Mecp2^em2Bird

A targeting vector, an sgRNA and CRISPR/Cas9 technology were used to modify the locus as follows: a loxP site, a neomycin resistance gene cassette, a transcriptional STOP cassette and a second loxP site, all inserted into intron 2, a modified exon 3, a modified exon 4, sequence for linker peptide GSSGSSG, and the EGFP gene. The modification to exon 3 is the deletion of sequence for codons 30-87 (splice variant e1) or 13-70 (splice variant e2). The modification to exon 4 is the replacement of sequence for codons 174-271 (approximately; includes the endogenous nuclear localization sequence (NLS))(splice variant e2) with sequence for linker peptide (GSSGSSG) and SV40 NLS (PKKKRKV), and the deletion of sequence for codons 313-484 (approximately)(splice variant e2). The neo-STOP cassette was subsequently removed through cre-mediated recombination. This leaves a knockin allele where the endogenous gene is replaced with a truncated version (with sequences immediately upstream of the methyl-CpG binding domain (MBD) and downstream of the NCor/SMRT interaction domain (NID) deleted and sequence between the two domains replaced with SV40 NLS sequence) fused to a fluorescent marker. The truncated protein retains 32% of the wild-type sequence. (J:252845)