Li Ming-Wen, Willis Brandon J, Evans Kristin D, Araiza Renee S, Lee Angus Yiu-Fai, Lloyd K C Kent
Mouse Biology Program, University of California, Davis, CA, 95618, USA,
Transgenic Res. 2015 Feb;24(1):99-108. doi: 10.1007/s11248-014-9819-7. Epub 2014 Jul 31.
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.
转基因小鼠品系的成功培育依赖于嵌合体中突变等位基因的种系传递（GLT）。当由于雄性不育、疾病或其他问题自然交配无法实现GLT时，可以从嵌合体中采集精子，并用于体外受精（IVF）等辅助生殖技术，以尝试“挽救”GLT。然而，尚未建立一种合理的、基于证据的方法来确定是否应对嵌合体进行这种特殊的努力。因此，在本研究中，我们评估了从通过囊胚或桑椹胚显微注射含有LacZ表达盒且未能实现GLT的基因靶向胚胎干细胞（ES）克隆产生的雄性嵌合体中采集的附睾精子的产量、质量和基因型。该分析结果使我们能够确定GLT失败的原因，将毛色嵌合现象与LacZ阳性精子的比例相关联，并测试通过IVF实现GLT的可能性。在415只嵌合体中，332只（80%）通过自然交配未产生后代（“不育”），而83只（20%）仅产生野生型后代（“可育”）。在332只不育嵌合体中，209只（63%）根本无法产生任何精子，48只（15%）精子质量极差，75只（23%）精子质量良好。这些结果表明，大多数通过自然交配未实现GLT的嵌合体是不育的，不育的主要原因是精子发生失败。对519只嵌合体的精子进行基因分型显示，毛色嵌合现象与LacZ阳性精子的平均百分比之间存在显著的正线性相关性（R(2)=0.95）。最后，使用来自可育和不育嵌合体的高质量、LacZ阳性精子进行IVF，在56个基因中有19个“挽救”了GLT。我们得出结论，与单独的毛色嵌合现象相比，对毛色嵌合现象以及精子质量和基因型进行评估可以更好地为选择用于IVF以挽救GLT的嵌合体提供依据。
