School of Life Science and Technology, Weifang Medical University, Weifang, Shandong 261053, China.
School of Life Science and Technology, Weifang Medical University, Weifang, Shandong 261053, China; and Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
Reprod Fertil Dev. 2021 Nov;33(14):772-781. doi: 10.1071/RD21199.
Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG-hCG, IAS-hCG and eCG-IAS-hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS-hCG and eCG-IAS-hCG methods. However, low fertilisation rates (36.3-38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG-hCG and IAS-hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.
生殖技术，如超排卵和体外受精（IVF），已广泛应用于基因修饰动物的产生。目前，在小鼠中超排卵的金标准是使用马绒毛膜促性腺激素（eCG）和人绒毛膜促性腺激素（hCG）的协同治疗。最近报道了一种使用抑制素抗血清（IAS）代替 eCG 的替代方法。在这里，我们评估了 C57BL/6J 和 B6D2F1 小鼠的不同超排卵策略。首先，我们发现使用 5 周龄的 C57BL/6J 和 4 周龄的 B6D2F1 供体可以获得更好的超排卵效果。然后，我们比较了 eCG-hCG、IAS-hCG 和 eCG-IAS-hCG 在两种小鼠品系中的不同剂量。使用 IAS-hCG 和 eCG-IAS-hCG 方法可以获得显著增加的卵母细胞数量。然而，当应用自然交配时，观察到受精率（36.3-38.8%）较低。我们随后证实，IVF 可以将受精率显著提高至 89.1%。最后，我们对 Scn11a 和 Kcnh1 基因座进行了 CRISPR-Cas9 介导的基因组编辑，使用 eCG-hCG 和 IAS-hCG 诱导的受精卵，通过自然交配或 IVF 受精，成功获得了突变幼崽。我们的结果表明，IAS 是一种有前途的超排卵试剂，并且使用 IAS 诱导的受精卵进行基因组编辑的效率不太可能受到影响。
