| Transgenic animal mammary gland bioreactor has been developed several years as a high-tech that produce active proteins in mammary gland of transgenic animals. The main technology is obtaining transgenic animals with various transgenic technologies which can high-level express foreign gene under mammary gland specific-expressed promoter in mammary gland of mammals, with that desired products can be obtained in milk. Dairy goat is an expected candidate to produce transgenic animal mammary gland bioreactor because of short pregnancy period, high-level milk production, high disease resistance, etc. The most component of Whey proteins in ruminant milk isβ-lactoglobulin. The regulatory elements of BLG have been used in construction of mammary gland specific-expressed vector. In general, the regulators of genes encoded milk protein exist in their 5' and 3' remote ends and introns, and common plasmids have limited capability. Thus, in most vector construction, several regulatory elements were assembled in artificial. So the vectors always lack sequences among regulatory elements, and these sequences may regulate genes expressing also. So, assembling the whole BLG locus of dairy goat including all regulator elements and foreign genes which encoding desired proteins into vector that has large capability is a alternative method besides "gene targeting" to produce transgenic animals that specific and high-level expressing foreign genes without "position effect" in mammary gland.To obtain the whole BLG locus of dairy goat, a library of dairy goat genomes and screening the library for BLG positive clones are needed. BAC is an expected vector to construct Eukaryotic nucleus genomic library because of its large capacity, high transform effect, less chimerism, easy to manipulate and recover, stable existing in host cells. Besides isolating intact gene, the genomic library is utilized in many other applications: the whole genome physical mapping; elucidating gene organization, function and expression pattern; positional cloning; long range DNA sequencing and anchoring; identification of putative cis-regulatory elements and comparative genomics research. Leukocytes were isolated from blood in jugular vein of Saanen dairy goat, and were embeded in LMP agarose gel. To obtain size collecting high molecular weight DNA, genomes were partially digested with HindIII followed by twice selecting with CHEF gel electrophoresis, and then DNA fragments in suitable size were recovered by electroelution for less damage. Vector pIndigoBAC-5 was dephosphorylated with HK Phosphatase. The partially digested genomic DNA was ligated with treated BAC vector at a 1:10 molar ratio. Liagtion mixtures were dialyzed for desalinization and concentration followed by electroporating the competent DH10B bacteria. Electroporations were plated on trays containing chloramphenicol, IPTG, and X-gal. White clones were picked with sterile toothpicks to construct the library. The insert size ranged from 25 to 140 kb, and the average insert size is 78 kb in the library which corresponds to approximately 1.33 genome equivalents. Two couples of primers were designed according to goat BLG gene sequences (accession No. Z33881) in GenBank, and PCR was performed to screen the library for positive clones containing entire BLG sequences. Two clones with 60kb, 82 kb inserted fragments which were larger than BLG regulatory sequences in GenBank were obtained. These inserted fragments can be used for constructing BAC-based mammary gland-specific expressing vectors.The method of obtaining HMW DNA, optimizing the condition of partially digestion and effected ligating and transforming system were studied, a BAC library construction system that suit our laboratory was established. Two positive clones containing BLG loci were isolated for later work.
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