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Genetic Background: Understanding its importance in mouse-based biomedical research A Jackson Laboratory Resource Manual This resource manual highlights the importance of using genetically well-defined mice for biomedical research. It briefly describes the following: • The importance of genetic background • Resources for helping researchers choose the appropriate mouse model • Proper nomenclature to communicate the genetic makeup of mouse models • The Jackson Laboratory’s Genetic Quality Control and Genetic Stability programs Cover Photos Front cover, left: JAX® Mice strain C3H/HeJ (000659) Front cover, middle: Technician displaying holders with straws in the liquid nitrogen storage tank in our Cryopreservation Repository. Front cover, right: JAX® Mice strain C57BL/6J (000664) Table of Contents Introduction......................................................................................................................... 1 Genetic Background Definition and Examples............................................................................................. 2 Genetic Background Makes a Difference.................................................................. 2 The Influence of 129 Substrain Backgrounds on Targeted Mutations.................. 4 Consequences of Using Inappropriate Backgrounds............................................... 4 Minimizing the Confounding Effects of Genetic Background............................... 5 How Substrains Arise................................................................................................... 5 Resources to Help You Choose Appropriate Models The Mouse Phenome Database................................................................................... 6 The JAX® Mice Database.............................................................................................. 6 The JAX® Mice Catalog................................................................................................ 6 JAX Technical Support................................................................................................. 6 Correct Nomenclature........................................................................................................ 7 How We Ensure Genetic Quality & Stability................................................................... 8 Our Genetic Quality Control Program...................................................................... 8 Our Genome Scanning Service................................................................................... 9 Our Genetic Stability Program................................................................................. 10 The Jackson Laboratory: Pioneer in Cryopreservation......................................... 11 Do Your Part to Lessen the Impact of Genetic Drift.............................................. 12 References........................................................................................................................... 13 Introduction The utility of the laboratory mouse as a research model of human biology is increasing every year. Following are some of the reasons: • The laboratory mouse is biologically similar to humans, is susceptible to many of the same diseases, is easy to maintain, reproduces quickly, and is very amenable to genetic manipulation and analysis. • The C57BL/6J strain was selected by the Mouse Genome Sequencing Initiative to be the first mouse strain to be sequenced. • Fifteen JAX® Mice strains, including DBA/2J and C3H/HeJ have been resequenced by the National Institute of Environmental Health Sciences Resequencing Project (NIH News 2006). Dense SNP maps of virtually the entire mouse genome for these strains are available from the Mouse Phenome Database (www.jax.org/phenome). This information facilitates comparative genomics among mouse strains, humans, and other sequenced species. • Mouse biology databases, such as the Mouse Genome Informatics Database (MGI, www.informatics.jax.org), the JAX® Mice Database (jaxmice.jax.org), the Mouse SNP Database (mousesnp.roche.com), and the Mouse Phenome Database (www.jax.org/phenome) are continually being expanded and improved. • The number of available mouse models, including congenics, consomics, recombinant inbred strains, spontaneous mutants, ENU-generated mutants, targeted mutants, and transgenics, is increasing almost exponentially. As the amount of mouse-based biomedical research increases, researchers must be more mindful than ever of the genetic makeup of the models they use. If research is to be reliable and reproducible over time and place, and, most importantly, if it is to have the greatest potential for improving human health, it must be conducted with mice of well-defined, stable, and clearly communicated genetic backgrounds. In the following pages, we discuss how these criteria can be met. The Jackson Laboratory 1 Genetic Background Definition and Examples You may occasionally see the following cautionary note on strain data sheets of some of our JAX® Mice models: This strain is on a genetic background different from that on which the allele was first characterized. