheme for newly established Cre lines. Some of the artifacts and problems that can be associated with Cre-driver mouse lines are also discussed, with emphasis on the importance of the characterization steps. Finally, the more a given Cre line is used, the more of these issues will be evaluated.

Investigators using Cre mouse lines are therefore encouraged to upload their information onto databases where all information is centralized and available to the scientific community, as exemplified by the Cre-X-Mice database (Nagy et al., 2009).

ACKNOWLEDGMENTS The authors acknowledge Fabrice Augé, Graziella Neau, Leila El Fertak, and the MCI histopathology service for technical assistance. We are indebted to Kristina Schoonjans and Charles Thomas for the phenotypic analysis of the Vil1-CreERT2 mouse line and to Jabier Gallego-Llamas for IHC studies. We wish to thank Jean-Louis Mandel, Guillaume Pavlovic, Lydie Venteo, and Thomas F. Vogt for critical reading of the manuscript. The “CreZoo” project at the MCI was initiated under the auspices of Professors Pierre Chambon and Daniel Metzger. This work was supported in part by an INCa grant and by the EUCOMM European integrated project.

LITERATURE CITED Akagi, K., Sandig, V., Vooijs, M., Van der Valk, M., Giovannini, M., Strauss, M., and Berns, A. 1997. Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res. 25:1766-1773. Argmann, C.A., Chambon, P., and Auwerx, J. 2005. Mouse phenogenomics: The fast track to “systems metabolism”. Cell Metab. 2:349360. Austin, C.P., Battey, J.F., Bradley, A., Bucan, M., Capecchi, M., Collins, F.S., Dove, W.F., Duyk, G., Dymecki, S., Eppig, J.T., Grieder, F.B., Heintz, N., Hicks, G., Insel, T.R., Joyner, A., Koller, B.H., Lloyd, K.C., Magnuson, T., Moore, M.W., Nagy, A., Pollock, J.D., Roses, A.D., Sands, A.T., Seed, B., Skarnes, W.C., Snoddy, J., Soriano, P., Stewart, D.J., Stewart, F., Stillman, B., Varmus, H., Varticovski, L., Verma, I.M., Vogt, T.F., von Melchner, H., Witkowski, J., Woychik, R.P., Wurst, W., Yancopoulos, G.D., Young, S.G., and Zambrowicz, B. 2004. The knockout mouse project. Nat. Genet. 36:921-924. Auwerx, J., Avner, P., Baldock, R., Ballabio, A., Balling, R., Barbacid, M., Berns, A., Bradley, A., Brown, S., Carmeliet, P., Chambon, P., Cox, R., Davidson, D., Davies, K., Duboule, D., Forejt, J., Granucci, F., Hastie, N., de Angelis, M.H., Jackson, I., Kioussis, D., Kollias, G., Lathrop, M., Lendahl, U., Malumbres, M., von Melchner, H., Müller, W., Partanen, J., Ricciardi-Castagnoli, P., Rigby, P., Rosen, B., Rosenthal, N., Skarnes, B., Stewart, A.F., Thornton, J., Tocchini-Valentini, G., Wagner, E., Wahli, W., and Wurst, W. 2004. The European dimension for the mouse genome mutagenesis program. Nat. Genet. 36:925-927.



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Birling, M.C., Gofflot, F., and Warot, X. 2009. Sitespecific recombinases for manipulation of the mouse genome. Methods Mol. Biol. 561:245263. Bookout, A.L., Cummins, C.L., Mangelsdorf, D.J., Pesola, J.M., and Kramer, M.F. 2006. High-throughput real-time quantitative reverse transcription PCR. Curr. Protoc. Mol. Biol. 73:15.8.1-15.8.28. Branda, C.S. and Dymecki, S.M. 2004. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6:7-28. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., and Wittwer, C.T. 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55:611-622. Champy, M.F., Selloum, M., Piard, L., Zeitler, V., Caradec, C., Chambon, P., and Auwerx, J. 2004. Mouse functional genomics requires standardization of mouse handling and housing conditions. Mamm. Genome 15:768-783. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. Chotteau-Lelievre, A., Dolle, P., and Gofflot, F. 2006. Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes. Methods Mol. Biol. 326:61-87. Collins, F.S., Rossant, J., and Wurst, W. 2007. A mouse for all reasons. Cell 128:9-13. Danielian, P.S., Muccino, D., Rowitch, D.H., Michael, S.K., and McMahon, A.P. 1998. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8:1323-1326. Dupe, V., Davenne, M., Brocard, J., Dolle, P., Mark, M., Dierich, A., Chambon, P., and Rijli, F.M. 1997. In vivo functional analysis of the Hoxa1 3 retinoic acid response element (3 RARE). Development 124:399-410. Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. 1996. Ligandactivated site-specific recombination in mice. Proc. Natl. Acad. Sci. U.S.A. 93:10887-10890. Feil, R., Wagner, J., Metzger, D., and Chambon, P. 1997. Regulation of Cre recombinase activity by mutated estrogen receptor ligandbinding domains. Biochem. Biophys. Res. Commun. 237:752-757. Forni, P.E., Scuoppo, C., Imayoshi, I., Taulli, R., Dastru, W., Sala, V., Betz, U.A., Muzzi, P., Martinuzzi, D., Vercelli, A.E., Kageyama, R., and Ponzetto, C. 2006. High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly. J. Neurosci. 26:9593-9602. Cre-Driver Mouse Lines

Gofflot, F., Chartoire, N., Vasseur, L., Heikkinen, S., Dembele, D., Le Merrer, J., and Auwerx, J.

2007. Systematic gene expression mapping clusters nuclear receptors according to their function in the brain. Cell 131:405-418. Hayashi, S. and McMahon, A.P. 2002. Efficient recombination in diverse tissues by a tamoxifeninducible form of Cre: A tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244:305-318. Indra, A.K., Warot, X., Brocard, J., Bornert, J.M., Xiao, J.H., Chambon, P., and Metzger, D. 1999. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: Comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 27:43244327. Kaelin, C.B., Gong, L., Xu, A.W., Yao, F., Hockman, K., Morton, G.J., Schwartz, M.W., Barsh, G.S., and MacKenzie, R.G. 2006. Signal transducer and activator of transcription (stat) binding sites but not stat3 are required for fasting-induced transcription of agouti-related protein messenger ribonucleic acid. Mol. Endocrinol. 20:2591-2602. Kellendonk, C., Tronche, F., Casanova, E., Anlag, K., Opherk, C., and Schutz, G. 1999. Inducible site-specific recombination in the brain. J. Mol. Biol. 285:175-182. Knoll, J.H., Lichter, P., Bakdounes, K., and Eltoum, I.-E. A. 2007. In situ hybridization and detection using nonisotopic probes. Curr. Protoc. Mol. Biol. 79:14.7.1-14.7.17. Lakso, M., Sauer, B., Mosinger, B. Jr., Lee, E.J., Manning, R.W., Yu, S.H., Mulder, K.L., and Westphal, H. 1992. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 89:62326236. Lewandoski, M. 2001. Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2:743755. Lifschitz-Mercer, B., Sheinin, Y., Ben-Meir, D., Bramante-Schreiber, L., Leider-Trejo, L., Karby, S., Smorodinsky, N.I., and Lavi, S. 2001. Protein phosphatase 2Calpha expression in normal human tissues: An immunohistochemical study. Histochem. Cell Biol. 116:31-39. Lobe, C.G., Koop, K.E., Kreppner, W., Lomeli, H., Gertsenstein, M., and Nagy, A. 1999. Z/AP, a double reporter for cre-mediated recombination. Dev. Biol. 208:281-292. Lusis, A.J., Yu, J., and Wang, S.S. 2007. The problem of passenger genes in transgenic mice. Arterioscler Thromb. Vasc. Biol. 27:2100-2103. Mandillo, S., Tucci, V., Hölter, S.M., Meziane, H., Banchaabouchi, M.A., Kallnik, M., Lad, H.V., Nolan, P.M., Ouagazzal, A.M., Coghill, E.L., Gale, K., Golini, E., Jacquot, S., Krezel, W., Parker, A., Riet, F., Schneider, I., Marazziti, D., Auwerx, J., Brown, S.D., Chambon, P., Rosenthal, N., Tocchini-Valentini, G., Wurst, W. 2008. Reliability, robustness, and reproducibility in mouse behavioral phenotyping: A crosslaboratory study. Physiol. Genomics 34:243255.

