Abstract
There has been a significant increase in the use of C57BL/6N-derived ES cells for the production of gene knockout mice. However, the potential for germline transmission (GLT) from chimeras on this genetic background has been observed to be highly variable. Using coat color as an indicator of somatic chimerism to infer the extent of chimeric contribution to the germ cell population, even highly agouti C57BL/6N-derived chimeras can fail to achieve GLT. We investigated the extent to which quantitative PCR genotyping for a marker gene expressed in gene targeted ES cells can be performed on DNA extracted from sperm present in copulatory plugs to determine the contribution of ES cells to the germ cells. We found that an objective assessment of sperm DNA from copulatory plugs combined with a subjective assessment of coat color chimerism can be used to accurately inform the selection of chimeras for breeding that are likely to achieve GLT. These results indicate that, compared to random selection of chimeras, including an analysis of copulatory plugs to set chimeras for breeding can help to reduce costs, minimize time, and facilitate research for projects requiring the production, selection, breeding, and testing of chimeras to generate gene-targeted mice.
This is a preview of subscription content, log in via an institution to check access.
References
Auerbach W, Dunmore JH, Fairchild-Huntress V et al (2000) Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. Biotechniques 29:1024–1028
Bradley A, Anastassiadis K, Ayadi A et al (2012) The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23:580–586
Collins FS, Rossant J, Wurst W et al (2007) A mouse for all reasons. Cell 128:9–13
Deb K, Reese J, Paria BC et al (2006) Methodologies to study implantation. Methods Mol Med 121:9–34
Hansen GW, Markesich DC, Burnett MB et al (2008) Large-scale gene trapping in C57BL/6N mouse embryonic stem cells. Genome Res 10:1670–1679
Hughes ED, Qu YY, Genik SJ et al (2007) Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line. Mamm Genome 18:549–558
Pettitt SJ, Liang Q, Rairdan XY et al (2009) Aqouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat Methods 7:493–495
Poueymirou WT, Auerbach W, Frendewey D et al (2006) F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol 25:91–99
Skarnes WC, Rosen B, West AP et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342
Wilson S, Sheardown SA (2011) Identification of germline competent chimeras by copulatory plug genotyping. Tansgenic Res 2:429–433
Acknowledgments
This research was funded by NIH/OD U42, Award No. U42OD012210 supporting the Mutant Mouse Regional Resource Center (MMRRC) at UC Davis and by the UC Davis Mouse Biology Program.
We would like to acknowledge Wing Kong and Charles Rawls who performed the chimera mating and sperm plug collection, Chelsea Hayes and Polly Pearce for their assistance with the subjective evaluation of coat color of chimeras for breeding, and Diana Young, Kayla Jager, Lia D’Angelo, Maggie Tam and Ron Basco who performed ES cell microinjection.
Author information
Authors and Affiliations
Corresponding author
Additional information
Angus Yiu-Fai Lee and Kristin Evans have contributed equally to this work.
Rights and permissions
About this article
Cite this article
Lee, A.YF., Evans, K., Willis, B. et al. Combining sperm plug genotyping and coat color chimerism predicts germline transmission. Transgenic Res 22, 1265–1272 (2013). https://doi.org/10.1007/s11248-013-9731-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-013-9731-6