Fact Sheet: Mutation Identified in C57BL/6JBomTac Strain

Summary

A mutation in the Y chromosome long arm (Yq) of the C57BL/6JBomTac strain (model # B6JBOM) has recently been identified.1 The mutation most likely occurred between 2008-2009, and it is present in both US and European colonies. Due to the highly repetitive nature of the mouse Y chromosome long arm (Yq), it is not possible to pinpoint the exact size of the mutation, but it appears that some of the genomic sequence has been deleted. Taconic is working with Y chromosome experts to assess the phenotypic impact of this mutation and will provide updates to this fact sheet as they are available. Taconic has observed no other notable unexpected phenotypes for a B6 substrain. We also have no reports from customers regarding unexpected phenotypes or study performance issues. The C57BL/6JBomTac strain will remain available for sale. The mutation is noted on the model webpage as a genetic characteristic of the strain.

The mouse Y chromosome

Copy number variation (CNV) in the mouse Y chromosome exists, is common and has an evolutionary origin. These CNVs can be associated with specific reproductive phenotypes.2

In mice, the Y chromosome long arm (Yq) consists almost entirely of massively amplified sequence, with three primary gene families. As reported in Soh et al. 2014, "The long-arm ampliconic sequence consists of a half-megabase unit amplified about 200 times. ...Amplification of the half-megabase unit results in> 96% of the mouse MSY sharing at least 98% intrachromosomal identity... This amplified sequence makes up 3% of the haploid male mouse genome. Within the long-arm amplicons are two regions of extended and outstanding identity: one pair of 7-Mb direct repeats of 99.999% identity, and a second pair of 4.5-Mb tandem repeats of 99.995% identity...".3 There are three primary gene families on the mouse Y chromosome long arm: Sly, Srsy, and Ssty. These genes are repeated multiple times, with "132, 197, and 317 copies of Sly, Srsy, and Ssty, respectively".3

Figure 1: Structure of mouse Y chromosome long arm

y chromosome long arm

The hugely repetitive nature of the mouse Y chromosome long arm (Yq) significantly complicates the investigation of genetic events in this region. In fact, the first complete sequence of the mouse Y chromosome was only published in 2014, the result of an effort that began in 2002.3

The putative mutation

A genetics company (GVG Genetic Monitoring) with new genetic markers using short tandem repeats (STRs) contacted Taconic to report a large deletion in the Y chromosome long arm of the C57BL/6JBomTac strain (model # B6JBOM). GVG also reported to Taconic that it did not identify any anomalous STR findings in any other chromosome of C57BL/6JBomTac. Taconic investigated this information regarding the Y chromosome long arm using several different testing platforms. The C57BL/6NTac, another B6 substrain at Taconic, was used as a comparison line for our investigation. GVG did not identify any anomalous STR findings in the C57BL/6NTac strain.1

STR results

We cannot independently confirm the reported deleted region. Our assessment is further restricted because much of this region is composed of complex repeat sequence. GVG mapped the putative deletion by analyzing 25 unique regions of the Y chromosome, using 13 positional markers and additionally 5 different STR-markers each targeting between 1-4 specific regions. GVG reported that the putative deletion covers the region from 6.57 to 46.73 Mbp, which is a large portion of the Y chromosome long arm (Yq).1

The putative deleted region consists primarily of amplified sequence of the three primary gene families Sly, Srsy, and Ssty along with the gene Rbm31y, which is present in two copies. There are also a number of predicted genes, non-coding RNA and partial precursor RNA sequences within the putative deleted region, but these have not been studied in detail. The list of genes identified in the putative deleted region is available upon request.

SNP results

We tested the C57BL/6JBomTac using a 143,000 SNP panel provided by the University of North Carolina Systems Genetics Core, which has 83 SNPs in the Y chromosome. As the most comprehensive SNP panel publicly available, we used this assay to assess the overall genetic integrity of the C57BL/6JBomTac. The SNP markers in the region of interest on the Y chromosome long arm are not unique and thus not informative with respect to a deletion in that region. All tested SNPs resulted as expected. This information was not conclusive as to the presence or absence of a deletion in the Y chromosome long arm, but did provide confirmation that other chromosomes of C57BL/6JBomTac are as expected.