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Mascrez, B., Mark, M., Dierich, A., Ghyselinck, N.B., Kastner, P., and Chambon, P. 1998. The RXRalpha ligand-dependent activation function 2 (AF-2) is important for mouse development. Development 125:4691-4707. Meseguer, A. and Catterall, J.F. 1987. Mouse kidney androgen-regulated protein messenger ribonucleic acid is expressed in the proximal convoluted tubules. Mol. Endocrinol. 1:535541. Metzger, D. and Chambon, P. 2001. Site- and timespecific gene targeting in the mouse. Methods 24:71-80. Nagy, A. 2000. Cre recombinase: The universal reagent for genome tailoring. Genesis 26:99109. Nagy, A., Mar, L., and Watts, G. 2009. Creation and use of a cre recombinase transgenic database. Methods Mol. Biol. 530:1-14. Pinto, D., Robine, S., Jaisser, F., El Marjou, F.E., and Louvard, D. 1999. Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines. J. Biol. Chem. 274:6476-6482. Rajewsky, K., Gu, H., Kuhn, R., Betz, U.A., Muller, W., Roes, J., and Schwenk, F. 1996. Conditional gene targeting. J. Clin. Invest. 98:600-603. Robine, S., Jaisser, F., and Louvard, D. 1997. Epithelial cell growth and differentiation. IV. Controlled spatiotemporal expression of transgenes: New tools to study normal and pathological states. Am. J. Physiol. 273:G759-G762. Sauer, B. and Henderson, N. 1989. Cre-stimulated recombination at loxP-containing DNA

sequences placed into the mammalian genome. Nucleic Acids Res. 17:147-161. Schmidt-Supprian, M. and Rajewsky, K. 2007. Vagaries of conditional gene targeting. Nat. Immunol. 8:665-668. Schwenk, F., Kuhn, R., Angrand, P.O., Rajewsky, K., and Stewart, A.F. 1998. Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acids Res. 26:1427-1432. Soriano, P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21:70-71. Thomas, C., Pellicciari, R., Pruzanski, M., Auwerx, J., and Schoonjans, K. 2008. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov. 7:678-693. Thomas, C., Gioiello, A., Noriega, L., Strehle, A., Oury, J., Rizzo, G., Macchiarulo, A., Yamamoto, H., Mataki, C., Pruzanski, M., Pellicciari, R., Auwerx, J., and Schoonjans, K. 2009. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab. 10:167-177. Vasioukhin, V., Degenstein, L., Wise, B., and Fuchs, E. 1999. The magical touch: Genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl. Acad. Sci. U.S.A. 96:8551-8556. Vooijs, M., Jonkers, J., and Berns, A. 2001. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep. 2:292-297. Weber, P., Metzger, D., and Chambon, P. 2001. Temporally controlled targeted somatic mutagenesis in the mouse brain. Eur. J. Neurosci. 14:1777-1783.



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Standardized Post-Mortem Examination and Fixation Procedures for Mutant and Treated Mice Cristina Antal,1,4 Stéphanie Muller,1 Olivia Wendling,1,2 Yann Hérault,1,2 and Manuel Mark1,2,3 1

Mouse Clinical Institute, Illkirch, France Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/Université Louis Pasteur, Illkirch, France 3 Hôpital Universitaire de Strasbourg, Strasbourg, France 4 Institut d’Histologie, Faculté de Médecine, Strasbourg, France 2

ABSTRACT A procedure for post-mortem examination (or necropsy) of mice is provided. The aim is to obtain a “holistic” picture of organs and systems at the anatomical and histological levels. The major issue is tissue preservation, which is achieved by rapid transfer into a fixative solution, usually neutral buffered formalin. Fixation is the first of the four basic steps in histopathological analyses of tissues, which also include embedding, sectioning, and staining. The protocols provided here describe routine methods for tissue fixation, as these methods are integral parts of any necropsy procedure. There is also a Strategic Planning section that addresses the overall approach to histopathological evaluation, as well as specifics such as age and gender of the mice, cohort size, and controls. Curr. C 2011 by John Wiley & Sons, Inc. Protoc. Mouse Biol. 1:17-53 Keywords: phenotyping r tissue collection r histology r necropsy r pathology

INTRODUCTION After establishing a phenotype in a mouse line through the use of in vivo tests, the subsequent challenge is to refine this phenotype at the organ, tissue, cell, and molecular (DNA, RNA, and protein) levels. This is most easily achieved after sacrificing the mice. This article focuses on collection of murine tissues for standard morphological analyses. A dissection for the purpose of post-mortem analysis, or necropsy, is undertaken to identify, at a macroscopic level (by the naked eye or with a dissecting microscope), morphological defects that characterize the mutant mouse and identify gross lesions that may contribute to morbidity and mortality. After the general condition and the body weight of the animal have been recorded, individual organs are removed, examined, sampled, and fixed in a systematic manner. All lesions should be described with regard to location, color, size, shape, consistency, distribution, and number or percent of involvement of a specific organ. Photographs of lesions provide documentation for records. Tissues that are collected for subsequent histopathological analyses require appropriate handling and preservation to prevent their deterioration. Once death has occurred, tissues undergo a process of self-digestion (autolysis). One major aim of necropsy is to avoid any unnecessary delay in collecting tissue samples, so that they can be optimally preserved through rapid immersion into a fixative solution. The article begins with a Strategic Planning section that addresses the approach to histopathological evaluation of mutant or treated mice. It then provides the information for collecting tissues to obtain a complete, standardized, macroscopic, and histological analysis of the mouse (see Basic Protocol 1). Proper fixation of murine tissues with formalin (see Basic Protocol 2) or Bouin’s solution (see Alternate Protocol) is also described. Current Protocols in Mouse Biology 1: 17-53, March 2011 Published online March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/9780470942390.mo100118 C 2011 John Wiley & Sons, Inc. Copyright

Post-Mortem Examination and Fixation of Mice

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STRATEGIC PLANNING NOTE: mutant mice are used hereafter as examples, but the proposed screen is applicable to mice treated with test compounds or tumor inducers as well. As histotechnology is time-consuming and labor-intensive, it is important to avoid unnecessary experiments while achieving the highest degree of comprehensiveness. To this end, it is best to perform a first-line screen followed by organ/pathology-specific targeted assays. An in-depth evaluation of organs that have been fixed, processed, and embedded in a systematic manner is used for first-line extended assays. Such assays involve readyto-use tissue samples and are rapidly applied after completion of the systematic screen. In contrast, secondary assays correspond to in-depth evaluations of organs requiring special fixation, tissue processing, and/or embedding procedures, and thus the generation of a new cohort of mice. A first-line histological screen is aimed at detecting the broadest array of tissue abnormalities in the mutant mouse line. Since each of the tissue defects represents a potentially invaluable clue toward understanding the physiological functions of the mutated gene, their inventory should be as exhaustive as possible. However, listing all the histological abnormalities in a given mouse is useless if they are not correlated with the genetic alteration. Thus, the overall goal of histopathology is to characterize the tissue lesions and ensure they are caused by the genetic alteration. This latter goal can only be achieved if proper control mice are analyzed along with those bearing the genetic alteration, and by having a comprehensive knowledge of the phenotype of the background or control strain to recognize true deviations from “normal.”

Importance of performing systematic analyses At least two scenarios can apply to mutant mouse lines entering a histological screen. The “black box” scenario corresponds to situations where the mutant mice have no overt clinical phenotype, when they die post-natally at variable ages for unknown reasons (Turgeon and Meloche, 2009), and/or when the expression domain of the mutated gene is widespread and a large number of tissues are potentially targeted. In contrast, a mutant mouse line entering a systematic histological screen may display consistent clinical and/or histological defects restricted to a single organ or to a small subset of organs expressing the gene under study. However, altering the functions of a single organ may have broad and unexpected secondary consequences on the whole organism. Thus, almost any mutant mouse is eligible for a systematic screen, even when it carries an organ-specific gene alteration generated through somatic mutagenesis. For instance, mice carrying targeted ablation of retinoid X receptors (RXRs) only in the epidermis develop a systemic syndrome, mimicking that observed in atopic dermatitis patients, including lymph node hyperplasia, splenomegaly, and eosinophilic infiltrates in the liver, lung, and heart (Li et al., 2005).

Post-Mortem Examination and Fixation of Mice

Optimal age for analyzing a mutant mouse line Mice are weaned and sexually mature at 3 and 6 weeks, respectively. Likewise, histogenesis is completed at 3 weeks in most tissues and at 6 weeks in reproductive organs. Although a 6-week-old mouse can be considered a young adult, necropsies and histological analyses are in general postponed until ∼4 months, which is the time required for the completion of in vivo phenotyping tests. Four month-old mice are sexually mature, do not have age-related pathology, and are in an age range for which data on inbred strains are readily available. It should be stressed that a number of mutations affect the propensity to develop age-related diseases. Thus, although the cost of maintaining aging mice is prohibitive, it is sometimes necessary to investigate old (i.e., >12 months) and even senescent (i.e., > 24 months) subjects for late-onset diseases such as degenerative tissue changes and cancers (Harvey et al., 1993; Fan et al., 1998; Huang et al., 2002;

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Kim et al., 2002; Khetchoumian et al., 2007). For instance, Trim24-null (Trim24-/- ) mutants show no histopathological abnormalities at 2 months of age. However, monitoring a large cohort of Trim24-deficient mice (n > 100) for a long time period allows detection of hepatic tumors at necropsy in 80% of male and 69% of female Trim24-null mice between months 9 and 29, as compared to only 4% of age-matched, wild-type control (Trim24+/+ ) mice (Khetchoumian et al., 2007). Conversely, if the mutants die before 4 months of age, it will be necessary to determine the window of lethality before selecting an age group for morphological evaluation. It may also be necessary to study the mutant line at several different ages to accurately follow the progression of the phenotypic changes, and thus understand the biological functions of the mutated gene.

