Download Genetic Background: Understanding its importance in mouse

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)
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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.
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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
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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).
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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]
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