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available. We include this note because the variety of genetic backgrounds and the mutations characterized and published on them are continually increasing. As a result, researchers must be more mindful than ever of the genetic backgrounds of the mouse models they use. As applied to a mutant mouse strain, genetic background refers to its genetic makeup (all its alleles at all loci) except the mutated gene of interest and a very small amount of other genetic material, generally from one or two other strains. As we shall see, that “other” genetic material can significantly influence a mutant strain’s phenotype. Correct strain nomenclature indicates what a mutant strain’s background is. For example, the genetic background of the targeted mutant strains NOD.129S7(B6)-Rag1tm1Mom/J (003729) and NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/Sz (004848) is NOD. However, the first strain carries a targeted mutation of the Rag1 gene, likely a few Rag1-linked alleles from 129S7-derived ES cells, and possibly some B6 alleles from crosses in the strain’s breeding history. In contrast, the second strain is a congenic (Cg) with more than one donor strain in its breeding history. It carries targeted mutations of the Rag1 and Prf1 genes and possibly some background alleles from those other strains. Similarly, the genetic background of transgenic strains FVB/N-Tg(MMTVneu)202Mul/J (002376) and FVB/N-Tg(MMTV-PyVT)634Mul/J (002374) is FVB/N. However, whereas the first strain carries an MMTVneu transgene, the second carries an MMTV-PyVT transgene. On the other hand, strains B6.129S7-Rag1tm1Mom/J (002216) and NOD.129S7(B6)-Rag1tm1Mom/J (003729) each have the same targeted mutation of the Rag1 gene, but on different backgrounds: C57BL/6J (B6) and NOD. Genetic Background Makes a Difference The technology for producing genetically engineered mice has been substantially refined, resulting in an ever-increasing number, variety, and availability of mutant mouse models. Generally, alleles of interest (such as spontaneous mutations, targeted mutations, transgenes, and congenic regions) are maintained on one to several backgrounds that are more vigorous, better characterized, more amenable to scientific experiments, reproduce better, display a phenotype better, or have some other advantages over other backgrounds. However, alleles are sometimes transferred to backgrounds that are not well characterized. In any case, inattention to a mutant’s genetic background can seriously confound research results. Each strain has unique background alleles that may interact with and modify the expression of a mutation, transgene, or other genetic insert. The likelihood of such modifier genes having a confounding effect is especially high in an uncharacterized background or in a segregating or mixed background of unspecified origin. Even in a well-characterized strain, undiscovered modifier genes may confound results, sometimes making them unexplainable. Such modifier genes are the reason why normal development and physiology often vary significantly among inbred strains. One of the first documented instances of the influence of genetic background on gene expression was the discovery that, on a B6 background, the diabetes (db) and obese (ob) mutations cause obesity and transient diabetes, but, on a C57BLKS/J (BKS) background, they cause obesity and overt diabetes (Coleman and Hummel 1973; Coleman 1978). (Fig. 1, page 3). Since those results were published, genetic background has been shown to influence the expression of many other genes, including the following: • The multiple intestinal neoplasia mutation (Min) of the adenomatous polyposis coli (Apc) gene (ApcMin). B6 mice heterozygous for the ApcMin mutation are very susceptible to developing intestinal polyps. Offspring of these mice mated with AKR/J, MA/MyJ, or CAST mice are significantly less susceptible, indicating that the latter three strains harbor strain-unique ApcMin modifier loci, The Jackson Laboratory 2 Genetic Background Genetic Background Makes a Difference (continued) named modifier of Min 1 (Mom1) (Moser et al. 1992; Dietrich et al. 1993). The AKR allele of Mom1 has been shown to actually contain two genes (MacPhee et al. 1995; Cormier et al. 1997, 2000). • The Prkdcscid mutation. On the NOD background, the Prkdcscid mutation is associated with low natural killer (NK) cell activity, no complement activity, impaired macrophage development, and impaired antigen presenting cell functions; on the BALB/c background, it is associated with high NK cell activity, high complement activity, normal macrophage development, and normal antigen presenting cell functions (Custer et al. 1985; Shultz et al. 1995). • Prkdcscid-associated leakiness (tendency to produce some functional B and T cells with age). Leakiness in Prkdcscid mutants is generally high on the B6 and BALB/c backgrounds, low on the C3H background, and very low on the NOD background (Shultz et al. 1995). • IL10-deficiency. On the B6 background, IL10-deficiency only slightly