Gender The question regarding the use of males or females in histological investigations is not trivial, as male physiology is not subject to the cyclical changes that, in females, can present another source of phenotypic variability. Upon initial consideration, one might imagine systematically analyzing males and restricting the analysis of female organs to those exhibiting sexual dimorphism (reproductive organs, adrenal glands, hypophysis, kidneys, and salivary glands). However, in mutant lines, gender-specific tissue alterations have been reported in organs that do not display sexually dimorphic histological features (Costet et al., 1998; Liu et al., 2000; Khetchoumian et al., 2007). Therefore, in a firstline histological screen, an equal number of males and females should be systematically analyzed. Control mice and the importance of comparative histopathology To interpret histological data, it is important to have a comprehensive knowledge of the phenotypic peculiarities and common lesions present in the inbred strains used to generate the experimental mouse line. The same holds true with respect to the environment of the experimental mouse line (pathogen status of the animal facility, chlorine or antibiotics in drinking water, and so on). This information must be taken into account to interpret possible discrepancies in data from different laboratories. Common pathologies of inbred strains are mentioned in Brayton et al. (2001) and Naf et al. (2002). It is mandatory, when designing a histological screen, to compare mutant mice to control mice that share the closest possible genetic background and that are bred under identical conditions. Size of cohort and the first-line phenotyping strategy The size of a mouse cohort should be sufficient to allow meaningful scientific interpretation of the data. Scientific statements are generally considered acceptable if they are characterized by an uncertainty of 10 MHz are generally selected for mouse echocardiography. This is necessary because of the small size of the heart and its rapid rate of contraction. In the authors’ core echo laboratory, there is one Siemens Sequoia C256 with a 13-MHz linear transducer, one GE Vivid7 with an i13L probe (14 MHz), and one VisualSonics Vevo770 with 30- and 40-MHz probes for mouse cardiac and vascular examination.

Mouse echo protocol 1. The mouse is injected intraperitoneally with 2.5% avertin, 0.012 ml/g body weight (300 mg/kg). Heart rates are monitored and generally maintained at 400 to 500 beats/min. 2. When using the Siemens Sequoia C256 or GE Vivid7, the chest hair is shaved and EKG needle leads are connected to the limbs for electrocardiogram gating. The mouse is then placed on a warm pad to maintain the body temperature ∼37◦ C. A rectal thermometer is inserted for monitoring the body temperature. 3. Warmed echo gel is placed on the shaved chest. The mouse heart is imaged with a 13-MHz linear transducer (Siemens Sequoia C256) or 14-MHz probe (GE Vivid7) while the mouse lies on the warm pad at a shallow left-side position. 4. When using the VisualSonics Vevo 770, due to the much higher probe frequency and interference from remaining hair, a depilatory lotion is applied to the chest to help facilitate complete removal of hair. The platform temperature of the equipment is set at 40◦ to 42◦ C, which is higher than optimal animal core temperature to help maintain the mouse core temperature at 37◦ C. The mouse is placed onto the warm plate in the supine position and the limbs are taped onto the metal EKG leads. For cardiac imaging, the 30-MHz transducer is used, while the 40-MHz transducer is utilized for vascular imaging. 5. By placing the transducer along the long-axis of LV, and directing it to the right side of the neck of the mouse, two-dimensional (2-D) LV long-axis is obtained. Then the transducer is rotated clockwise by 90◦ , and the LV short-axis view is visualized. The diagrams showing the positions and directions of the transducer for basic mouse echo views are demonstrated in Figure 1. 2-D-guided LV M-mode at the papillary muscle level is recorded from either the short-axis view and/or the long-axis view. Transmitral inflow Doppler spectra are recorded in an apical four-chamber view by placing the sample volume at the tip of the mitral valves. Angle correction can be used for accurate flow velocity measurements. Doppler waveforms from other regions of the heart can be recorded as needed.

Echocardiography in Mice


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A

AO LV

LA

B

LV

Figure 1 Diagrams for basic mouse echocardiography views. A shows the position and direction (small arrow) of probe (upper left) for LV long-axis view (upper right). B demonstrates the position and direction (small arrow) of probe (lower left) for LV short-axis view (lower right). LA, left atrium; AO, aorta.

6. After the scanning is finished, the residual echo gel is removed, and the mouse is returned to the cage for recovery. 7. Echo images are downloaded and analyzed offline using Scion images software or an echo work station. At least three beats need to be measured and averaged for the interpretation of any given measurement. From the authors’ experience, at least five mice are normally needed per experimental group to show statistically significant relevance.

CONSIDERATIONS IN MURINE ECHOCARDIOGRAPHY To obtain consistent, reproducible echocardiographic data, in addition to obtaining good images, the condition of the animals must be controlled. Therefore, adhere to the following parameters during echo scanning. 1. The mouse body temperature should be carefully monitored and maintained at 37◦ C during the entire procedure. 2. Since cardiac function is closely related to heart rate, the heart rate should be controlled at a similar level within each strain of mice. Therefore, the choice of anesthetic agent, dose, and dosing interval should be carefully reproduced and considered. From the authors’ experience, the variation of HR within 100 bpm for a strain/set of experiments should be acceptable. Echocardiography in Mice

3. Echo measurement time should be similar after anesthesia to minimize the effects of changes in anesthetic levels with time on echocardiography parameters.

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ECHO MEASUREMENTS LV systolic function LV interventricular septal thicknesses (IVS), LV internal dimensions (LVID), and posterior wall thicknesses (PW) at diastole and systole (IVSd, LVIDd, PWd, and IVSs, LVIDs, PWs, respectively) are measured from M-mode images at the level of the papillary muscles. An example of LV M-mode in mice is displayed in Figure 2A. LV ejection fraction (EF), LV fractional shortening (FS), and LV posterior wall thickening (PWT) are calculated by using the following formulas (Gardin et al., 1995; Tanaka et al., 1996; Syed et al., 2005; Tsujita et al., 2005): EF (%) = 100 × [(LVIDd3 – LVIDs)3 )/LVIDd3 ] FS (%) = 100 × [(LVIDd – LVIDs)/LVIDd] PWT (%) = 100 × [(PWs – PWd)/PWd] LV ejection fraction (EF) and LV fractional shortening (FS) are measured for evaluation of LV global systolic function. When the LV contracts without regional wall motion

A

B

Sa

IVSd

IVSs

LVIDd

LVIDs

PWd

C

PWs

Ea ET

Aa

IVCT IVRT

LV fractional shortening (FS%)

Figure 2 Images of echocardiographic measurements in mice. (A) LV M-mode, allows for assessment of LV systolic function. IVSd, LVIDd, PWd, and IVSs, LVIDs, PWs are LV interventricular septum thicknesses, LV internal dimensions and LV posterior wall thicknesses at diastole and systole, respectively. (B) Doppler of transmitral inflow most often used for evaluation of LV diastolic function. E and A are peak velocities at early and late filling, respectively. IVRT and IVCT are isovolumetric relaxation and contraction time. ET is LV ejection time. (C) Tissue Doppler waveform obtained in LV posterior wall, used for assessing regional wall motion abnormality. Ea and Aa were two waveforms at early and late diastolic phases. Sa is the peak wall motion velocity in systole.

40

FVB C57BL/6J

35

*

30 25

*

*

20

*

15 Baseline

1W-TAC

2W-TAC

3W-TAC

Figure 3 Comparing fractional shortening (FS) in two strains of mice (FVB, C57BL/6J) before and after 1, 2, and 3 weeks of pressure overload induced by transverse aortic constriction (TAC). FS was significantly decreased in C57BL mice (square) even 1 week after TAC. However, in FVB mice (triangle) FS was maintained at normal levels even after 2 weeks of TAC. *p >A. Since diastolic dysfunction progresses rapidly in mice, multiple different Doppler patterns may exist in the same group of surgically modeled or genetically altered mice and this may lead to misinterpretation of the stage of diastolic dysfunction. Thus, confirming Doppler measurements by other methods such as tissue Doppler, color M-mode Doppler, or pressure measurements, is essential. The Doppler parameters at baseline and 2 weeks after TAC in FVB mice were measured; diastolic dysfunction was evident in the echocardiogram, as reflected by decreased A wave velocity, increased E/A ratio, and an increased index (IVRT + IVCT)/ET, implying increased stiffness of LV after TAC as seen in Table 2. However, systolic function in these same FVB mice was maintained 2 weeks after TAC (Figure 3). Tissue Doppler imaging Tissue Doppler imaging (TDI) is tissue motion velocity obtained from the mitral annulus or LV posterior wall from the myocardium, which normally consists of three basic waveforms: two in early and late diastole (Ea and Aa, respectively), and one in systole (Sa). Decreased Ea/Aa ratio indicates diastolic dysfunction. Importantly, these values are influenced to a lesser extent by loading conditions (Schaefer et al., 2003). A TDI example is demonstrated in Figure 2C. Color M-Mode Doppler Color M-Mode Doppler flow propagation of transmitral inflow (Vp) is obtained by placing the M-mode cursor through the center of the mitral inflow, which is guided by color Doppler. Decreased Vp implies impaired LV relaxation, as correlated to pulse wave Doppler parameters (Schmidt et al., 2002; Tsujita et al., 2005). Myocardial performance index Pulse wave Doppler or tissue Doppler–derived myocardial performance index (MPI) is a useful index for assessing cardiac systolic and diastolic function in mice. It can be calculated by using the ratio of isovolumetric contraction and relaxation time to ejection



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Table 2 LV Diastolic Function: Transmitral Doppler Parameters at Baseline and at 2 Weeks after TAC in FVB Micea

Baseline

2 weeks TAC

E wave velocity (cm/sec)

54.2±2.7

61.9±3.5

A wave velocity (cm/sec)

43.8±2.8

28.0±4.1b

E/A

1.26±0.06

2.5±0.4b

DT (msec)

18.6±1.7

21.9±2.3

IVRT (msec)

17.5±0.4

14.8±0.7b

R-R (msec)

133±3.4

136±5.8

ET of LVOT (msec)

53.3±1.0

48.9±1.3b

(IVRT + IVCT)/ET

0.43±0.03

0.59±0.05b

11

10

n

a All the values are mean ± SE. DT, deceleration time; IVRT, isovolumetric

relaxation time; R-R, interval between R waves in EKG; ET, LV ejection time; n, number of mice. b p < 0.05 versus baseline. After 2 weeks of TAC, there was clearly LV diastolic dysfunction as reflected by a decrease in A wave velocity, increase in E/A ratio as well as myocardial performance index (IVRT + IVCT)/ET.

time (IVRT + IVCT)/ET. Increased MPI indicates diastolic dysfunction. Since this index is based on the ratio of several portions within the same cardiac cycle, MPI is independent from heart rate and LV shape (Broberg et al., 2003; Schaefer et al., 2005).

