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Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification
JoVE 3155 11/18/2011

BRC Current Technology October 2020


14. Generation of chimeric mice with spermatozoa fully derived from embryonic stem cells

Analysis of lethal gene function without
conditional knockout mouse

Generation of chimeric mice

Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo, but generation and maintenance of these mice is generally time and effort-consuming. To overcome these drawbacks, we established a strategy to produce F0 generation chimeric mice with tissues or cells fully derived from embryonic stem cells (ESCs) with lethal gene mutations. We microinjected Cas9 mRNA and three different guide RNAs targeting the two exons of the Nanos3 gene (sgNanos3), which is essential for germ cell development, into wild-type mouse zygotes. We produced male chimeric mice by injecting male ESCs with a Dnmt3b mutation, which normally causes embryonic death, into the sgNanos3-treated blastocysts. The chimeric mice produced F1 pups derived exclusively from the mutant ESCs, suggesting that the chimeras had spermatozoa fully derived from the ESCs. This result revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach could be applied for analyses in other combinations of organs and lethal genes.


Reference K Miura et al. “Generation of chimeric mice with spermatozoa fully derived from embryonic stem cells using a triple-target CRISPR method for Nanos3Biol Reprod, DOI: 10.1093/biolre/ioaa176