Detection of individual genes in the putative deleted region

The Rbm31y locus exists in only two copies on the Y chromosome, both in the putative deleted region of the Y chromosome long arm. This gene is thus informative in the investigation of the reported deletion. Genetic testing via PCR shows that the Rbm31y gene is not present in C57BL/6JBomTac, confirming that a mutation of some type exists.

Expression analysis of genes present in the Y chromosome in C57BL/6JBomTac

The three primary gene families on the mouse Y chromosome long arm, Sly, Srsy, and Ssty are repeated in 100+ copies each2 The Sly, Ssty1, and Ssty2 genes would be present on the Y chromosome long arm of C57BL/6JBomTac, but the gene dosage is likely to be lower compared to other B6 substrains if there is a deletion.4

It has been reported in the literature that various known deletions of the Y chromosome affect expression of genes that are present in several copies on Y. We tested the expression of various genes that are in the Y chromosome repeats of the putative deleted region. Expression of all Y chromosome genes tested show a significant difference between C57BL/6JBomTac and C57BL/6NTac. However, the most dramatic change is the absence of expression of Rbm31y, which is completely absent in C57BL/6JBomTac (as expected per the genetic testing results above). Genes present in chromosomes other than the Y (Prm1, Acvr1 and Zfy2) show little difference in expression between C57BL/6NTac and C57BL/6JBomTac. These results support the existence of a deletion resulting in lowered gene dosage for highly repeated genes in Yq.

Biological impact of the mutation

Sex ratios

Changes in gene dosage of Sly or the X-chromosome homolog Slx are associated with altered sex ratios. Mice with partial Y chromosome deletions produce more female than male offspring.4 The C57BL/6JBomTac strain displayed a biased sex in pups weaned for the period 2009 through 2016 -- more female than male mice were weaned. The sex ratio for another B6 substrain at Taconic, C57BL/6NTac, is more balanced with a small bias towards males (see Table 3).
Table 1: Sex Ratio in Production Barriers for C57BL/6JBomTac and C57BL/6NTac

Male/Female Sex Ratio

Year C57BL/6JBomTac C57BL/6NTac
2004 1.12 1.07
2005 1.1 1.04
2006 1.1 1.04
2007 1 0.94
2008 0.99 0.9
2009 0.76 0.99
2010 0.83 1.01
2011 0.77 1.02
2012 0.79 1
2013 0.8 1.03
2014 0.87 1.04
2015 0.77 1
2016 0.82 1.03
The M/F sex ratio became biased in the barriers in 2009

Fertility

Literature focused on other known Y chromosome deletions has outlined impacts on fertility. Taconic has observed very minimal impact to fertility in the C57BL/6JBomTac strain. However, IVF using frozen sperm gives much lower yields than when using fresh sperm, so when cryopreserving, we recommend using embryos.

C57BL/6JBomTac breeding performance

At Taconic, breeding performance is measured by the Production Efficiency Index (PEI). This index is calculated as the total pups weaned per total female breeders during a defined period, and it is a direct measure of the number of pups per breeding female. We calculated the PEI for 2016 of all Expansion Colonies for Taconic MPF colonies. The PEI for C57BL/6JBomTac is comparatively similar to C57BL/6NTac (Table 4).

Table 2: PEI comparison between Expansion Colonies of C57BL/6NTac and C57BL/6JBomTac
Formal Nomenclature Health Status PEI
C57BL/6JBomTac MPF 0.74
C57BL/6NTac MPF 0.76

Fertilization rates in C57BL/6JBomTac

IVF experiments using C57BL/6JBomTac sperm have shown fertilization rates comparable to C57BL/6NTac (see Table 4).

Data was collected using sperm of males 12-14 weeks of age.

Table 3: 2015 Fertilization Rates for C57BL/6JBomTac and C57BL/6NTac Sperm
Formal Line Name Fertilization Rate
C57BL/6JBomTac 85 ± 6 %
C57BL/6NTac 88 ± 7%

Sperm analysis

Analysis of sperm morphology is underway. Those results will be reported here when available.

Other biological impacts

Taconic has observed no other notable unexpected phenotypes for a B6 substrain. We also have no reports from customers regarding unexpected phenotypes or study performance issues.