increases susceptibility to inflammatory bowel disease (IBD); in the 129/SvEv, BALB/c, and C3H/HeJBir backgrounds, it greatly increases IBD susceptibility (Beckwith et al. 2005). Many phenotypic differences among substrains can only be explained by the presence of as yet undiscovered modifiers. Following are several examples: • Differences in tissue rejection among related 129 strains (Simpson et al. 1997) • Differences in susceptibility to proteoglycan-induced arthritis and spondylitis among C3H substrains (Glant et al. 2001) • Differences in fear responses between C57BL/6J and C57BL/6N substrains (Radulovic et al. 1998; Stiedl et al. 1999) • Differences in the effects of anesthetics on cardiac function between C57BL/6J and C57BL/6N substrains (Roth et al. 2002) • Differences in the electroconvulsive thresholds between C57BL substrains (Yang et al. 2003) Diabetes db/db (Leprdb) Obesity ob/ob (Lepob) •C57BL/6J (B6.Cg-m +/+ Leprdb/J) obesity with transient diabetes • C57BL/6J (B6.V-Lepob/J) obesity with transient diabetes • C57BLKS/J (BKS.Cg-m +/+ Leprdb/J) obesity with overt diabetes • C57BLKS/J (BKS.V-Lepob/J) obesity with overt diabetes Figure 1. On a B6 background, the diabetes (db) and obese (ob) mutations cause obesity and transient diabetes, but, on a C57BLKS/J (BKS) background, they cause obesity and overt diabetes. The Jackson Laboratory 3 Genetic Background The Influence of 129 Substrain Backgrounds on Targeted Mutations In the last ten years, the influence of various 129 substrain backgrounds on gene expression has become particularly apparent. Because embryonic stem (ES) cells derived from 129 mice colonize germlines so efficiently, they are the most widely used cell lines for producing targeted mutants. Simpson et al. (1997) found that protein markers, simple sequence length polymorphic marker alleles, tail skin graft acceptance, and coat color alleles vary among seventeen 129 substrains and twelve 129-derived ES cell lines. Threadgill and colleagues (Threadgill et al. 1997) found evidence that strain 129X1/SvJ is significantly different from other 129 substrains and suggested that it should be classified as a recombinant congenic strain (129cX/Sv) derived from 129/Sv and an unknown strain, “X.” The “X” was added to reflect this hypothesized contamination (Festing et al. 1999). Recently, Petkov and colleagues (Petkov et al. 2004b), using a panel of SNPs, determined that the 129X1/SvJ substrain likely has genetic contributions from C57BL/6J on chromosomes 5, 7, 14, 18, and 19, and from BALB/cJ on chromosomes 7, 8, 10, 18, 19, and X, suggesting that the “X” is an F1 hybrid between C57BL/6J and BALB/cJ. Both Simpson and Threadgill found that ES cell lines from the numerous 129 substrains were being used with little attention to their genetic differences. Indeed, Threadgill and his colleagues concluded that confusing results of their own experiments involving targeted Egfr alleles are due to genetic differences in the 129 substrains they used. In another context, Hogan et al. (1994) invoked differences between 129 substrains to explain why 129X1/SvJ is a high ovulator in response to exogenous gonadotropins, whereas 129P3/J and 129P1/ReJ are low ovulators. Consequences of Using Inappropriate Backgrounds As mentioned earlier, using mouse models with inappropriate genetic backgrounds can produce unreliable results. This risk is more than theoretical, as several examples have already been reported, including the following: •Wasted efforts because of a mix-up in AL/N substrains (Bailey 1982) 4 The Jackson Laboratory • Confounded results due to lack of awareness of 129 substrains (Hogan et al. 1994; Threadgill et al. 1997) • Dubious results because of inattention to C57BL substrain differences (Specht et al. 2001; Wotjak 2003) Many other instances have likely been unreported or undetected. Genetic Background Minimizing the Confounding Effects of Genetic Background Although you may not be able to eliminate the confounding effects of genetic background in your research, you can minimize them considerably by observing the following practices: • Use mutants with genetically well-defined backgrounds (Silver 1995; Linder 2001, 2006; Yoshiki and Moriwaki 2006) • Use appropriate controls. If a mutation arose spontaneously or was induced on a well-characterized inbred strain, the inbred strain is likely coisogenic with and therefore the best control for the mutant harboring that mutation (Silver 1995; Linder 2001, 2006) • When possible, construct congenic, targeted mutation, transgenic, and other genetically altered strains that are coisogenic to controls (Silver 1995; Linder 2006) • Construct congenics and transgenics on well-defined backgrounds, such as B6 and FVB (Silver 1995; Linder 2006) • Construct targeted mutation strains on well-defined ES cell lines, derived either from B6 or well-defined 129 strains (Simpson et al. 1997; Threadgill et al. 1997; Linder 2006) • If possible, analyze mutations on several backgrounds. If the mutation is maintained on a mixed genetic background, analyze it in hybrids of the two progenitor strains (Silver 1995; Linder 2001, 