LV regional function LV wall thickening As mentioned above, LV wall thickening is measured from several regions of the LV wall and is a basic index for evaluation of LV regional systolic function (Thibault et al., 2007). This can be a critical measurement in a heart with dysynchronous contraction, for example, after a myocardial infarction, where one LV wall might exhibit enhanced function, while the other wall may not contract at all, or even paradoxically. Tissue Doppler imaging and strain rate Systolic waveform (Sa) is a measurement of regional LV wall systolic motion velocity as obtained by tissue Doppler and represents regional wall contraction. Strain rate (SR) is the relative change of length of myocardial tissue over time, and as such it can be measured using TDI. TDI and SR have been demonstrated to be sensitive methods for the detection of LV regional wall contractile changes associated with aging, exercise, cardiac toxic drugs, or myocardial ischemia (Sebag et al., 2005; Thibault et al., 2007; Derumeaux et al., 2008; Jassal et al., 2009).


Two-dimensional speckle tracking echocardiography Two-dimensional (2-D) speckle tracking echocardiography (STE), also known as realtime strain rate, is a novel method for the assessment of LV segmental function by tracking the speckle motion in a 2-D echocardiography imaging. Briefly, LV short axis view is acquired at a high frame rate, e.g., over 200 frames/sec, and specific software is needed to measure the radial and circumferential strain and strain rate for each segment of the LV wall. The feasibility of 2-D-STE in mice has been tested (Peng et al., 2009). Compared to the strain rate derived from TDI, which is Doppler angle dependent and can only be obtained in the anterior and posterior LV segments, 2-D speckle tracking has the ability to assess all segments in radial and circumferential strain components. However,

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due to the thin LV wall and very high heart rate in mice, the application of the (2-D) speckle tracking technique in mice needs to be improved.

VASCULAR ULTRASOUND IN MICE Coronary flow reserve in mice Coronary reserve (CR) is the ratio of maximal coronary flow under hyperemia to baseline coronary flow. Therefore, monitoring the coronary flow is essential for the measurement of CR. High-resolution echocardiography machines make it possible for the measurement of coronary reserve in mice (Wikstrom et al., 2005, 2008; Saraste et al., 2006; Hartley et al., 2008). Since CR derived from coronary flow velocity (CFVR) correlates very well with the CR derived from volumetric coronary flow (CFR) in mice, it is acceptable to simply use CFVR for determination of CR (Wikstrom et al., 2008). In the authors’ laboratory, the high-resolution ultrasound machine VisualSonics Vevo770, with a probe frequency of 30 MHz or 40 MHz, is used for this measurement. The proximal left coronary artery (LCA) is visualized in a modified parasternal LV long-axis view, and Doppler spectrum of LCA is recorded at baseline, and under hyperemic conditions induced by infusing adenosine (160 μg/kg/min) for at least 3 min. From the Doppler spectrum of the left coronary artery, mean diastolic velocity and peak diastolic velocity are measured at baseline (CFVbaseline ) and following maximal coronary vasodilation induced by adenosine infusion (CFVhyperemia ). Coronary reserve based on coronary flow velocity is calculated using the following formula: CFVR= CFVhyperemia /CFVbaseline . Simultaneously, left main coronary artery diameter (d) is measured in the modified LV short-axis view. Cross-sectional area (A) of LCA is calculated as A = π × d 2 /4. Velocity time integral (VTI) of LCA is obtained from Doppler. Blood flow of LCA (CF) = VTI × A × HR. HR is heart rate. Coronary reserve from blood flow is calculated as CFR = CFhyperemia /CFbaseline . CR measured in normal 129SVJ mice in the authors’ laboratory by maximum velocity and by volumetric blood flow are 2.28 ± 0.1 and 2.68 ± 0.15, respectively (Gao et al., 2008a,b). These are similar to those from previously reported studies (Wikstrom et al., 2005, 2008).

Other vessels in mice With the high frequency probe (30 to 40 MHz), mouse carotid arterial lumen, length, and Doppler waveform can also be studied. Williams et al. (2007) verified the feasibility by measuring the pulse wave velocity in mouse carotid artery. Using the same technique, the aortic arch and abdominal aorta can also be visualized in mice (Feintuch et al., 2007; Luo et al., 2007). In TAC mice, measuring the flow velocity through the banded site can help assess the pressure gradient between LV and aorta non-invasively. In general, the following steps can be used to scan the vessels: first, place the probe along the course of the vessel of interest to obtain the long-axis images for lumen, length, and wall thickness measurements, and then tilt the probe to direct the ultrasound beam along the direction of blood flow to record the Doppler signals.

MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY Myocardial contrast echocardiography (MCE) is performed with the aid of intravenously injected contrast agents (micro-bubbles) to enhance the myocardial image for evaluation of myocardial perfusion and the perfusion defect in myocardial ischemia experiments. The feasibility of MCE in mice has been demonstrated by several groups (Mor-Avi et al., 1999; Scherrer-Crosbie et al., 1999; French et al., 2006; Kaufmann et al., 2007; Raher et al., 2007). The high resolution of the VisualSonics echocardiography machine makes



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it easier to perform this function in mice. One might predict that it will be more difficult with echo machines of lower resolution.

STRESS ECHOCARDIOGRAPHY IN MICE In mice, stress echocardiography is generally performed with administration of pharmacologic agents under anesthesia. In the authors’ echo laboratory, echocardiography is often performed for the purpose of monitoring cardiac response to sympathomimetic amines, e.g., isoproterenol or dobutamine. For example, the protocol for isoproterenol in the authors’ laboratory is as follows: 1. A jugular vein catheter is inserted in advance for drug infusion. 2. A Harvard infusion/withdrawal pump is used for drug infusion and set to deliver isoproterenol at 0.01, 0.02, and 0.04 μg/kg/min. 3. The isoproterenol solution is prepared to deliver a final concentration of 0.01 μg/kg/min using an infusion speed of 2 μl/min. When preparing the solutions, it is important to take into account the body weight for each mouse. 4. The mouse is anesthetized using 2.5% avertin, as described above, and LV 2-D and M-mode images are obtained at baseline. 5. The catheter is connected to a 100-μl syringe prefilled with the isoproterenol solution. The syringe diameter in the infusion pump is input and the infusion speed is set at 2 μl/min. The first dose is infused at 0.01 μg/kg/min for 5 min. The echo images are recorded at 5 min of infusion. 6. The next dose is switched to dose at 0.02 μg/kg/min by adjusting the infusion speed to 4 μl/min, and increased again to 0.04 μg/kg/min by increasing the infusion speed to 8 μl/min. Echo is recorded after 5 min of infusion for each of these dosages. 7. After completing all of the doses, the echo data are analyzed offline. LV M-mode images at baseline and after isoproterenol infusion are compared in Figure 4A-B. LV fractional shortening is increased with increasing isoproterenol dose in FVB mice as shown in Figure 4D.

COMMENTARY Background Information


Multiple methods for cardiac imaging have been developed over the years for the visualization and assessment of cardiac function. Among these cardiac echocardiography, micro CT (Nahrendorf et al., 2007), PET scan (Kreissl et al., 2006), and contrast-enhanced cardiac MRI are included (Slawson et al., 1998; Wiesmann et al., 2001; Yang et al., 2004). However, due to the cost and frequent need for contrast material in cardiac MRI and CT and the ease of echocardiography, echo remains the most frequently used modality for the routine evaluation of cardiac function in mice. In performing echocardiography in mice, care must be taken to control the heart rate, body temperature, and the level of anesthesia. Once the animal has been properly prepared and good images are obtained, both sys-

tolic and diastolic cardiac function can be accurately measured and compared for the monitoring of cardiac pathophysiology, as well as the effectiveness of any intervention.

Acknowledgements This work has been supported in part by NIH grants HL033107, HL069020, AG027211, HL101420, HL093481, HL059139, HL095888, DK083826, and HL102472.

Literature Cited Asai, K., Yang, G.P., Geng, Y.J., Takagi, G., Bishop, S., Ishikawa, Y., Shannon, R.P., Wagner, T.E., Vatner, D.E., Homcy, C.J., and Vatner, S.F. 1999. Beta-adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic G(sα) mouse. J. Clin. Invest. 104:551-558.

