[Study on a human tissue-type plasminogen activator mutant cDNA knocked-in the beta-casein gene site of murine ES cells].

A murine beta-casein gene targeting vector was constructed using the cloned genomic sequence. The short arm was 2.7 kb including mouse beta-casein gene 5' flanking sequence, exon1, intron1 and partial exon2. The long arm is a 3.4 kb fragment including partial intron2, exon3 approximately 7, intron3 approximately 6 and partial intron7. The human t-PA mutant cDNA was subcloned in the exon2 and fused with the mice beta-casein signal peptide sequence. The positive selective marker neo was placed in the middle of intron2. A tk negative selective marker was just outside the short arm. TC-1 ES cells were cultured and amplified on G418 resistant feeder layer. The linearized targeting construct DNAs of 45 microg were introduced into 2 x 10(7) ES cells by electroporation. Totally 192 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days. The colonies were amplified and subjected to genomic DNA preparation. The genomic DNAs were digested with EcoR I and used for Southern blot analysis. A probe inside the 5' homologous arm was used for hybridization. A 9.8 kb band was found in wild type, but the band was shift down from 9.8 kb to 6.6 kb in the beta-casein gene targeted allele because a new EcoR I site was introduced into the exon2 along with the human t-PA mutant gene. There were 9.8 kb and 6.6 kb bands in targeted ES cells. One clone of targeted ES cells with correct homologous recombination events was obtained among 78 analyzed clones. It lays foundation for gene targeted mice making.