A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing
Xue Zhao A # , Johnny X. Huang
A B # * , Hailong Zhang A , Xueyang Gong A , Jinhua Dong A , Hong-Lin Ren C and Zengshan Liu A C
+ Author Affiliations
- Author Affiliations
A School of Life Science and Technology, Weifang Medical University, Weifang, Shandong 261053, China.
B Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
C Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Xi An Da Lu 5333, Changchun 130062, China.
* Correspondence to: johnny.xiao.huang@gmail.com
# These authors contributed equally to this paper
Handling Editor: Xiaolong Wang
Reproduction, Fertility and Development 33(14) 772-781 https://doi.org/10.1071/RD21199
Published online: 9 November 2021
© 2021 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND)
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.
Keywords: assisted reproductive technology, CRISPR, embryo manipulation, fertilisation, genome editing, inhibin, in vitro fertilisation, superovulation.
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