A Successive Three-step ‘Gap-repair’ Method to Generate the mWAP-hLF Hybrid Gene Locus

Abstract:

To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin(hLF) genomic sequence is directed by the up&down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step ‘Gap-repair’ method. First, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Then using ‘Gap-repair ’method mediated by Red recombination system of l-prophage in Escherichia coli, in the first step, the 8 kb 3￠ flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5￠ flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5￠ & 3￠ flanking region of mWAP gene locus was constructed. The result was confirmed by PCR、restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.