| In this paper, we directly cloned 2 248 bp 5′flank regulatory sequence of β-lactoglobulin gene from goat by PCR which was used as promoter to regulate human lactoferrin gene cDNA expressing in goat mammary epithelial cells for verifying the rationality of expressional vector and expressional efficiency of foreign gene. The recombinant plasmid pBLIG containing human lactoferrin gene cDNA was transfected into goat mammary epithelial cells after liposome transinfection. Positive cells were selected by G418 and PCR. The positive cells were used as nucleus dour to create transgenic cloned embryos by nuclear transfer techniques. The foreign gene in transgenic cloned embryos was detected by PCR and observation of EGFP. The object of detecting the foreign gene was to examine foreign gene integration and expression in positive transgenic cloned embryos in their early stages. The results of this research were as following: 1. The purified goat mammary epithelial cells kept normal characteristics until fifteenth passages. The cells could propagate and form dome-like construction which looked like nipple and it was called milk orb; majority of the cells were short shuttle-like or guboidal which looked like beehive; part of the cells looked like big round flat cakes; part of the cells were long. More microvilli appeared on the surface of the fifteenth epithelial cells, mitochondria and rough endoplasmic recticulums were rich in the cytoplasms and the cell cytoplasms had plenty of fatty droplets, which showed that the cells had vigorous secretive activity. Analysis on chromosome of the goat mammary epithelial cells indicated that the cells were unchanged during cultivation in vitro. Supernatant proteins of goat mammary epithelial cells were separated by SDS-PAGE and found that the supernatant of goat mammary epithelial cells had three stripes in accordance with three standard protein stripes. The results proved that the cultured epithelial cells in this research were goat mammary epithelial cells and they could express goat casein when cultured in vitro. 2. For the purpose of constructing mammary gland specific expressional vector, the goat β-lactoglobulin gene 5′flanking fragment (2 248 bp ) was cloned by PCR amplification. It consisted in the first exon and the first intron. After PCR product recovered and purified, the aim fragment was cloned at T site of pMD 18-T Vector. The positive recombination plasmid was marker as pBLG. The fragment was sequenced and compared with goat BLG gene which had published and found that the homology was 99.70 %. The nucleotide acid sequence was analyzed by compute, many factors or protein binding sites and response elements were found such as STAT5(MGF)binding site, CCAAT/enhancer binding protein binding site, SP1 factor binding site, Milk box , TBF factor binding site, RARE, TRE, MSBF and NF-1 binding sites, et al. It suggested that the cloned regulatory element could direct extra genes to express specifically in mammary epithelial cells, and it could be used to construct mammary gland-specific expressional vector. 3. pBLG and pEGFP-c1 were digested with restrictive enzyme AseI and NheI, the aim fragment was connected with T4 DNA ligase, the positive recombination plasmid was marker as pEB. pEB and pLTF were digested with HindIII and NheI, the aim fragment was connected with T4 DNA ligase, the positive recombination plasmid was marker as pBL. pBL and pIRES were digested with SalI and HindIII, the aim fragment was connected with T4 DNA ligase, the positive recombination plasmid was marker as pBLIE. PBLIE and pEGFP were digested with SalI and BamHI, the aim fragment was connected with T4 DNA ligase, so the mammary gland-specific expressional vector was constructed and was marker as pBLIG, which consisted in regulatory element (goat β-lactoglobulin 5′flank regulatory sequence), aim gene(hLF gene), IRES, report gene (EGFP), antibiotics gene (neo) and polyA sequence. 4. The recombinant plasmid pBLIG containing human lactoferrin gene cDNA was transfected into goat mammary epithelial cells after liposome transinfection. Positive cells were selected by G418 and PCR. Positive cells were proliferated and induced by hormone. The result of Western blotting analysis on cultured cell supernatant showed that transfected cells could expressed the exogenic gene and secreted hLF protein, whose MW was 58kDa. Positive cells were cultured in vitro and the transgenic goat mammary epithelial cells were established which could be used as nucleus dour to create transgenic goats by nuclear transplant techniques. 5. The transgenic cloned embryos were created effectively by means of electro-fusion, which used the transgenic goat mammary epithelial cells as nucleus dour. SOFaa medium plus 10 % NGS was better combination in goat transgenic cloned embryos culturing in vitro , the ratio of blastocyst could reach 15.91 %(7/44). 6. The foreign aim gene in transgenic goat cloned embryos was detected through PCR and the result showed that hLF gene and EGFP gene were combined in the chromosome of the cloned embryos successfully. EGFP was expressed gradually after 8～16 cells periods in most embryos. Part of the cloned embryos expressed EGFP at 2-cell period, and EGFP expressed more and more with development of the embryos, which reflected success of the transgenic identification by application of fluorescent phenomena. The ability to visualize, track and quantify events in living cells of EGFP could be used for check positive transgenic cloned embryos in their early stages .
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