Efficiency comparison of B6(Cg)-Tyrc−2j /J and C57BL/6NTac embryos as hosts for the generation of knockout mice

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.

3R measures in facilities for the production of genetically modified rodents

“…In general, the mouse strain from which the ES cell line was originally generated should be at least as vigorous as the host embryo's strain to take a dominant role in embryonic development. For instance, ES cell clones of inbred strains (e.g., C57BL/6) are injected with high success rate into host embryos of inbred (e.g., C57BL/6 or BALB/c), but not outbred strains; ES cell lines of F1 hybrids (e.g., B6D2F1 or B6129F1) can also be successfully injected into or aggregated with outbred embryos (e.g., CD1) 42,[60][61][62][63][64][65][66][67] . Combinations of ES cell lines and host embryos leading to the expression of different coat colors (e.g., 'black' C57BL/6 ES cells in 'white' BALB/c) are usually employed to identify chimeras with a substantial ES cell contribution.…”