| This study was undertaken to explore the feasibility of constructing a vector to integrate human TPO gene to as 1-casein locus of bovine genome and transfecting this vector to bovine somatic cells through lipofectamine. Consulting beta-casein sequences of bovine, ovine and goat, a pair of primers was selected to amplify the 5 'regulation region of beta-casein gene of buffalo and ovine through LA-PCR, which are about 4.5Kb, 4.1Kb separately. These two fragments were cloned into the pMD-18T vector for the partial sequencing and restriction enzyme analysis. Homologous sequence alignment indicated 98% homology with that of sequences in GeneBank and the restriction enzyme digestion results were consistent with the design. Two TPO gene bovine somatic cells targeting vectors, ploxp-6.0-B|3CN-TPO-1.78 and ploxp-6.0-SpCN-TPO-1.78, had been constructed, which are about 25Kb and are consisted of five parts: ploxp skeleton, 6.0Kb homologous left arm, 1.78Kb homologous right arm, 5.5Kb tpo gene and 4.1Kb, 4.5Kb 5' regulation region. Analysis by LA-PCR and restriction enzymes digestion proved that the genetargeting vectors were identical to the design. The lined ploxp-6.0-SpCN-TPO-1.78 wastransfected into the fetal bovine fibroblast cells by lipofectamine and the transfected cellswere selected by G418 and GANC, a few of Aanti-neomycin cells were gotten after 24 daysof selection culture.In conclusions, 1) LA-PCR and Ex Taq can be used to amplify the 5'regulation region of buffalo and ovine. 2) Two 25Kb human TPO gene bovine somatic cells targeting vectors can be constructed by linking buffalo and ovine 5' regulation region (promotor), human TPO gene (purpose gene) and bovine as 1-casein gene homologous elements (targeted gene) into the ploxp plasmid vector without de-phosphatization. 3) Aanti-neomycin cell clone can be obtained by targeting these vectors (ploxp-6.0-SpCN-TPO-1.78) into bovine fetal fibroblasts through lipofectamine and long time culture selection.
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