In general, the Y chromosome is thought to be involved primarily in regulating male reproduction. Most Y chromosome genes are expressed only in the male germline.2 The idea that the Y chromosome is a regulator beyond the reproductive system is a relatively recent concept and an emerging field of study. There are limited published reports examining the impact of murine Y chromosome deletions outside of reproduction. There is a single report that suggests a role of the Y chromosome in B and NK cells.5

Taconic is working with mouse geneticists with expertise in the Y chromosome to more fully assess the biological impact of this deletion. Updates will be reported here as they become available.

Conclusions

The C57BL/6JBomTac strain carries a mutation in the Y chromosome, most likely a deletion. Despite extensive analysis, Taconic is not able to confirm the size of the deleted region, but it may comprise the region between 6.57 to 46.73 Mbp of the Y chromosome long arm (Yq), according to STR testing by GVG. Taconic has confirmed that the Rbm31y locus is deleted in the Y chromosome. The biological impact of this mutation appears to be mild, producing a somewhat female-biased sex ratio of pups, but normal fertility and reproduction.

Next steps

Taconic will provide updates to this fact sheet as they become available. The C57BL/6JBomTac strain will remain available for sale both in the US and Europe. We will assist you with questions and concerns about how this might impact your studies. Researchers with questions may contact the following Taconic staff for a consultation:

Dr. Ana Perez, Global Director, Genetics Sciences and Compliance, ana.perez@taconic.com, +1 (518) 697-3984

Dr. Megan MacBride, Portfolio Director, megan.macbride@taconic.com, +1 (518) 697-3933

Are other B6 strains at Taconic affected?

No. The C57BL/6NTac strain does not carry this mutation. Taconic has many GEM lines on B6 backgrounds, but none on the C57BL/6JBomTac substrain. Thus no commercial GEM lines are affected.

Are other strains or stocks at Taconic affected?

We have no evidence that other stocks or strains at Taconic have mutations in the Y chromosome. Taconic has tested C57BL/6NTac, FVB/NTac, NOD/MrkTac, 129S6/SvEvTac and C3H/HeNTac with a 143,000 SNP panel and no abnormalities were detected. Taconic is pursuing whole genome sequencing of its inbred strains. Data for some strains is now being analyzed and will be available in the future. Due to the nature of biological processes, it is possible that during DNA replication, spontaneous mutations arise. To decrease the potential for spontaneous mutations becoming fixed in a population, Taconic has cryopreserved stock for all its inbred strains. Taconic refreshes all inbred strains from cryopreserved stock every 5 years or 10 generations, whichever comes first. The initial cryopreservation for each strain took place over a number of years. The C57BL/6JBomTac strain was first cryopreserved in 2010.

Why did Taconic not detect the mutation sooner?

The Taconic genetic quality program has several components, including major quality control programs in genetic integrity and strain harmonization. These two programs are designed to maintain genetic integrity of each strain and ensure that animals obtained from one Taconic site are as genetically similar as possible to those obtained from other Taconic sites.

Additionally, Taconic maintains cryopreserved stocks of its main inbred strains in order to avoid genetic drift. As spontaneous mutations are not preventable, best practices in strain management dictate that a strain be genetically refreshed through recovery of embryos from a set stock with some frequency. Taconic follows this best practice, and maintains cryopreserved stocks of its main inbred strains in order to avoid genetic drift. The C57BL/6JBomTac strain was refreshed in 2015 using embryos from a 2010 cryopreservation. Unfortunately, the refreshed C57BL/6JBomTac stock also contained the deletion, indicating that the mutation occurred prior to 2010.

Spontaneous mutations occur in inbred mouse strains on an ongoing basis. This is a natural process that cannot be prevented and can be difficult or impossible to detect. Taconic maintains an extensive genetic monitoring program for all commercial lines using single nucleotide polymorphism (SNP) panels at varying densities during the maintenance of the breeding colonies (96 GenMon SNP Panel) and including genetic characterization profile of embryo derived refreshed colonies (1,449 SNP Panel). The majority of SNP panels available have poor or no coverage for the Y chromosome due to its highly repetitive nature and the subsequent challenge in testing of this region. The standard panel of 1,449 SNPs used by Taconic for genetic monitoring does not include any SNPs on the Y chromosome. The 96 GenMon SNP Panel includes one Y chromosome SNP marker, which is present in the C57BL/6JBomTac. However, the mutation is outside of this marker. Thus our routine genetic monitoring did not detect this deletion. Taconic is in the process of developing additional higher density SNP panels to enhance its genetic monitoring program.

