Figure 1.
Genetic design of the hypersensitive Chrna2L9′S/L9′S mouse line. (A) The generation of the hypersensitive Chrna2L9′S/L9′S mouse line has been described in the Materials and Methods. Briefly, a leucine to serine substitution was engineered using gene synthesis. The targeting vector was electroporated into 129S4/SvJae embryonic stem (ES) cells and homologous recombinants were confirmed through DNA sequencing (B) and Southern blot analysis (using the probe denoted by the single red line) (C). Targeted ES cells were microinjected into C57Bl/6J blastocyst embryos and implanted in pseudopregnant female mice, with the DNA sequence of the germline transmitted offspring confirming the AGC (Serine)/CTC (Leucine) heterozygous genomic modification (denoted by *). Genotyping of mice of was through tail biopsy, MyTaq HS Red Mix (Bioline) with PCR primers (A2L9S_1.10, A2L9S_1.11, denoted by arrows flanking the 34 bp FRT Site) designed upstream of and downstream from the deleted PGK-NEO, as described (FRT site denoted by the single arrow).