C57BL/6N Albino/Agouti Mutant Mice as Embryo Donors for Efficient Germline Transmission of C57BL/6 ES Cells

Abstract

We generated C57BL/6NTac mice carrying a tyrosinase loss-of function mutation and a reversion of the nonagouti locus to agouti. This strain has a high superovulation response, allows visual detection of chimeric coat color contribution of C57BL/6 ES-cells and provides a simplified breeding format that generates black G1 offspring of pure inbred C57BL/6 background in one step, providing the ideal host for genetically manipulated C57BL/6 ES cells.

Comparison of BALB/c and B6-albino mouse strain blastocysts as hosts for the injection of C57BL6/N-derived C2 embryonic stem cells

“…Zevnik et al (2014) also reported a yield of 2.7 blastocysts per donor BALB/ cJBomTac female mouse. It has been reported that these numbers can be increased if embryos are harvested at 2.5 dpc and cultured overnight.…”

“…The wide availability of robust genetically modified ES cell lines has shifted the focus from improving the efficiency of introducing mutations into ES cells, to optimizing protocols for the successful introduction of targeted ES cells into mouse germlines. Conventional microinjection into inbred or outbred albino blastocysts has been the standard method of injection with ES cells derived from the B6 strain (Lemckert et al 1997;Auerbach et al 2000;Schuster-Gossler et al 2001;Poueymirou et al 2007;Zevnik et al 2014), although the injection of 8-cell embryos increased the contribution of B6 ES cells to the resulting chimeras (Poueymirou et al 2007). Aggregation of B6 ES cells with outbred host morulae has been demonstrated to produce similar results without the need for sophisticated microinjection equipment (Auerbach et al 2000;Gertsenstein et al 2010), and various host embryo alternatives have been used in efforts to improve the efficiency of generating genetically modified mouse models (Pacholczyk et al 2008;Taft et al 2013;Zevnik et al 2014).…”

“…Conventional microinjection into inbred or outbred albino blastocysts has been the standard method of injection with ES cells derived from the B6 strain (Lemckert et al 1997;Auerbach et al 2000;Schuster-Gossler et al 2001;Poueymirou et al 2007;Zevnik et al 2014), although the injection of 8-cell embryos increased the contribution of B6 ES cells to the resulting chimeras (Poueymirou et al 2007). Aggregation of B6 ES cells with outbred host morulae has been demonstrated to produce similar results without the need for sophisticated microinjection equipment (Auerbach et al 2000;Gertsenstein et al 2010), and various host embryo alternatives have been used in efforts to improve the efficiency of generating genetically modified mouse models (Pacholczyk et al 2008;Taft et al 2013;Zevnik et al 2014). In general, BALB/c and B6-albino inbred strains have been found to be more effective host embryos compared with other inbred or outbred strains for producing chimeras by conventional injection of B6 ES cells into blastocysts (Ledermann and Burki 1991;Auerbach et al 2000;Schuster-Gossler et al 2001).…”

Genetic quality: a complex issue for experimental study reproducibility

“…These targeted ES cells and mouse strain resources are available in high quality from major mouse repositories such as KOMP, MMRRC, EMMA, The Jackson Laboratory and RIKEN BRC (Birling et al 2021 ). While early ES cell lines were primarily derived from the 129 strains, ES cell lines with high germline transmission rates are now available for C57BL/6J or C57BL/6N substrains (Pettitt et al 2009 ; Tanimoto et al 2008 ; Hansen et al 2008 ; Zevnik et al 2014 ) and some other inbred strains (Bouabe and Okkenhaug 2013 ). Chimeras of 129 ES cells and C57BL/6 host blastocysts are traditionally crossed with the pertinent C57BL/6 substrain due to the poor breeding performance of the 129 strains.…”

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

“…Taft et al ( 2013 ) developed a “Perfect Host” in which germ cells were ablated in early development, thus resulting in fully ESC-derived chimeric male offspring. Zevnik et al ( 2014 ) reported that the C57BL/6N Albino/Agouti strain not only had a high superovulation response, but also provided a simplified breeding format as a result of identifiable coat color. Although those novel approaches led to some progress, there were also some drawbacks.…”