alexa Rescue of germline transmission from chimeras by IVF after sperm analysis.
Reproductive Medicine

Reproductive Medicine

Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biology

Author(s): Li MW, Willis BJ, Evans KD, Araiza RS, Lee AY

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Successful production of genetically modified mouse lines is dependent on germline transmission (GLT) of mutant alleles from chimeras. When natural mating fails to achieve GLT due to male infertility, sickness, or other problems, sperm can be harvested from chimeras and used for assisted reproductive technologies such as in vitro fertilization (IVF) to attempt to "rescue" GLT. However, a rational, evidence-based approach to determine if such extraordinary efforts should be attempted on a chimera has not been established. Therefore, in the present study we assessed the production, quality and genotype of epididymal sperm harvested from male chimeras generated by blastocyst or morula microinjection of gene targeted embryonic stem (ES) cell clones containing a LacZ expression cassette and that failed to achieve GLT. Results of this analysis enabled us to determine the cause of GLT failure, correlate coat color chimerism with the proportion of LacZ-positive sperm, and test the likelihood of achieving GLT by IVF. In 415 chimeras, 332 (80%) produced no offspring by natural mating ("infertile"), while 83 (20%) produced only wildtype offspring ("fertile"). Of the 332 infertile chimeras, 209 (63%) failed to produce any sperm whatsoever, 48 (15%) had extremely poor quality sperm, and 75 (23%) had good quality sperm. These results indicate that most chimeras that do not achieve GLT by natural mating are infertile, and the primary cause of infertility is failed spermatogenesis. Genotyping of sperm from 519 chimeras revealed a significant positive linear correlation between coat color chimerism and mean percentage of LacZ-positive sperm (R(2) = 0.95). Finally, IVF using good quality, LacZ-positive sperm from fertile and infertile chimeras "rescued" GLT for 19 out of 56 genes. We conclude that an assessment of coat color chimerism together with sperm quality and genotype can better inform the selection of chimeras for IVF to rescue GLT than coat color chimerism alone.

This article was published in Transgenic Res and referenced in Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biology

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