Detection of spontaneous mutations in inbred strains is challenging. Unless there is an observable phenotype, nothing less than genome sequencing would detect most spontaneous mutation. Taconic has already begun a whole genome sequence program of its most popular inbred strains to add to the available genetic data on these strains. For more information on the Taconic genetic monitoring program see https://www.taconic.com/quality/genetic-integrity.

Why did Taconic not detect a sex ratio discrepancy in barrier production colonies?

Taconic does monitor sex ratios of all breeding colonies. A 55% female to 45% male offspring ratio was observed in the C57BL/6JBomTac breeding colonies. Our genetic monitoring program did not detect any abnormalities with the C57BL/6JBomTac strain. Additionally, there were no phenotypic anomalies reported for this line when used in studies and when used as the background strain for various GEM lines. Given the observations available to us at the time, the shift in sex ratio was not considered substantial and was considered to be a normal trait of the C57BL/6JBomTac strain.

How do I know if my line carries the mutation?

If you purchased C57BL/6JBomTac mice from Taconic from 2009 until now, we expect that the mice you received have the mutation. If you used C57BL/6JBomTac to maintain a GEM line, the mutation may or may not be present depending on the details of your mating scheme. If you wish to analyze whether the mutation is present in your GEM line, we recommend testing your line by one of the two following methods:

  • Detection of Rbm31y gene (please complete the short form below to access the document):



  • Testing via the GVG STR Panel. Please note Taconic cannot independently verify the STR results reported by GVG Genetic Monitoring, but this panel is available for use by the public.

My experiments are working fine. Do I need to do anything?

It is likely that the mutation does not affect your organ system of interest unless you are studying reproduction. If you are in doubt, we suggest you use another B6 substrain available for comparison to confirm that there are no differences.

I have a knockout on the C57BL/6BomTac background. Can I just start mating to C57BL6/NTac or C57BL/6J?

You can transfer your mutation of interest to another B6 substrain background, but we would advise performing multigenerational backcrosses to assure you have a consistent, congenic background. Mixed substrain backgrounds can confound experiments.

Are there any other chromosomal deletions or mutations that I should be aware of?

Spontaneous mutations are a natural process that cannot be prevented. Various mutations have become fixed in commonly used inbred strains, and as whole genome sequencing becomes more common, additional mutations are likely to be discovered. For example, in B6 substrains, the following mutations are known:

Mutation
Substrain Vendor NntC57BL/6 Snca deletion Crb1rd8 Mmrn1 deletion Cyfip2M1N Dock2Hsd Nlrp12 mutation
C57BL/6NTac Taconic Yes Yes
C57BL/6JBomTac Taconic
C57BL/6NHsd Envigo Yes Yes Yes
C57BL/6JOlaHsd Envigo Yes Yes
C57BL/6RccHsd Envigo
C57BL/6J Jax Yes Yes
C57BL/6NJ Jax Yes Yes
C57BL/6NCrl CRL Yes Yes
Reference - 6 7 8 9 10 11 12

I made a KO mouse on a C57BL6/NTac strain and then backcrossed it to a C57BL/6JBomTac strain and lost my phenotype. Is that due to the deletion?

This is unlikely, as no problems with organ systems have been reported with C57BL/6JBomTac. This could be due to many other reasons.

I made a KO mouse on a BALB/c strain and then backcrossed it to a C57BL/6JBomTac strain and lost my phenotype. Is that due to the deletion?

No, this is likely due to different genetic modifiers of each strain. There are many reports in the scientific literature that changing the genetic background alters the phenotype of a mutation.

Given this discovery, will you now monitor for Y chromosome deletions in all your other strains of mice?

Yes, as technology becomes available and new markers are developed, Taconic is constantly assessing new platforms/assays to improve strain monitoring.

Are other chromosomes subject to the same kind of mutation?

Spontaneous mutations of various types, including deletions, have been reported in various laboratory mouse strains on other chromosomes.