2006) • Consider the effects of environmental factors such as noise, light, home cage environment, handling, and diet on gene expression and behavior (Crawley et al. 1997; Bailey et al. 2006) • In all your research communications, describe your mouse models with correct nomenclature (Linder 2006) How Substrains Arise Substrains are genetic variants of an inbred strain. They arise for the following reasons: • Most commonly, genetic drift following separation of a colony from its parent colony for more than 20 generations (10 generations in the sub-colony plus the 10 that simultaneously pass in the parent colony) (Silver 1995) • Residual heterozygosity or incomplete inbreeding at the time of separation from progenitors (Bailey 1977, 1982; Silver 1995) • Undetected spontaneous mutations that become fixed in a colony (Radulovic et al. 1998; Sluyter et al. 1999; Stiedl et al. 1999; Specht et al. 2001; Roth et al. 2002; Wotjak 2003) • Undetected genetic contamination (Naggert et al. 1995) • Deliberate and subsequent unrecorded and/or forgotten outcrossing of strains for specific experimental purposes (Bailey 1977, 1982; Simpson et al. 1997; Threadgill et al. 1997; Wotjak 2003) The Jackson Laboratory 5 Resources to Help You Choose Appropriate Models Many Jackson Laboratory resources can help you choose the most appropriate genetic backgrounds and controls for your research. The Mouse Phenome Database The Mouse Phenome Database (MPD, www.jax.org/ phenome) is the product of an international collaboration (The Mouse Phenome Project) and is maintained at The Jackson Laboratory. It is particularly useful for helping researchers select appropriate background strains. It contains the following: (www.jax.org/phenome) • Detailed protocols on how the phenotypes were measured • Tools for manipulating, downloading, statistically analyzing, and displaying the raw data The MPD is continually updated with data generated by contributing scientists. • Hundreds of baseline measurements of phenotypes for a set of 40 commonly used and genetically diverse inbred mouse strains The JAX® Mice Database (www.jax.org/jaxmice) The JAX® Mice Database is the most comprehensive source of information on JAX® Mice strains. For each strain, there is a strain data sheet, which includes a strain description, mating systems, colony maintenance, H2 haplotype, generation number, strain development, related strains, references, animal health reports, research applications, and suggested controls. The strain information page (www.jax.org/jaxmice/info) has links to detailed descriptions of inbred, hybrid, genetically engineered, wild-derived, recombinant inbred, recombinant congenic, chromosomal substitution strains, and chromosomal aberrations. The JAX® Mice Catalog The JAX® Mice Catalog has a great deal of information on JAX® Mice strains and can help you select the strains and controls that best support your research. It features sections on Technical Knowledge Resources and Quality Control Programs, and appendices on H2 haplotypes, JAX® Services, and literature. The JAX® Mice Catalog and Price Lists may be requested online at www.jax.org/jaxmice/literature. JAX Technical Support Our technical support personnel are the best in the field. They are eager to help you select the appropriate JAX® Mice strains and controls for your research. 6 The Jackson Laboratory Contact them at 1-800-422-6423 or via the website at micetech.jax.org. Correct Nomenclature By using correct strain nomenclature, scientists can accurately communicate with their colleagues, compare and draw conclusions across experiments, locate the institutions or researchers that maintain strains, and accurately document data in animal facilities, research laboratories, and publications. For these reasons, the International Committee on Standardized Genetic Nomenclature for Mice established guidelines that accurately describe the following: • Mouse models, including inbred, transgenic, targeted mutation, spontaneous mutation, congenic, wild-derived inbred, recombinant inbred, consomic, and recombinant congenic strains, strains with chromosome aberrations and hybrid mice • Mutations, including type, alleles, mode of inheritance, and transgenes (including the species of origin) and • Genetic background, relation to other strains, and laboratory of origin It is particularly important that substrains be properly named. An inbred mouse colony that is found to be genetically different or separated from its parent colony for 20 or more generations should be given substrain status. It should be designated by the name of the parent strain followed by a forward slash and a substrain symbol that may be a number and/or the Laboratory Registration Code of either the individual or institution that maintains or generated the substrain. For example, DBA/1J, DBA/1LacJ, and DBA/2J are substrains of DBA: numbers 1 and 2 identify the substrains, Lac is the Laboratory Registration Code for Laboratory Animal Centre at Carshalton, U.K., and J is the Laboratory Registration Code for The Jackson Laboratory. When successive substrains arise, substrain symbols accumulate. For