Generation and Characterization of Knockout Mice Targeting REV7 gene, which is involved in DNA damage tolerance

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In the present study, we aimed to generate and analyze REV7 knockout mice. We carried out the construction of a knockout vector targeting the middle region of REV7, exon 3 to 6, in which REV7 interacts various proteins. The knockout vector was introduced into ES cells, and the cells were incubated in the selection medium with G418. About 150 G418-resistant colonies were cloned, and their genomic DNAs were extracted and used for PCR screening to identify homologous recombinants. However, we could not find homologous recombinants. Then we constructed another knockout vector using a different plasmid vector and introduced the vector into ES cells. About 750 G418-resistant clones were isolated and screened for identification of homologous recombinants. Despite many times of screening using various PCR condition, we could not find homologous recombinants in these clones. Possible reasons for the failure to identify homologous recombinants were ; 1) the genomic region cloned into the knockout vectors might not be suitable for homologous recombination, 2) PCR screening was not appropriate for identification of homologous recombinants in this case. For this reason, we decided to make the other knockout construct again using the different region of REV7 genome for homologous recombination. We constructed a new knockout vector targeting the region from exon 1 to exon 6 of REV7. We will introduce the new vector into ES cells soon and advance our project to generate REV7 knockout mice.