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Broberg, C.S., Pantely, G.A., Barber, B.J., Mack, G.K., Lee, K., Thigpen, T., Davis, L.E., Sahn, D., and Hohimer, A.R. 2003. Validation of the myocardial performance index by echocardiography in mice: A noninvasive measure of left ventricular function. J. Am. Soc. Echocardiogr. 16:814-823. Depre, C., Wang, Q., Yan, L., Hedhli, N., Peter, P., Chen, L., Hong, C., Hittinger, L., Ghaleh, B., Sadoshima, J., Vatner, D.E., Vatner, S.F., and Madura, K. 2006. Activation of the cardiac proteasome during pressure overload promotes ventricular hypertrophy. Circulation 114:18211828. Derumeaux, G., Ichinose, F., Raher, M.J., Morgan, J.G., Coman, T., Lee, C., Cuesta, J.M., Thibault, H., Bloch, K.D., Picard, M.H., and ScherrerCrosbie, M. 2008. Myocardial alterations in senescent mice and effect of exercise training: A strain rate imaging study. Circ. Cardiovasc. Imaging 1:227-234. Droogmans, S., Lauwers, R., Cosyns, B., Roosens, B., Franken, P.R., Weytjens, C., Bossuyt, A., Lahoutte, T., Schoors, D., and Van Camp, G. 2008. Impact of anesthesia on valvular function in normal rats during echocardiography. Ultrasound Med. Biol. 34:1564-1572. Du, J., Liu, J., Feng, H.Z., Hossain, M.M., Gobara, N., Zhang, C., Li, Y., Jean-Charles, P.Y., Jin, J.P., and Huang, X.P. 2008. Impaired relaxation is the main manifestation in transgenic mice expressing a restrictive cardiomyopathy mutation, R193H, in cardiac TnI. Am. J. Physiol. Heart Circ. Physiol. 294:H2604-H2613. Feintuch, A., Ruengsakulrach, P., Lin, A., Zhang, J., Zhou, Y.Q., Bishop, J., Davidson, L., Courtman, D., Foster, F.S., Steinman, D.A., Henkelman, R.M., and Ethier, C.R. 2007. Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling. Am. J. Physiol. Heart Circ. Physiol. 292:H884-H892. French, B.A., Li, Y., Klibanov, A.L., Yang, Z., and Hossack, J.A. 2006. 3D perfusion mapping in post-infarct mice using myocardial contrast echocardiography. Ultrasound Med. Biol. 32:805-815. Gao, S., Yan, L., Hong, C., Zhao, X., Chen, L., Shen, Y.T., Vatner, S.F., and Vatner, D.E. 2008a. Enhanced coronary reserve mediated by nitric oxide in adenylyl cyclase type 5 knockout. Circulation 118:S 367. Gao, S., Yan, L., Hong, C., Zhao, X., Chen, L., Shen, Y.V., Vatner, S.F., and Vatner, D. 2008b. Reduced coronary reserve as a mechanism in betaadrenergic receptor mediated cardiomyopathy and its rescue by adenylyl cyclase type 5 knockout. Circulation 118:S 562.

Vatner, S.F., and Sadoshima, J. 2009. Genetic inhibition of calcineurin induces diastolic dysfunction in mice with chronic pressure overload. Am. J. Physiol. Heart Circ. Physiol. 297:H1814H1819. Guellich, A., Gao, S., Hong, C., Yan, L., Wagner, T.E., Dhar, S.K., Ghaleh, B., Hittinger, L., Iwatsubo, K., Ishikawa, Y., Vatner, S.F., and Vatner, D.E. 2010. Effects of cardiac overexpression of type 6 adenylyl cyclase affects on the response to chronic pressure overload. Am. J. Physiol. Heart Circ. Physiol. 299:H707-H712. Hart, C.Y., Burnett, J.C. Jr., and Redfield, M.M. 2001. Effects of avertin versus xylazineketamine anesthesia on cardiac function in normal mice. Am. J. Physiol. Heart Circ. Physiol. 281:H1938-H1945. Hartley, C.J., Reddy, A.K., Madala, S., Michael, L.H., Entman, M.L., and Taffet, G.E. 2008. Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy. Ultrasound Med. Biol. 34:892-901. Iwase, M., Bishop, S.P., Uechi, M., Vatner, D.E., Shannon, R.P., Kudej, R.K., Wight, D.C., Wagner, T.E., Ishikawa, Y., Homcy, C.J., and Vatner, S.F. 1996. Adverse effects of chronic endogenous sympathetic drive induced by cardiac GS alpha overexpression. Circ. Res. 78:517524. Iwase, M., Uechi, M., Vatner, D.E., Asai, K., Shannon, R.P., Kudej, R.K., Wagner, T.E., Wight, D.C., Patrick, T.A., Ishikawa, Y., Homcy, C.J., and Vatner, S.F. 1997. Cardiomyopathy induced by cardiac Gs alpha overexpression. Am. J. Physiol. 272:H585-H589. Jassal, D.S., Han, S.Y., Hans, C., Sharma, A., Fang, T., Ahmadie, R., Lytwyn, M., Walker, J.R., Bhalla, R.S., Czarnecki, A., Moussa, T., and Singal, P.K. 2009. Utility of tissue Doppler and strain rate imaging in the early detection of trastuzumab and anthracycline mediated cardiomyopathy. J. Am. Soc. Echocardiogr. 22:418424. Kaufmann, B.A., Lankford, M., Behm, C.Z., French, B.A., Klibanov, A.L., Xu, Y., and Lindner, J.R. 2007. High-resolution myocardial perfusion imaging in mice with high-frequency echocardiographic detection of a depot contrast agent. J. Am. Soc. Echocardiogr. 20:136-143. Kreissl, M.C., Wu, H.M., Stout, D.B., Ladno, W., Schindler, T.H., Zhang, X., Prior, J.O., Prins, M.L., Chatziioannou, A.F., Huang, S.C., and Schelbert, H.R. 2006. Noninvasive measurement of cardiovascular function in mice with high-temporal-resolution small-animal PET. J. Nucl. Med. 47:974-980.

Gardin, J.M., Siri, F.M., Kitsis, R.N., Edwards, J.G., and Leinwand, L.A. 1995. Echocardiographic assessment of left ventricular mass and systolic function in mice. Circ. Res. 76:907-914.

Luo, J., Fujikura, K., Homma, S., and Konofagou, E.E. 2007. Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts. Ultrasound Med. Biol. 33:12061223.

Gelpi, R.J., Gao, S., Zhai, P., Yan, L., Hong, C., Danridge, L.M., Ge, H., Maejima, Y., Donato, M., Yokota, M., Molkentin, J.D., Vatner, D.E.,

Mor-Avi, V., Korcarz, C., Fentzke, R.C., Lin, H., Leiden, J.M., and Lang, R.M. 1999. Quantitative evaluation of left ventricular function in a



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transgenic mouse model of dilated cardiomyopathy with 2-dimensional contrast echocardiography. J. Am. Soc. Echocardiogr. 12:209-214. Nahrendorf, M., Badea, C., Hedlund, L.W., Figueiredo, J.L., Sosnovik, D.E., Johnson, G.A., and Weissleder, R. 2007. High-resolution imaging of murine myocardial infarction with delayed-enhancement cine micro-CT. Am. J. Physiol. Heart Circ. Physiol. 292:H3172H3178. Odashima, M., Usui, S., Takagi, H., Hong, C., Liu, J., Yokota, M., and Sadoshima, J. 2007. Inhibition of endogenous Mst1 prevents apoptosis and cardiac dysfunction without affecting cardiac hypertrophy after myocardial infarction. Circ. Res. 100:1344-1352. Odley, A., Hahn, H.S., Lynch, R.A., Marreez, Y., Osinska, H., Robbins, J., and Dorn, G.W. 2nd 2004. Regulation of cardiac contractility by Rab4-modulated beta2-adrenergic receptor recycling. Proc. Natl. Acad. Sci. U.S.A. 101:70827087. Ohno, M., Cheng, C.P., and Little, W.C. 1994. Mechanism of altered patterns of left ventricular filling during the development of congestive heart failure. Circulation 89:2241-2250. Peng, Y., Popovic, Z.B., Sopko, N., Drinko, J., Zhang, Z., Thomas, J.D., and Penn, M.S. 2009. Speckle tracking echocardiography in the assessment of mouse models of cardiac dysfunction. Am. J. Physiol. Heart Circ. Physiol. 297:H811-H820. Peter, P.S., Brady, J.E., Yan, L., Chen, W., Engelhardt, S., Wang, Y., Sadoshima, J., Vatner, S.F., and Vatner, D.E. 2007. Inhibition of p38 alpha MAPK rescues cardiomyopathy induced by overexpressed beta 2-adrenergic receptor, but not beta 1-adrenergic receptor. J. Clin. Invest. 117:1335-1343. Raher, M.J., Thibault, H., Poh, K.K., Liu, R., Halpern, E.F., Derumeaux, G., Ichinose, F., Zapol, W.M., Bloch, K.D., Picard, M.H., and Scherrer-Crosbie, M. 2007. In vivo characterization of murine myocardial perfusion with myocardial contrast echocardiography: Validation and application in nitric oxide synthase 3 deficient mice. Circulation 116:1250-1257. Roth, D.M., Swaney, J.S., Dalton, N.D., Gilpin, E.A., and Ross, J. Jr. 2002. Impact of anesthesia on cardiac function during echocardiography in mice. Am. J. Physiol. Heart Circ. Physiol. 282:H2134-H2140. Rottman, J.N., Ni, G., Khoo, M., Wang, Z., Zhang, W., Anderson, M.E., and Madu, E.C. 2003. Temporal changes in ventricular function assessed echocardiographically in conscious and anesthetized mice. J. Am. Soc. Echocardiogr. 16:1150-1157. Rottman, J.N., Ni, G., and Brown, M. 2007. Echocardiographic evaluation of ventricular function in mice. Echocardiography 24:83-89. Echocardiography in Mice

Saraste, A., Kyto, V., Saraste, M., Vuorinen, T., Hartiala, J., and Saukko, P. 2006. Coronary flow reserve and heart failure in experimental coxsackievirus myocarditis. A transthoracic

