Efficiency comparison of B6(Cg)-Tyrc\u22122j /J and C57BL/6NTac embryos as hosts for the generation of knockout mice

Yu’E Ma,Jing Wang,Yichen Zhu,Zhiwei Liu,Wenni Cao,Lijie Xiang,Man Gao,Jie Zhang,Lei He,Fei Zhou,Wenjing Zhu

doi:10.1007/s11248-021-00248-9

Abstract

Careful selection of the host embryo is critical to the efficient production of knockout (KO) mice when injecting mouse embryonic stem (mES) cells into blastocysts. B6(Cg)-Tyrc−2j/J (B6 albino) and C57BL/6NTac (B6NTac) strains of mice are widely used to produce host blastocysts for such procedures. Here, we tested these two strains to identify an appropriate match for modified agouti C57BL/6N (JM8A3.N1) mES cells. When comparing blastocyst yield, super-ovulated B6NTac mice produced more injectable blastocysts per female than B6 albino mice (8.2 vs. 5.4). There was no significant difference in birth rate when injected embryos were transferred to the same pseudopregnant recipient strain. However, the live birth rate was significantly higher for B6NTac blastocysts than B6 albino blastocysts (62.7% vs. 50.2%). In addition, the proportion of pups exhibiting high-level and complete chimerism, as identified by coat color, was also significantly higher in the B6NTac strain. There was no obvious difference in the efficiency of germline transmission (GLT) when compared between B6NTac and B6 albino host embryos (61.5% vs. 63.3% for mES clones; 64.5% vs. 67.9% for genes, respectively), thus suggesting that an equivalent GLT rate could be obtained with only a few blastocyst injections for B6NTac embryos. In conclusion, our data indicate that B6NTac blastocysts are a better choice for the microinjection of JM8A3.N1 mES cells than B6 albino blastocysts.

Highlights

The knockout (KO) mouse model plays a critical role in the new era of functional genomics and is widely used to study the role of specific genes
We compared the yield of embryos after superovulation for the B6 albino with B6NTac strains (Table 1); 8-cell and morulae embryos with normal morphology were counted and cultured in vitro for further development
KO mice are usually generated by the injection of genetically manipulated ES cells into blastocysts

Summary

Introduction

The knockout (KO) mouse model plays a critical role in the new era of functional genomics and is widely used to study the role of specific genes. A global scientific collaborative initiative, the International Knockout Mouse Consortium (IKMC), planned to use C57BL/6N-derived ES cells to generate KO models for every mouse gene. A repository of mutant mES cells has been established to serve the global research community. We hold the responsibility to distribute the resource and to generate as many more mouse models as possible using the mES cells. There have been several developments that aim to improve the efficiency of generating a genetically modified model using mES cells. The 8-cell embryo injection technique has been demonstrated to produce more chimeras and fully ESC-derived mice (Guo et al 2014; Tokunaga and Tsunoda 1992; Ramırez et al 2009). Selecting an appropriate culture medium has been shown to improve the survival of embryos and ES cells and elevates the production of founders with high-level chimerism and potential for germ line transmission (Gertsenstein et al 2010; Ramırez et al 2009)

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