example, A/HeJ is a substrain held first by Heston and now maintained at The Jackson Laboratory. Related inbred strains (strains with a common origin but separated before F20) are given symbols that indicate this relationship (for example, NZB and NZW; NOD and NON). For more detailed information on mouse strain nomenclature, consult the following: • The Jackson Laboratory Mouse Nomenclature Home Page (www.informatics.jax.org/nomen) • JAX® Mice Nomenclature Articles and Announcements (www.jax.org/jaxmice/info/nomenclature) • Interactive Nomenclature Tutorial (www.jax.org/jaxmice/ request/nomenclature) The Jackson Laboratory 7 How We Ensure Genetic Quality & Stability Even if you choose the proper genetic background, consider substrain differences, and clearly communicate the genetic background of the mouse models you use, your research results can still be confounded by genetic contamination and genetic drift. As the repository for over 4,000 JAX® Mice strains and the distributor of these mice to biomedical researchers worldwide, we at The Jackson Laboratory are committed to supplying you with the most genetically well-defined mouse models possible. We ensure the genetic quality of our mice through the following two programs: 1. A rigorous Genetic Quality Control (GQC) program 2. An innovative Genetic Stability Program (GSP) Our Genetic Quality Control Program Our Genetic Quality Control (GQC) program is designed to detect and prevent the spread of genetic contamination. Genetic contamination is the accidental introduction of genes from one inbred mouse strain into the genome of a second inbred strain. The most common cause of genetic contamination is human error, such as inadvertent outcrossing or strain mislabeling. It can also result from deliberate outcrossing. In any case, it occurs quickly and is relatively easy to detect. Three examples follow: • Inadvertent outcrossing of C57BL/6J to DBA/2J, resulting in the C57BLKS strain (Naggert et al. 1995) • Deliberate outcrossing of the 129 strain (Simpson et al. 1997; Threadgill et al. 1997) • Genetic contamination (affecting several chromosomes) with either FVB or an FVB-like strain in two NIA contract colonies of C57BL/6 (www.nia.nih.gov) The purpose of our GQC program is to prevent such mistakes. Continually improved over the past 30 years, the program is founded on the following five components: 1. Rigorous breeding protocols • We limit the number of generations attained in our foundation and expansion stocks to less than 10 generations from the main pedigree line • We isolate foundation, expansion, and production stocks from each other • We maintain detailed pedigrees of our foundation and expansion stocks • We systematically refresh our production stocks with mice from foundation stocks • We adhere to proven mouse husbandry practices 8 The Jackson Laboratory 2. Systematic screens of all stocks for phenotypic variations, including coat color, body size, and behavior 3. SNP-genotyping Foundation Stocks (Self Perpetuating) [Pedigreed] Pedigreed Expansion Stocks [Pedigreed] Production Stocks Pooled Production Stocks Investigators Figure 2. Relationship between foundation stocks, pedigreed expansion stocks, and production stocks at The Jackson Laboratory. How We Ensure Genetic Quality & Stability Our Genetic Quality Control Program (continued) Between the 1960s and 2003, our GQC program used biochemical (isoenzyme) variants to distinguish among different strains of mice. In 2003, after extensive testing, we converted to single nucleotide polymorphisms (SNPs). Although SNPs are the most abundant type of polymorphism, until the mouse genome was sequenced, few mouse SNPs were mapped or available in public databases. Additionally, the feasibility of using them as genetic markers had not been established. Dr. Petko Petkov and his colleagues at The Jackson Laboratory demonstrated that SNP-genotyping with a panel of only 28 carefully selected SNPs can distinguish between virtually all JAX® Mice strains. (Petkov et al. 2004a; Petkov et al. 2004b) The panel has the following advantages: • It is reliable, simple, quick, and inexpensive • It is amenable to high throughput • It is suitable for both large and small scale animal facilities • It may be used to type mice before they are used as breeders Using this panel has greatly facilitated monitoring the genetic integrity of JAX® Mice. For example, we formerly used erythrocyte antigen (Ea) assays to distinguish strains like C57BL/6J and C57BL/10J, which type identically for 23 isoenzymes, are both black, have the same major histocompatibility (H2) haplotype, but have different Eas. However, we no longer use the Ea assay because several SNP markers can distinguish between C57BL/6J, C57BL/10J, and other closely related strains. Occasionally, we use other assays to either verify SNP results or to type closely related strains that SNP-genotyping cannot distinguish. For example, only a hemolytic complement (Hc) assay can distinguish between congenic strains B10.D2-Hc1 H2d H2-T18c/nSnJ and B10.D2-Hc0 H2d H2-T18c/oSnJ (whereas B10.D2-Hc1 H2d H2-T18c/nSnJ expresses hemolytic complement, B10.D2-Hc0 H2d H2-T18c/oSnJ does not). For details on other molecular markers we use, see the following website: www.jax.org/jaxmice/geneticquality/monitoring 4. Verification of mutant alleles in mutant stocks Our molecular genotyping lab uses allele-specific assays, PCR-genotyping, and other assays to verify mutant genotypes of genetically engineered and cloned spontaneous mutations. Experienced technicians verify the visible phenotype of many spontaneous mutant colonies. 