Doppler echocardiography study. Am. J. Physiol. Heart Circ. Physiol. 291:H871-H875. Schaefer, A., Klein, G., Brand, B., Lippolt, P., Drexler, H., and Meyer, G.P. 2003. Evaluation of left ventricular diastolic function by pulsed Doppler tissue imaging in mice. J. Am. Soc. Echocardiogr. 16:1144-1149. Schaefer, A., Meyer, G.P., Hilfiker-Kleiner, D., Brand, B., Drexler, H., and Klein, G. 2005. Evaluation of tissue Doppler Tei index for global left ventricular function in mice after myocardial infarction: Comparison with pulsed Doppler Tei index. Eur. J. Echocardiogr. 6:367-375. Scherrer-Crosbie, M. and Thibault, H.B. 2008. Echocardiography in translational research: Of mice and men. J. Am. Soc. Echocardiogr. 21:1083-1092. Scherrer-Crosbie, M., Steudel, W., Ullrich, R., Hunziker, P.R., Liel-Cohen, N., Newell, J., Zaroff, J., Zapol, W.M., and Picard, M.H. 1999. Echocardiographic determination of risk area size in a murine model of myocardial ischemia. Am. J. Physiol. 277:H986-H992. Schmidt, A.G., Gerst, M., Zhai, J., Carr, A.N., Pater, L., Kranias, E.G., and Hoit, B.D. 2002. Evaluation of left ventricular diastolic function from spectral and color M-mode Doppler in genetically altered mice. J. Am. Soc. Echocardiogr. 15:1065-1073. Sebag, I.A., Handschumacher, M.D., Ichinose, F., Morgan, J.G., Hataishi, R., Rodrigues, A.C., Guerrero, J.L., Steudel, W., Raher, M.J., Halpern, E.F., Derumeaux, G., Bloch, K.D., Picard, M.H., and Scherrer-Crosbie, M. 2005. Quantitative assessment of regional myocardial function in mice by tissue Doppler imaging: Comparison with hemodynamics and sonomicrometry. Circulation 111:2611-2616. Semeniuk, L.M., Severson, D.L., Kryski, A.J., Swirp, S.L., Molkentin, J.D., and Duff, H.J. 2003. Time-dependent systolic and diastolic function in mice overexpressing calcineurin. Am. J. Physiol. Heart Circ. Physiol. 284:H425H430. Slawson, S.E., Roman, B.B., Williams, D.S., and Koretsky, A.P. 1998. Cardiac MRI of the normal and hypertrophied mouse heart. Magn. Reson. Med. 39:980-987. Stypmann, J. 2007. Doppler ultrasound in mice. Echocardiography 24:97-112. Syed, F., Diwan, A., and Hahn, H.S. 2005. Murine echocardiography: A practical approach for phenotyping genetically manipulated and surgically modeled mice. J. Am. Soc. Echocardiogr. 18:982-990. Tan, T.P., Gao, X.M., Krawczyszyn, M., Feng, X., Kiriazis, H., Dart, A.M., and Du, X.J. 2003. Assessment of cardiac function by echocardiography in conscious and anesthetized mice: Importance of the autonomic nervous system and disease state. J. Cardiovasc. Pharmacol. 42:182190. Tanaka, N., Dalton, N., Mao, L., Rockman, H.A., Peterson, K.L., Gottshall, K.R., Hunter, J.J.,

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Chien, K.R., and Ross, J. Jr. 1996. Transthoracic echocardiography in models of cardiac disease in the mouse. Circulation 94:1109-1117.

using high-resolution color Doppler echocardiography and ultrasound biomicroscopy. J. Am. Coll. Cardiol. 46:720-727.

Thibault, H., Gomez, L., Donal, E., Pontier, G., Scherrer-Crosbie, M., Ovize, M., and Derumeaux, G. 2007. Acute myocardial infarction in mice: Assessment of transmurality by strain rate imaging. Am. J. Physiol. Heart Circ. Physiol. 293:H496-H502.

Wikstrom, J., Gronros, J., and Gan, L.M. 2008. Adenosine induces dilation of epicardial coronary arteries in mice: Relationship between coronary flow velocity reserve and coronary flow reserve in vivo using transthoracic echocardiography. Ultrasound Med. Biol. 34:10531062.

Tsujita, Y., Kato, T., and Sussman, M.A. 2005. Evaluation of left ventricular function in cardiomyopathic mice by tissue Doppler and color M-mode Doppler echocardiography. Echocardiography 22:245-253. Vatner, S.F. and Braunwald, E. 1975. Cardiovascular control mechanisms in the conscious state. N. Engl. J. Med. 293:970-976. Vatner, S., Takagi, G., Asai, K., and Shannon, R.P. 2002. Cardiovascular physiology in mice: Conscious measurements and effects of anesthesia. In Cardiovascular Physiology in the Genetically Engineered Mouse (R.A. Walsh and B.D. Hoit, eds.) pp. 257-275. Springer Netherlands. Wiesmann, F., Ruff, J., Engelhardt, S., Hein, L., Dienesch, C., Leupold, A., Illinger, R., Frydrychowicz, A., Hiller, K.H., Rommel, E., Haase, A., Lohse, M.J., and Neubauer, S. 2001. Dobutamine-stress magnetic resonance microimaging in mice: Acute changes of cardiac geometry and function in normal and failing murine hearts. Circ. Res. 88:563-569. Wikstrom, J., Gronros, J., Bergstrom, G., and Gan, L.M. 2005. Functional and morphologic imaging of coronary atherosclerosis in living mice

Williams, R., Needles, A., Cherin, E., Zhou, Y.Q., Henkelman, R.M., Adamson, S.L., and Foster, F.S. 2007. Noninvasive ultrasonic measurement of regional and local pulse-wave velocity in mice. Ultrasound Med. Biol. 33:1368-1375. Yan, L., Vatner, D.E., O’Connor, J.P., Ivessa, A., Ge, H., Chen, W., Hirotani, S., Ishikawa, Y., Sadoshima, J., and Vatner, S.F. 2007. Type 5 adenylyl cyclase disruption increase longevity and protects against stress. Cell 130:247258. Yang, X.P., Liu, Y.H., Rhaleb, N.E., Kurihara, N., Kim, H.E., and Carretero, O.A. 1999. Echocardiographic assessment of cardiac function in conscious and anesthetized mice. Am. J. Physiol. 277:H1967-H1974. Yang, Z., Berr, S.S., Gilson, W.D., Toufektsian, M.C., and French, B.A. 2004. Simultaneous evaluation of infarct size and cardiac function in intact mice by contrast-enhanced cardiac magnetic resonance imaging reveals contractile dysfunction in noninfarcted regions early after myocardial infarction. Circulation 109:11611167.



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Imaging Cancer in Mice by PET, CT, and Combined PET-CT Francisca Mulero,1 Luis E. Donate,1 and Manuel Serrano1 1

Spanish National Cancer Research Centre (CNIO), Madrid, Spain

ABSTRACT The possibility of imaging tumors in live mice has opened new opportunities for cancer research, particularly regarding the ability to perform longitudinal studies in combination with a therapeutic intervention. Here, we detail methods to optimize visualization of murine tumors by positron emission tomography (PET), computed tomography (CT), C 2011 by John Wiley & and combined PET-CT. Curr. Protoc. Mouse Biol. 1:85-103 Sons, Inc. Keywords: cancer r mouse models r positron electron tomography r computed tomography

INTRODUCTION Here we describe protocols for two imaging techniques, namely, positron emission tomography (PET) and computed tomography (CT), applied to cancer research in mouse models. PET detects the uptake of radiolabeled probes by tumors. Current PET technology for mice is of moderate spatial resolution (∼1 mm), but this is compensated for by its unparalleled sensitivity in detecting tumors (Wang et al., 2006). Standard PET technology currently exploits the high glucose avidity of cancer masses by the use of labeled analogs of glucose. The capacities of PET are rapidly expanding to measure other functional properties of tumors, such as cellular proliferation, hypoxia, or apoptosis (Massoud and Gambhir, 2003). On the other hand, CT allows the visualization of anatomical structures with high resolution (∼50 μm), but the ability to identify tumors depends on the differential absorption of radiation between the tumor and its surrounding tissue, and this is not always sufficient to guarantee high sensitivity. The combination of PET and CT overcomes the intrinsic limitations of each technology, combining the high sensitivity of PET with the high resolution of CT, thus offering an unprecedented ability to identify tumors, their functional status, and their dynamics (Massoud and Gambhir, 2003).

Positron emission tomography (PET) PET alone or in combination with CT (PET-CT) has become an important imaging technique for monitoring tumor dynamics in mice, particularly in response to anticancer drugs or other therapeutic interventions (Abbey et al., 2004; Dearling et al., 2004). PET devices detect high-energy gamma rays emitted from within the subject. Natural biological molecules can be labeled with a positron-emitting isotope. Positrons annihilate upon collision with nearby electrons emitting two gamma rays of high energy (511 keV) in exact opposite directions. The two gamma rays must be simultaneously detected at the crystals of the detector; this is what is called “a coincidence.” Positron-emitting isotopes frequently used include 15 O, 13 N, 11 C, and 18 F; the last is employed as a substitute for hydrogen. Other, less commonly used positron emitters are 14 O, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, and 68 Ga. Most of these isotopes are produced in cyclotrons (Strijckmans, 2001) and then incorporated through the appropriate chemical reactions into the desired molecule of biological interest.

