Assessment of three generations of mice derived by ICSI using freeze-dried sperm.
Assessment of three generations of mice derived by ICSI using freeze-dried sperm.
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DOI：
10.1017/s0967199409005292
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发表时间：
2009-08
期刊：
影响因子：
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通讯作者：
Lloyd KC
中科院分区：
文献类型：
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作者：
Li MW;Willis BJ;Griffey SM;Spearow JL;Lloyd KC
尽管先前已经证明了使用冷冻干燥精子通过卵胞浆内精子注射（ICSI）获得小鼠，但对其生存能力，健康状况和生育能力的全面分析尚未得到证实。本研究的目的是确定使用在4 °C下储存1-2个月的冷冻干燥精子进行ICSI的程度，所述冷冻干燥精子来自近交系（C57 BL/6 J）或杂交系（B6 D2 F1/J）遗传背景的小鼠，所述冷冻干燥精子导致小鼠及其两代后代的基因组不稳定性和/或表型异常。使用新鲜和冻干精子的ICSI的受精率（每个注射的卵母细胞的2个细胞的数量）在小鼠品系内和之间相似，尽管冻干精子衍生的胚胎比新鲜精子衍生的胚胎在体外（C57 BL/6 J和B6 D2 F1/J）和活产幼崽（仅B6 D2 F1/J）中发育成囊胚的数量少。然而，一旦出生，使用两种小鼠品系中的冷冻干燥精子通过ICSI获得的小鼠是健康的，生殖健康的。与自然交配（对照）组相比，使用冷冻干燥精子的ICSI衍生后代的两代后代（F2和F3）在窝仔数、断奶率和性别比方面无重大差异。此外，没有证据表明ICSI或冷冻干燥诱导基因组不稳定性，如通过与两种亲本基因型相比时衍生小鼠和后代的微卫星分析所确定的，也没有三代后代中任何一代的病理变化的数量或类型的差异。我们的结论是，可行的，健康的和基因组稳定的小鼠可以通过ICSI使用冷冻干燥的小鼠精子在冰箱中储存至少2个月。此外，由于与胚胎和精子冷冻保存相比，冷冻干燥是一种更简单，更经济的技术，因此本研究的结果证明了进一步研究的合理性，以继续开发和增强遗传改变小鼠的保存，储存和共享技术。
Although the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 °C for 1–2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocysts in vitro (C57BL/6J and B6D2F1/J) and liveborn pups in vivo (B6D2F1/J only). Nevertheless, once born, mice derived by ICSI using freeze-dried sperm in both mouse strains were healthy and reproductively sound. No major differences in litter size, weaning rate, and sex ratio were noted in the two generations of progeny (F2 and F3) of ICSI-derived offspring using freeze-dried sperm compared with that in the natural mating (control) group. Further, there was no evidence that either ICSI or freeze drying induced genomic instability, as determined by microsatellite analysis of the derived mice and subsequent generations when compared with both parental genotypes, nor were there differences in the number or types of pathological changes in any of the three generations of progeny. We conclude that viable, healthy and genomically stable mice can be derived by ICSI using freeze-dried mouse sperm stored in the refrigerator for at least 2 months. Further, because freeze drying is a simpler and more economical technique compared with embryo and sperm cryopreservation, the results of this study justify additional research to continue to develop and enhance the technique for the preservation, storage, and sharing of genetically altered mice.