A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing

Abstract

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.

Synchronization of the ovulation and copulation timings increased the number of in vivo fertilized oocytes in superovulated female mice

“…This technique will apply to genetically modified mice production and technology development for infertility treatment. (8)(9)(10)(11)(12)(13)(14) weeks old) treated with IASe were divided into three groups: pre ovulation (0-10 h after hCG injection), during ovulation (10-15 h after hCG injection), and post ovulation (15-19 h after hCG injection). These female mice were then allowed to copulate with male mice in each period.…”

“…Previously, we developed an improved superovulation method (ultrasuperovulation) using inhibin antiserum and eCG (IASe), which produced approximately 100 oocytes from a single C57BL/6 (B6) female mouse [ 13 ]. However, ovulation induced by IASe only resulted in approximately 20 fertilized oocytes, despite the large number of ovulated oocytes in the oviductal ampulla [ 14 ]. This result suggests that a limited number of sperm reach the ampulla in natural mating.…”