人凝血因子Ⅸ乳腺特异表达载体的构建及细胞转染

doi:10.3864/j.issn.0578-1752.2014.04.017

Abstract

Abstract: 【Objective】 Human coagulation factor Ⅸ (hFIX) plays a key role in blood coagulation and is important for clinical treatment of hemophilia. The objective of this study is to construct human coagulation factor Ⅸ (hFIX) mammary expression vector and test the expression of hFIX in porcine mammary epithelial, and obtain hFIX-transgenic porcine mammary epithelial and female porcine fetal fibroblast cells to prepare to produce hFIX mammary specific expression transgenic pigs .【Method】Total RNA was extracted from human fetal liver tissues and human coagulation factor Ⅸ (hFIX) cDNA was amplified by RT-PCR followed the instructions of RNAiso Reagent and Prime Script RT-PCR Kit from TAKARA. PCR was performed to amplify bovine growth hormone (BGH) polyA fragment. Both hFIX cDNA and BGH polyA fragments were cloned to pbCSN2-RC plasmid, located after the bovine beta-Casein (CSN2) promoter, to achieve mammary specific expression vector pbCSN2-hFIX-pA-RC with neomycin- resistance and red fluorescence genes. Porcine mammary epithelial cells were obtained by culturing 1mm3 mammary tissue cubes from the breast tissue of lactation pigs that slaughtered in the local slaughterhouse and purifying with time-controlled trypsinization. The chromosome analysis was performed on the derived cells, and the cells with normal number of chromosomes were transfected with pbCSN2-hFIX-pA-RC by lipofection technology. The transfected porcine mammary epithelial cells were screened by neomycin-resistance and red fluorescent expression. The expression of hFIX was further confirmed by real-time PCR. To only transfect the female fibroblasts, the SRY PCR was performed on the porcine fibroblasts cultured in the lab to determine the sex of the cells. Finally, the confirmed vector was transferred to porcine fetal fibroblast cells by lipofection technology. 【Result】 The results of PCR and restricted endonucleases digestion analysis showed that the hFIX cDNA and BGH PolyA were cloned into pbCSN2-RC, and the pbCSN2-hFIX-pA-RC with double screening labels was successfully constructed. After screening by G418 and red fluorescent expression of transfected porcine mammary epithelial cells, the expression of hFIX directed by bovine β-casein in porcine mammary epithelial cells was confirmed by real-time PCR in the positive cells. This result indicated that the vector pbCSN2-hFIX-pA-RC has the ability to direct the hFIX mammary specific expression. Then the constructed vector was transferred into female porcine fetal fibroblasts. After screening, the positive hFIX-transgenic female porcine fetal fibroblast cells were obtained successfully. 【Conclusion】The bovine β-casein promoter can successfully direct the mammary specific expression of exogenous gene. This research has laid a massive foundation for cultivating mammary gland bioreactor by somatic cell nuclear transfer.

Key words: human coagulation factor Ⅸ (hFIX) , mammary specific expression vector , porcine mammary epithelial cells , porcine fetal fibroblast cells , transfection

HAN Xue-Jie, SA Ri-Na, LIANG Hao, LI Xue-Ling, LI Rong-Feng. Construction of Human CoaguLation Factor Ⅸ Mammry Expression Vector and Transfection[J].Scientia Agricultura Sinica, 2014, 47(4): 769-778.

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