Zscan4c^tm1.1Kom

Most of exon 2 (from the ATG start codon onwards) and intron 2 was replaced with the emerald GFP gene and polyA site, a loxP site and an FRT site flanked neomycin resistance gene cassette. The neo cassette was removed through subsequent flp-mediated recombination. This creates a reporter and null allele with the target gene knocked-out and the GFP under the control of its promoter. Because of the high sequence similarity between the parologs in the gene cluster where the target gene is located, the targeted mutation strategy was combined with an endonuclease-mediated mutation strategy. The targeting vector was transfected into ES cells together with a CRISPR/CAS9 vector encoding two sgRNAs directed to the proximal half of intron 2. (J:241396)