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 lambda-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.
Animals ; Bioreactors ; DNA Repair ; genetics ; Genetic Engineering ; methods ; Humans ; Hybridization, Genetic ; Lactoferrin ; genetics ; Mammary Glands, Animal ; metabolism ; Mice ; Mice, Transgenic ; Milk Proteins ; genetics