Current Protocols in Mouse Biology 1: 85-103, March 2011 Published online March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/9780470942390.mo100137 C 2011 John Wiley & Sons, Inc. Copyright

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Labeled molecular probes are administered to the subject, and PET imaging monitors their distribution and concentration. The half-lives of many of the positron-emitting isotopes used for PET imaging are relatively short (e.g., 18 F has a half life of 110 min) and, therefore, the administration of the probe to the subject must take place relatively quickly (Gambhir, 2002). Commercial PET radiopharmacies are capable of ensuring the provision of commonly used PET tracers on a daily basis. The focus of this protocol will be on [18 F]FDG (fluorodeoxyglucose), which currently is the most widely used radiocompound for PET imaging. For human [18 F]FDG PET-CT studies, standard optimization protocols are well established (Schelbert, 1998). In the case of mouse PET-CT studies, previous researchers have pioneered efforts towards the optimization of mouse anesthesia and handling (Toyama et al., 2004; Fueger et al., 2006). The physiological uptake arising from organs with a high metabolic rate, mainly the brain and the myocardium, can mask the uptake of glucose by the tumors. Also, kidneys, urinary bladder, and to a lesser extent colon and gall bladder, are involved in the physiological elimination of [18 F]FDG and, therefore, may transiently produce [18 F]FDG signals. In addition, the dietary state, the ambient temperature, or the muscle activity can modify [18 F]FDG uptake by normal tissues and these, in turn, may affect tumor detection. The basal metabolic rate per unit body weight in mice is approximately 7-fold higher than that of humans and, therefore, the effect of dietary state and ambient temperature on [18 F]FDG biodistribution in mice is more pronounced than in humans (Fueger et al., 2006). [18 F]FDG uptake by muscle and brown adipose tissue (BAT) increases with the stress of the mice and with lower environmental temperature. Therefore, the stress inflicted on the mice during handling should be minimized as much as possible and temperature should be controlled. Finally, to increase the tumor/background rate, high glucose levels should be avoided because high glycemia can outcompete the [18 F]FDG probe.

Computed tomography (CT) Images in computed tomography (CT) are based on the differential absorption of X-rays by tissues of different composition, including tumors (Dilmanian et al., 1997; Dendy and Heaton, 1999). Volumetric data are acquired through a low-energy X-ray source of 30 to 50 kV, i.e., lower energy than in human CT scanners (115 to 120 kV), and a detector rotating around the animal, thus generating a three-dimensional rendering of the mouse that can be subsequently analyzed by sections along the three axes. This allows the identification of organs, anatomical structures, and tumors, as well as the acquisition of their volumetric data (Paulus et al., 2001; Berger et al., 2002; Holdsworth and Thornton, 2002). Unlike magnetic resonance imaging (MRI), CT has a relatively poor soft-tissue contrast, often making it necessary to administer an iodinated contrast agent to delineate organs or tumors. Mouse CT images are registered on high-resolution phosphor screen/CCD (coupledcharged device) detectors to optimize image quality. The completion of a scan of an entire mouse at a resolution of 200 μm takes ∼15 min. Higher-resolution (50-μm) images are achievable at longer scanning times. Three factors limit the spatial resolution of the system: the sampling rate of the pixel, the size of the X-ray source, and blurring on the phosphor screen. It is noteworthy that the radiation dose is not negligible (0.6 Gy per scan at 200-μm resolution; 5% of the lethal median dose, LD50 , for mice), which limits the repeated imaging of the same animal.


Basic Protocol 1 describes the protocol to perform PET. Basic Protocol 2 details the protocol for CT. Finally, Basic Protocol 3 explains the combined, multimodal, PET-CT imaging of tumors.

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CAUTION: The premises hosting the equipment needed for PET and CT studies must have all the necessary permits according to the applicable regulations. All personnel (investigators and technicians) must be qualified as radioactivity operators and be in the possession of the corresponding diplomas. Radiation safety equipment needed will include syringe and vial radioactivity protectors, shielded scales, shielded waste bins, and shielded screens. NOTE: Prior to commencing with any procedures involving the use of rodents, investigators are required to be trained in the proper use and care of small experimental animals and to be in the possession of the corresponding “Ethics Committee” approvals. These protocols must conform to the applicable regulations regarding the humane use and care of laboratory animals.

IMAGING BY POSITRON EMISSION TOMOGRAPHY (PET) The imaging of tumors at inner locations is more challenging than the imaging of subcutaneous tumors. Our interest lies in studying spontaneous tumors in genetically modified mice and, thus, we have optimized the procedures outlined in the following protocol to the particulars of visualizing tumors at internal body locations, such as the lung or the pancreas. Briefly, the mice are fasted the night before, anesthetized prior to the administration of the [18 F]FDG dose, and kept under anesthesia during the whole period of probe uptake and imaging, ensuring at all times that the mice are warm. The standardization of mouse handling and of anesthesia usage is essential to ensure data reproducibility and comparability.

BASIC PROTOCOL 1

Materials Genetically modified mouse models bearing spontaneous tumors in any body location Special mouse diets as necessary Diazepam (5 mg/ml in flip-top vial; see recipe) Isoflurane Oxygen [18 F]FDG (0.01 to 0.1 μg/ mCi), delivered daily from a local cyclotron (e.g., 40 mCi of [18 F]FDG of 95% to 99% radiochemical purity in 1 ml of physiological saline solution buffered at pH 6.0, for ∼10 PET scans) Physiological saline: 0.9% (w/v) NaCl Lacryvisc Gel 10 G (3 mg/ml carbomere in benzalconium chloride, commercially available from Alcon, http://www.alcon.com) Infrared heating lamp Isoflurane/oxygen-based anesthesia system fitted with an induction chamber and inhalation masks for mice Dose calibrator (also known as activimeter): e.g., VDC-505 dose calibrator from Veenstra Instruments (http://www.dosecalibrator.com/) PET-CT imaging system: e.g., eXplore Vista PET-CT, GE Healthcare (Fig. 1A); Argus PET-CT, SEDECAL (http://www.sedecal.com/) 1-cc tuberculin syringes 30-G needles Heating pads: e.g., Gaymar Mul-T-Pads (http://www.gaymar.com/) Heating pump to maintain temperature of heating pads: e.g., Gaymar TP600 (http://www.gaymar.com/) eXplore Vista PET-CT MMWKS software for image acquisition, processing, and analysis Workstation (e.g., Dell PowerEdge) for image acquisition, processing, and analysis meeting the following specifications:



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PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB processor PE1950 PCIX Riser (2 slots) PE1950 Bezel Assembly 2 GB FB 667 MHz Memory (2 × 1 GB dual rank DIMMs) Dell Studio XPS Desktop 435 MT PC (for 3DOSEM image reconstruction) meeting the following specifications: Processor: Intel Core i7 Quad CPU 940 4 × 2.93 GHz Memory: 6144 MB (6 × 1024) 1067 MHZ DDR3 Graphics: ATI Radeon HD 3450 256 Mb GDDR2 Fast mice 1. Fast mice overnight prior to the procedure (see exceptions below). The specific idiosyncrasy of each genetically modified mouse strain will have to be borne in mind when deciding whether fasting is appropriate and, if so, for how long. For example, a 4- to 6-hr fast may be more appropriate for some strains or disease conditions for which an overnight fast is too stressful.

a. For thoracic tumors, such as lung tumors or pulmonary metastases, mice can be fed a specifically formulated commercial high-fat diet (such as, for example, diet D12451 from Research Diets, with 45% of total calories from fat) or, alternatively, sunflower seeds, which are rich in vegetable fats, during the 24 hr prior to the day of analysis. In this case, mice will not be fasted during the night previous to the analysis. This will decrease glucose uptake by the myocardium (the so called “FDG-robbing effect”), eliminating, to a great extent, the interferences arising from the high uptake of the heart under standard feeding conditions. b. For abdominal tumors, such as those of the stomach, pancreas, colon, etc., it is absolutely essential that the mice be kept in the absence of food for at least 4 hr previous to PET exploration. Water will be always supplied. c. For brain tumors, there is no need for any special diet or fasting prior to the exploration. The feeding conditions of the mice in this case are the standard ones. d. Fasting prior to the exploration can be omitted in those mice with a compromised health status. This results in a slight decrease in the quality of the image but, in turn, it enhances the chances of mice surviving the procedure. Fasting increases the ratio of uptake between the tumor and the surrounding healthy tissue. In addition, it diminishes the uptake by the BAT by decreasing diet-induced thermogenesis. In case of diabetic mice, a glucose determination test will be done prior to the procedure and, if needed, insulin can be administered to reduce hyperglycemia (reducing uptake competition of the probe with circulating glucose). It must be borne in mind that this will result in an increase of [18 F]FDG uptake in the striated muscle, rendering a noisier image (showing, for instance, hotspots in the limbs).

Sedate and anesthetize mice 2. Transport mice to the imaging unit the day before the exploration, prepare the diet, and determine their weight in order to prepare the adequate doses of sedatives and anesthetics. 3. Administer an intraperitoneal (i.p.) injection of a muscle relaxant (diazepam) at a dose of 3 mg/kg, 20 to 60 min before commencing the procedures. This will decrease FDG uptake by muscles.

4. Place an infrared lamp above the cage to keep the mice warm. Imaging Cancer in Mice by PET, CT and PET-CT

Normal body temperature in mice should be 36◦ to 37◦ C, and this should be monitored closely with a rectal probe (Fig. 1B, C). Temperatures higher than 40◦ C imply risk of dehydration. Temperatures lower than 35◦ C may result in hypothermia.