5. Phenotypic deviant search The Mouse Mutant Resource phenotypic deviant search detects and prevents spontaneous mutations that cause visible phenotypes from becoming fixed in JAX® inbred strains. Animal care technicians send mice from breeding units with deviant phenotypes to a bi-weekly clinic. Scientists determine which deviant phenotypes are likely caused by a mutation and test them for heritability. Breeding units with deviant mice are removed from the breeding colonies, and the parent strains are closely monitored to see if the phenotypes recur. Our Genome Scanning Service Can Confirm your Strain’s Genetic Identity By using a panel of over 2,000 single nucleotide polymorphic (SNP) markers, our Genome Scanning Service can confirm and monitor your strain’s genetic background. Service technicians can work with you to design a protocol that meets your needs. Contact JAX® Services at [email protected]. The Jackson Laboratory 9 How We Ensure Genetic Quality & Stability Our Genetic Stability Program Our Genetic Stability Program (GSP) is designed to limit genetic drift, a cumulative change in the genetic makeup of an organism over time. Genetic drift in inbred mouse colonies happens slowly, subtly, and is difficult to detect and control. It is caused by the same factors that lead to substrain divergence: • Separation of a sub-colony from its parent colony for more than 20 generations (10 generations in the parent colony plus the 10 that simultaneously pass in the sub-colony) • Undetected spontaneous mutations that become fixed in a colony • Residual heterozygosity in or incomplete inbreeding of a colony before it is separated from its progenitors (Bailey 1977, 1982) Following are seven examples of genetic drift: • At least 40 C57BL substrains develop between 1930 and 1970 (Fig. 3): although some of these substrains are due to deliberate outcrossing, most are probably due to maintaining colonies separate from the originating colony for more than 10 generations • Histocompatibility variants exist within A, AKR, BALB/c, CBA, C3H, C57BL, C57L, DBA, and WG strains (Bailey 1982) • Substrains C57BL/6N, C57BL/6Nmg, and C57BL/6JKun are phenotypically different from each other and from the C57BL/6J founder line (Radulovic et al. 1998; Sluyter et al. 1999; Stiedl et al. 1999; Roth et al. 2002; Wotjak 2003) • C57BL/6JOlaHsd, a substrain of C57BL/6J, has a spontaneous deletion encompassing part of the alphasynuclein (Snca) gene and the entire multimerin-1 (Mmrn1) gene (Specht and Schoepfer 2001, 2004; Wotjak 2003) • A deletion of the killer cell lectin-like receptor, subfamily D, member 1 gene (Klrd1) on Chromosome 6 is identified in JAX® Mice strain DBA/2J (Wilhelm et al. 2003) Figure 3. At least 40 C57BL substrains develop between 1930 and 1970, some of which are due to genetic drift (adapted from Bailey 1977 by Dr. Michael V. Wiles, The Jackson Laboratory). 10 The Jackson Laboratory How We Ensure Genetic Quality & Stability Our Genetic Stability Program (continued) “This insidious evolution of the inbred genotype is known as genetic drift. It is capable of subverting the conclusions reached about comparable research results coming from different laboratories when each uses its own subline of the same inbred strain.” (Bailey 1977) To further limit genetic drift in our mouse colonies, we implement a three-component Genetic Stability Program: We have already cryopreserved embryos from JAX® Mice strains 129S1/SvImJ (002448), C3H/HeJ (000659) C57BL/6J (000664), DBA/2J (000671), FVB/NJ (001800), NOD/ShiLtJ (001976), and NOD.CB17-Prkdcscid/J (001303) (Taft et al. 2006). Limiting genetic drift in these strains is particularly important because each of them, except NOD.CB17Prkdcscid/J (001303), has been resequenced. C57BL/6J was sequenced by the Mouse Genome Sequencing Consortium in 2003 and FVB/NJ, BALB/cByJ, and 13 other JAX® Mice strains were resequenced by the National Institute of Environmental Health Sciences, as part of their 15-strain Resequencing Project (JAX® Notes 2005). Embryos derived from brother sister matings • We minimize the number of generations attained in our foundation and production stocks (see GQC section). • We use highly skilled and experienced technicians to oversee breeding in those stocks (see GQC section). • We use a unique cryopreservation approach to virtually stop genetic drift in the most commonly used inbred strains (see below). The cryopreservation component of our GSP was initiated in 2003 and entails cryopreserving supplies of embryos from broadly used strains (if technically feasible to do so) and refreshing our foundation stocks with these embryos about every five generations (Fig. 4). To obtain enough mice for infusion that span only one or two generations, we rapidly expand the strain of interest by in vitro fertilization. Establishment of frozen bank Re-establish Foundation about every five generations Frozen Stock Sufficient for up to 25 Years Foundation Stock Expansion • A spontaneous deletion of two ion channel genes, Kcnq2 and Chrna4, in a C57BL/6J substrain generates a mouse model of epilepsy (Yang et al. 2003). • C3H/HeJ mice are homozygous for a paracentric inversion in Chromosome 6 (JAX® Notes 2003). Figure 4. The Jackson Laboratory cryopreserves supplies of embryos from several widely-used strains and refreshes foundation stocks with these embryos about every five generations. The Jackson Laboratory: Pioneer in Cryopreservation for Over 30 Years The technique of cryopreserving mouse embryos was first reported by Whittingham et al. (1972). Later, Whittingham and Dr. Bailey of The Jackson Laboratory (Whittingham 1974; Bailey 1977) suggested that cryopreservation could be used to solve the problem of genetic drift in inbred mouse colonies. Indeed, it has been shown to stop genetic drift in embryos of outbred stocks (Goto et al. 2002). During the last 30 years, the Jackson Laboratory has successfully cryopreserved over 6,500 mouse strains. The Jackson Laboratory 11 How We Ensure Genetic Quality & Stability Do Your Part to Lessen the Impact of Genetic Drift You can lessen the impact of genetic drift on mouse-based biomedical research by practicing the following: • Obtain mice from a reliable breeding source. • If you maintain your own private colonies of these mice, periodically obtain new breeding stock from your supplier. • Although colonies of inbred mice expanded from our breeding stock can be maintained either by sibling or non-sibling matings, they may develop into substrains if they are expanded beyond ten generations. • Avoid comparing results from substrains that either arose early in a strain’s inbreeding regimen or that have been long separated. • Use proper nomenclature to describe your mouse models (see Nomenclature section). • Include a detailed description of the genetic background of the mice you use in all your communications. • When possible, use a common genetic background so that your experiments can be replicated. “. . . the constant tendency of genes to evolve even in the absence of selective forces. Genetic drift is fueled by spontaneous neutral mutations that disappear or become fixed in a population at random. Inbred lines separated from a common ancestral pair can drift rapidly apart from each other.” (Silver 1995). 12 The Jackson Laboratory References Bailey DW. 1977. Genetic drift: the problem and its possible solution by frozen-embryo storage. Ciba Found Symp 291-303. Bailey DW. 1982. How pure are inbred strains of mice? Immunol Today 3:210-14. Bailey KR, Rustay NR, Crawley JN. 2006. Behavioral phenotyping of transgenic and knockout mice: practical concerns and potential pitfalls. ILAR J 47:124-31. Beckwith J, Cong Y, Sundberg JP, Elson CO, Leiter EH. 2005. Cdcs1, a major colitogenic locus in mice, regulates innate and adaptive immune response to enteric bacterial antigens. Gastroenterology 129:1473-84. Coleman DL. 1978. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14:141-8. Coleman DL, Hummel KP. 1973. The influence of genetic background on the expression of the obese (Ob) gene in the mouse. Diabetologia 9:287-93. Cormier RT, Bilger A, Lillich AJ, Halberg RB, Hong KH, Gould KA, Borenstein N, Lander ES, Dove WF. 2000. The Mom1 AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64. Oncogene 19:3182-92. Cormier RT, Hong KH, Halberg RB, Hawkins TL, Richardson P, Mulherkar R, Dove WF, Lander ES. 1997. Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis. Nat Genet 17:88-91. Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ, Maxson SC, Miner LL, Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R. 1997. Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology (Berl) 132:107-24. Custer RP, Bosma GC, Bosma MJ. 1985. Severe combined immunodeficiency (SCID) in the mouse. Pathology, reconstitution, neoplasms. Am J Pathol 120:464-77. Dietrich WF, Lander ES, Smith JS, Moser AR, Gould KA, Luongo C, Borenstein N, Dove W. 1993. Genetic identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell 75:631-9. Festing MF, Simpson EM, Davisson MT, Mobraaten LE. 1999. Revised nomenclature for strain 129 mice. Mamm Genome 10:836. Glant TT, Bardos T, Vermes C, Chandrasekaran R, Valdez JC, Otto JM, Gerard D, Velins S, Lovasz G, Zhang J, Mikecz K, Finnegan A. 2001. Variations in susceptibility to proteoglycan-induced arthritis and spondylitis among C3H substrains of mice: evidence of genetically acquired resistance to autoimmune disease. Arthritis Rheum 44:682-92. Goto K, Muguruma K, Kuramochi T, Shimozawa N, Hioki K, Itoh T, Ebuduro M. 2002. Effects of cryopreservation of mouse embryos and in vitro fertilization on genotypic frequencies in clolonies. Mol Reprod Dev 62:307-11. Hogan B, Beddington R, Costantini F, Lacy E. 1994. Manipulating the mouse embryo: a laboratory manual, 2nd ed. Cold Spring Harbor (NY). JAX® Notes. 