Is it possible that only the individual mice that you have tested are subject to the deletion? Is it possible that some mice don't have the deletion?

This would have been possible for animals that were sold between 2008--2009 when the mutation arose in the C57BL/6JBomTac Foundation Colony (the source colony for all C57BL/6JBomTac stock). However, once the mutation was fixed in the Foundation Colony, all C57BL/6JBomTac production sites would have received breeders from the Foundation Colony and would therefore carry the mutation.

References:
1Fischer M, Kosyakova N, Liehr T, Dobrowolski P. (2016) Large deletion on the Y-chromosome long arm (Yq) of C57BL/6JBomTac inbred mice. Mamm Genome. 2016 Oct 31.

2Toure A, Clemente EJ, Mahadevaiah SK, Ojarikre OA, Ball PA, Reynard L, Loveland KL, Burgoyne PS, Affara NA. Identification of novel Y chromosome encoded transcripts by testis transcriptome analysis of mice with deletions of the Y chromosome long arm. Genome Biol (2005) 6:R102.1-R102.15.

3Soh YQ, Alföldi J, Pyntikova T, Brown LG, Graves T, Minx PJ, Fulton RS, Kremitzki C, Koutseva N, Mueller JL, Rozen S, Hughes JF, Owens E, Womack JE, Murphy WJ, Cao Q, de Jong P, Warren WC, Wilson RK, Skaletsky H, Page DC. (2014) Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes. Cell. 159(4):800-13.

4Ward MA, Burgoyne PS. (2006) The effects of deletions of the mouse Y chromosome long arm on sperm function--intracytoplasmic sperm injection (ICSI)-based analysis. Biol Reprod. 74(4):652-8.

5Sun S, Horino S, Itoh-Nakadai A, Kawabe T, Asao A, Takahashi T, So T, Funayama R, Kondo M, Saitsu H, Matsumoto N, Nakayama K, and Ishii N. (2013) Y chromosome-Linked B and NK Cell Deficiency in Mice. J Immunol 190:6209-6220.

6Freeman, H. C., Hugill, A., Dear, N. T., Ashcroft, F. M., & Cox, R. D. (2006). Deletion of nicotinamide nucleotide transhydrogenase. Diabetes, 55(7):2153-2156.

7Specht, C. G., & Schoepfer, R. (2001). Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neuroscience, 2:11.

8Mattapallil, M. J., Wawrousek, E. F., Chan, C. C., Zhao, H., Roychoudhury, J., Ferguson, T. A., Caspi, R. R. (2012). The Rd8 Mutation of the Crb1 Gene Is Present in Vendor Lines of C57BL/6N Mice and Embryonic Stem Cells, and Confounds Ocular Induced Mutant Phenotypes rd8 Mutation in Vendor B6 Mice and ES Cells. Investigative ophthalmology & visual science, 53(6):2921-2927.

9Gajović, S., Mitrečić, D., Augustinčić, L., Iaconcig, A., Muro, A. F. (2006). Unexpected rescue of alpha-synuclein and multimerin1 deletion in C57BL/6JOlaHsd mice by beta-adducin knockout. Transgenic research, 15(2),:255-259.

10Kumar, V., Kim, K., Joseph, C., Kourrich, S., Yoo, S. H., Huang, H. C., Vitaterna, M.H., Pardo-Manuel de Villena, F., Churchill, G., Bonci, A., Takahashi, J. S. (2013). C57BL/6N mutation in cytoplasmic FMRP interacting protein 2 regulates cocaine response. Science, 342(6165): 1508-1512.

11Mahajan, V. S., Demissie, E., Mattoo, H., Viswanadham, V., Varki, A., Morris, R., & Pillai, S. (2016). Striking Immune Phenotypes in Gene-Targeted Mice Are Driven by a Copy-Number Variant Originating from a Commercially Available C57BL/6 Strain. Cell reports, 15(9):1901-1909.

12Ulland TK, Jain N, Hornick EE, Elliott EI, Clay GM, Sadler JJ, Mills KA, Janowski AM, Volk AP, Wang K, Legge KL, Gakhar L, Bourdi M, Ferguson PJ, Wilson ME, Cassel SL, Sutterwala FS. (2016). Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment. Nat Commun. 7:13180.