Studies On The Transgenic Cloning And Genetic Analysis Of Guangxi Buffalos

Firstly, methods for isolation and in vitro culture of buffalo fetal fibroblasts (BFF) were establishd, and karyotype stability and mycoplasma contamination of BFF were detected during passages. Large number of high vitality cells could be obtained by tissue explant culture and trypsin digestion. Karyotype analysis of 5th, 15th, 25th, 30th passage BFF showed that more than 75% cells had normal karyotype, and there were no significant difference among different passages (P>0.05). Two pair of nested-PCR primers were designed to detect mycoplsma contamination of BFF, and no contamination was found in 5th, 15th, 25th, 30th passages cells. Thus, the present culture system is stable enough to serve BFF cells for somatic cell cloning and transgenic studies.Factors affecting the proliferation and colony formation of BFF cells cultured in low density were studied. The rate of colony formation was less than 20% when BFF cells were cultured in low density. Insulin could stimulate proliferation of BFF cells and more colonies were formed. Addition of cell conditioned medium also could improve the growing of BFF cells. Qualified serum could increase the colony formation in vitro than that of homemade serum. Poly-lysine was also beneficial to the form of the colony when used as growing media, but agar and glutin were harmful to the growing of BFF cells.The transfection efficiency of electroporation and lipofectin mediated methods was compared. The efficiency of electroporation was line up with the electrical field intensity in which 20%～50% of cells died when the field intensity was set at 40 to 50V/mm. Employment of 8μL lipofectin and 2μg DNA resulted in more cells transfected. Quality of DNA and state of cell were associated with transfection efficiency of lipofectin. At the same culture condition, lipofectin reagent method can obtain more transgenic colonies.The growing status of transgenic cells selected by G418 was investigated in this study. Buffalo fetal fibroblasts were transfected with lined plasmid pCE-EGFP-IRES-Neo-dNdB by Lipofectin reagent. Then anti-G418 cell colonies were collected after two weeks of selecting culture and continued culturing to enlarge the cell amount for detecting cytobiology characterization. The results showed that the proliferation vitality of anti-G418 cell colony was decreased, morphology changing can be seen with normal cells as control which grown on coverslips after hematoxylin and eosin staining. Apoptosis symptom also can be detected, though chromosome karyotype had no distinct difference with nomal cells. These results stated clearly that the long time screening in G418 could do harm to the transgenic cells to a certain degree.4.1kb 5' promoter region of sheepβ-casein gene from the far upstream sequence to the part of exon2 was amplified by the long distance-PCR technique, the human 5.5kb thrombiotin gene was obtained with the same technique. We insert these two segments into ploxp vector after cutting with restriction enzymes. Green fluorescent protein (GFP) gene sequence came from plasmid pCE-EGFP-IRES-Neo-dNdB and then inserted into ploxp-CN-TPO. Enzyme cutting analysis and PCR testing results showed that the transgenic vector ploxp-EGFP-CN-TPO was constructed as designed advance. BFF cells were transfected with the lined ploxp-EGFP-CN-TPO, and 35 anti-G418 cell colonies were harvested after G418 selection. GFP signals was detected in 4 colonies and got 1 positive transgenic cell colony after PCR proving. The positive transgenic cells were used as donors to reconstruct embryos by micromanipulate and fusion method. The development rate of embryos derived from transgenic cells were significant lower than normal cells (P<0.05). The GFP signal could be detected after 4-cell stage of reconstructed transgenic embryos, which means that foreign gene can express normally in the transgenic embryos.Twenty three microsatellite loci were selected to observe the genetic variation of Guangxi swamp buffalo population. Nineteen primer pairs amplified discrete products and gave polymorphic band patterns on a panel of 60 buffaloes. The mean effective number of alleles per polymorphic marker was 4.0189, mean heterozygosity per polymorphic marker ranged from 0.1852 to 0.4722, mean polymorphism information contents (PIC) for Guangxi buffalo was 0.6639, eighteen microsatellite loci displayed high polymorphism. Then genome source of cultured cell was analyzed using nineteen microsatellite loci, results indicated that cultured cell and donor buffalo had same genome pattern. Thus we conclude that eighteen microsatellite loci can be used in identify the genetic relationship of buffaloes.To investigate the polymorphism of mitochondrial DNA in Guangxi swamp buffalo, the entire mitochondrial D-loop region of 66 water buffaloes of four different breeds were analyzed, 58 mitochondrial haplotypes with 147 polymorphic sites were detected. The haplotypes of swamp buffaloes and river buffaloes formed two different clusters. Using bovine D-loop sequences as outgroup, three phylogenetic trees were constructed with NJ, ML and Bayes method respectively. These trees all showed that swamp buffaloes and river buffaloes belong to two different clusters distinctly, and two small clusters were observed in the swamp cluster of the three. By median joining network analysis, three different clusters also be found, the high diverged clusters between swamp and river buffalo, and two little clusters appear in the whole swamp cluster. The mismatch distribution analysis showed that there may be two different indepent events in the evolution history of buffaloes. Pooled with 211 released D-loop sequences of Genbank, we detected 129 haplotypes with 158 polymorphic sites in 878bp multi-aligned region. The phylogenetic tree and median joining network analysis also showed that there were three clusters in total buffaloes, two swamp buffalo's clusters and one high diverged river swamps. Two obviously wave crests appeared in the mismatch distribution analysis, which means that there maybe two different population expanding event in water buffaloes. We conclude based on the above results, that river and swamp buffalos decented from two different domestication events, and there were at least two ancestor gene pools in the domestication of swamp buffaloes.