2003. Chromosomal inversion discovered in C3H/HeJ mice. JAX® Notes 491:15. JAX® Notes. 2005. NIEHS to Sequence 15 JAX® Mice Strains. JAX® Notes 496:3 Linder CC. 2001. The influence of genetic background on spontaneous and genetically engineered mouse models of complex diseases. Lab Anim 30:34-9. Linder CC. 2006. Genetic variables that influence phenotype. ILAR J 47:132-40. MacPhee M, Chepenik KP, Liddell RA, Nelson KK, Siracusa LD, Buchberg AM. 1995. The secretory phospholipase A2 gene is a candidate for the Mom1 locus, a major modifier of ApcMin-induced intestinal neoplasia. Cell 81:957-66. Moser AR, Dove WF, Roth KA, Gordon JI. 1992. The Min (multiple intestinal neoplasia) mutation: Its effect on gut epithelial cell differentiation and interaction with a modifier system. J Cell Biol 116:1517-26. Naggert JK, Mu JL, Frankel W, Bailey DW, Paigen B. 1995. Genomic analysis of the C57BL/Ks mouse strain. Mamm Genome 6:131-3. NIH News. 2006. Mouse DNA to Aid Biomedical Research. NIEHS PR #06-17, October 25 (www.niehs.nih.gov/oc/news/snp2.htm). Petkov PM, Cassell MA, Sargent EE, Donnelly CJ, Robinson P, Crew V, Asquith S, Harr RV, Wiles MV. 2004a. Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse. Genomics 83:902-11. Petkov PM, Ding Y, Cassell MA, Zhang W, Wagner G, Sargent EE, Asquith S, Crew V, Johnson KA, Robinson P, Scott VE, Wiles ME. 2004b. An efficient SNP system for mouse genome scanning and elucidating strain relationships. Genome Res 14:1806-11. The Jackson Laboratory 13 References Radulovic J, Kammermeier J, Spiess J. 1998. Generalization of fear responses in C57BL/6N mice subjected to one-trial foreground contextual fear conditioning. Behav Brain Res 95:179-89. Roth DM, Swaney JS, Dalton ND, Gilpin EA, Ross J. 2002. Impact of anesthesia on cardiac function during echocardiography in mice. Am J Physiol Heart Circ Physiol 282: H2134-40. Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, Tennent B, McKenna S, Mobraaten L, Rajan TV, Greiner DL, Leiter EH. 1995. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 154:180-91. Silver L. 1995. Mouse Genetics. Oxford. p 394. Simpson EM, Linder CC, Sargent EE, Davisson MT, Mobraaten LE, Sharp JJ. 1997. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat Genet 16:19-27. Sluyter F, Marican CC, Crusio WE. 1999. Further phenotypical characterisation of two substrains of C57BL/6J inbred mice differing by a spontaneous single-gene mutation. Behav Brain Res 98:39-43. Specht CG, Schoepfer R. 2001. Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2:11. Specht CG, Schoepfer R. 2004. Deletion of multimerin-1 in alphasynuclein-deficient mice. Genomics 83:1176-8. Stiedl O, Radulovic J, Lohmann R, Birkenfeld K, Palve M, Kammermeier J, Sananbenesi F, Spiess J. 1999. Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behav Brain Res 104:1-12. Taft RA, Davisson M, Wiles MV. 2006. Know thy mouse. Trends Genet 22:649-53. Threadgill DW, Yee D, Matin A, Nadeau J, Magnuson T. 1997. Genealogy of the 129 inbred strains: 129SvJ is a contaminated inbred strain. Mamm Genome 8:390-3. Whittingham DG, Leibo SP, Mazur P. 1972. Survival of mouse embryos frozen to -196 degrees and -269 degrees C. Science 178:411-4. Whittingham DG. 1974. Embryo banks in the future of developmental genetics. Genetics 78:395-402. Wilhelm BT, Landry JR, Takei F, Mager DL. 2003. Transcriptional control of murine CD94 gene: differential usage of dual promoters by lymphoid cell types. J Immunol 171:4219-26. 14 The Jackson Laboratory Wotjak CT. 2003. C57BLack/BOX? The importance of exact mouse strain nomenclature. Trends Genet 19:183-4. Yang Y, Beyer BJ, Otto JF, O’Brien TP, Letts VA, White HS, Frankel WN. 2003. Spontaneous deletion of epilepsy gene orthologs in a mutant mouse with a low electroconvulsive threshold. Hum Mol Genet 12:975-84. Yoshiki A, Moriwaki K. 2006. Mouse phenome research: implications of genetic background. ILAR J 47:94-102. Acknowledgements Senior Editor and Technical Writer: Ray Lambert, M.S. Many people helped to compile, write, and lay out this manual. Special thanks to Muriel Davisson, Ph.D., Beverly Day, B.A., Leah Rae Donahue, Ph.D., Christian Gilbert, B.S., Michael Greene, M.B.A., Mike Kirby, B.A., Chip Leighton, M.B.A., Linda Neleski, Mike Sasner, Ph.D., Laura Trepanier, M.S., Alicia Valenzuela, M.S., Ray VonderHaar, Ph.D., and Michael V. Wiles, Ph.D. “ Just as the purity of the chemical assures the pharmacist of the proper filling of the doctor’s prescription, so the purity of the mouse stock can assure a research scientist of a true and sure experiment.” —Dr. Clarence Cook Little founder of The Jackson Laboratory Founded in 1929, The Jackson Laboratory is a nonprofit biomedical research institution dedicated to leading the search for tomorrow’s cures. Our mission: We discover the genetic basis for preventing, treating and curing human disease, and we enable research and education for the global biomedical community. Phone: 1-800-422-6423 1-207-288-5845 SN Customer Service: [email protected] Technical Support: [email protected] 8/08 | www.jax.org/jaxmice JAX® is a registered trademarks of The Jackson Laboratory.