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A

B

C

Figure 1 Equipment at a PET-CT facility for mice. (A) PET-CT machine (eXplore Vista, GE Healthcare). (B) Monitor of vital constants (VisionVet, RGB) registering ECG, temperature, and respiratory frequency. (C) Mouse fitted with electrodes and monitoring probes being introduced into a PET-CT machine.

5. Hold the mouse carefully in your hand and bring its tail closer to the infrared lamp (∼5 cm) during 3 to 4 min, taking care not to expose the rest of the body to excessive heating. This procedure results in vasodilation at the tail, thus facilitating injection of [18 F]FDG through the caudal veins.

6. Once the tail veins have dilated, introduce the mice into the anesthesia chamber where a deep anesthesia will be induced by inhalation of 2% isoflurane in 100% oxygen at a rate of 1 liter/min. In our hands, inhaled isoflurane is more effective and less harmful than other anesthetic agents, although noninhalant anesthetics can be used. In this last instance, our recommendation is i.p. injection of a mixture containing ketamine (200 mg/kg) and xylazine (10 mg/kg). Protocols for anesthesia may vary by institution, so consult your veterinary staff.



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Figure 2 Administration of the probe. Tail vein injection of the radioactive probe into a mouse with anesthetic mask and using a shielded syringe.

Administer the radiolabeled probe 7. Transfer anesthetized mice to the injection area and, using a tuberculin syringe and a 30-G needle, inject them with [18 F]FDG in the tail (Fig. 2) at a dose of 500 μCi in a volume of 0.2 ml physiological saline (0.9% NaCl), calibrating the dosage using an activimeter. The injection of 500 μCi per mouse corresponds to 5 to 50 ng of [18 F]FDG, which is very low compared to the normal glycemia and therefore without pharmaceutical effect. It is advisable to make the first attempt at injection in a distal location on the tail (Fig. 2). In case of failure, this will make it possible to repeat the injection in more proximal positions. In case of excessive radioactive decay, it may not be possible to administer a 500-μCi dose, in which case we advise injection of as much activity as possible in a maximum volume of 0.2 ml. It is not worthwhile to inject less than 100 μCi because it will result in poor-quality images. To ensure reproducibility, it is advisable to use the same dose and volume in all the mice, in order to be able to later compare the results of the quantification of [18 F]FDG uptake. All the procedures involving handling of the [18 F]FDG must be carried out with double-gloved hands and using radioprotection devices to maximize the safety of the operator.

8. Once injected, maintain mice under anesthesia during the duration of the [18 F]FDG uptake time, which is 45 min. Imaging Cancer in Mice by PET, CT and PET-CT

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During this period, [18 F]FDG distributes all over the body. For that reason, it is paramount to keep the mice under the best possible conditions, which include minimal stress achieved by anesthesia and sedation (with the added benefit of minimizing muscle uptake), and body temperature around 36◦ to 37◦ C, aided by the infrared lamp (to minimize BAT uptake). Current Protocols in Mouse Biology

IMPORTANT NOTE: In case of brain tumors, or if the health of the mice is severely compromised, perform the injection and uptake in the absence of anesthesia. In this case, mice will be kept in their cage with appropriate shield protection and visible warning signs. For PET-CT imaging, the CT image acquisition (15 min) is carried out during the [18 F]FDG uptake period. In this manner, wait for 30 min after [18 F]FDG administration and then carry out the CT study; the PET study will be performed immediately after the CT one is finished.

Perform image acquisition 9. Before placing mice on the exploration table, administer eye lubricant (Lacryvisc Gel 10G) to mice to avoid lesions in the cornea while the mice are anesthetized, since under these conditions the blinking reflex is lost. During image acquisition, mice must be anesthetized with a mask providing 2% isoflurane in 100% oxygen at 1 liter/min. 10. Set the heating pads on the exploration table of the PET machine at a constant temperature of 37◦ C. Place a blotter on top of the heating pad to collect possible urine from the mouse. When studying nude mice or mice of compromised health, cover the mice with a blanket of plastic bubbles to avoid heat dissipation as much as possible.

11. Place mice on the exploration table as stretched out as possible to minimize organ superposition. Mice should be fixed to the exploration table with adhesive tape even if they are anesthetized, to minimize involuntary movements (Fig. 1C). The size of the bed depends on the dimensions of the detector and on the number of crystals (in the case of the eXplore Vista PET-CT, one bed position corresponds to 47 mm). Always make sure that the exploration table is free of possible residues of urine from a previous mouse, since urine will contain radioactive probe and will produce false zones of probe hyper-uptake. A new blotter will always be used for each exploration.

12. Perform image acquisition as described below. The duration of the PET exploration will be ∼20 to 30 min. The standard setup values are as follows: 20 to 30 min for whole acquisition in 1 bed position, and 45 min for whole acquisition in 2 bed positions (bed positions are units of measurement in imaging studies that refer to the length of the static field of view). In mice with compromised health, PET acquisition can be reduced to 10 min in 1 bed position. PET acquisition times below 10 min will result in poor-quality images. If only 1 bed is registered, focus must be on the anatomical area of interest. If the likely location of the primary tumor is unknown or extension studies are needed because of possible metastasis, register 2 beds, ensuring that the whole body of the mouse is explored.

a. Perform a blank test. This test consists in performing an empty acquisition, without any radiation. It must be done daily, before any real work is carried out, to assess the correct functioning of the glass detectors of the PET equipment. If the system is working properly, as determined by the blank test, then the user can start the normal operation of the PET. The software program MMWKS VISTA CT (Pascau et al., 2006) is launched, the option “PET ACQ” is ticked, and a static study of 5 min duration is selected, without any isotope.

b. Decide in advance the anatomical area of interest because, if possible, it is preferable to perform 1 bed position, which should take between 20 and 30 min. If it is necessary to explore the entire body, then perform 2 bed positions (sufficient for a mouse of standard size), and PET acquisition time in this case should not exceed 45 min.



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c. Divide the acquisition into two parts. Select or create the folder where the study will be saved and create what is known as a scout (the scout delimitates the area to be studied), or use a scout previously created (if a CT of the same mouse has already been performed, a scout for this given mouse will already be available). Once the area to be studied has been determined, select the kind of PET study to be carried out, define the duration of the procedure, the isotope to be used, and the energy window of the isotope of choice. In the authors’ instance the isotope is 18 F and the window is fluorine.

d. There are some predefined PET acquisition protocols currently available. Static (1 bed): This protocol allows for one bed position; it requires previous knowledge of the anatomical region of interest (i.e., abdominal or thoracic). This is the protocol of choice for a standard tumor study. While acquisition is in progress, take notice of the number of coincidences; this value should be between 200 and 500. This serves as a quality control for the injection of the radiolabeled compound. The number of coincidences is equivalent to the number of detected photons arising from radioactive disintegrations. If that number of coincidences is not reached, it will not be possible to perform PET acquisitions. In this last instance, the user should inject another dose of the probe again, so as to have coincidence events to be detected. Whole-body (≥2 beds): This protocol allows the acquisition of two or more bed positions. Usually, two bed positions suffice for the complete exploration of the entire body of a mouse of average size. With the eXplore Vista PET-CT, it is possible to study rats, since it is prepared to admit settings of more than two bed positions. This would be the protocol of choice when studying adult mice for which the anatomic location of the tumor is not known or when the user wants to investigate the possible existence of distant metastases. In this instance, the user can either set the same duration time for each one of the two beds or set a larger time for one of the beds, depending on the area of interest to be studied or the area for which he/she would like to have higher counting statistical values, but never exceeding a total exploration time of 45 min. Dynamic studies: This protocol may cover one or more beds. These are the protocols of choice for studies of probe kinetics and biodistribution, and also for gating studies. Dynamic studies need to predefine the periodicity and duration of each frame. In the case of gating, images are acquired concurrently with respect to an external signal, such as breathing or electrocardiogram (ECG; Fig. 1B, C). After acquisition, during image processing, frames are grouped according to the external signal. ECG and respiratory signals are obtained from an external monitoring device (Fig. 1B, C). Respiratory gating registers inhaling and exhaling, and this is of relevance in CT studies of lung tumors (see Basic Protocol 2). The feasibility of separating systole from diastole by means of the ECG-gated trigger signal is of relevance in PET studies, since this makes it possible to carry out studies of cardiac functionality (imaging systole and diastole) and to calculate the ejection fraction.

e. Perform the PET exploration setting the type of isotope to 18 F and set the lower energy threshold to 150 KeV. 13. Once PET acquisition is finished, retrieve mice out of the equipment, disconnect the anesthesia, and take mice to a warm cage where they will wake up on their own. Ensure that the mice are placed in a way that it facilitates their recovery and that their breathing path is free of any obstacles (pellets of food, shreds of litter, etc.). Keep the cage containing the mice in a shielded isolation rack. If it is not required that the mice be returned to their housing cages, they may be kept overnight at the imaging facility to allow for a complete radioactivity decay and returned to their housing cells the next day. Imaging Cancer in Mice by PET, CT and PET-CT

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Reconstruct PET image 14. Choose an option for image reconstruction. Three options for image reconstruction are available in the eXplore Vista PET-CT equipment: 2D-FBP (filtered back projections), 2D-OSEM (ordered-subsets expectation maximization), and 3D-OSEM. The authors’ preference is 3D-OSEM with the number of iterations set to eight and employing random and scatter correction. In this manner, we achieve a good imaging quality with reasonable computing resources and usage and an acceptable calculation running time (it must be borne in mind that an independent PC is needed to run 3D-OSEM). The average running time for a 3D-OSEM calculation (eight iterations, random and scatter correction) in a Dell Studio XPS Desktop 435MT PC is

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