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| Construction of a Vector pEGFP-loxp-Lox2272 and Optimization of Procedure for Selecting Transgenic Bovine Fetal Fibroblasts |
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Abstract Green fluorescent protein (GFP) is an ideal marker for cell sorting. Integrating green fluorescent Protein (GFP) into bovine Y chromosome is the key for sorting X and Y sperm by GFP. In this study, one loxp site and one Lox2272 site were inserted into the vector pEGPP-N3 through special designed primers amplified by high-fidelity enzyme and the vector pEGFP-loxp-Lox2272 was constructed. The bovine fetal fibroblasts were transfected with linearized pEGFP-loxp-Lox2272 by LipofectamineTM (Lip) 2000. The results revealed that the best transfected effect was mediaed by 3 μL Lip 2000 and the best concentration of G418 for screening transgenic cell clones was 600 μg/mL. This study provided a useful method for constructing vectors and important reference for screening bovine gene-targeted cell clones.
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Received: 28 January 2011
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HUANG Xing, YAN Ai-Fen, DENG Ting-Xian, OUYANG Hong-Jia, LIU Lian, FENG Juan, ZHU Xiang-Xing, NIE Qing-Hua, TANG Dong-Sheng, ZHANG Xi-Quan. Construction of Zinc Finger Nuclease-induced Targeting Vector of Luchuan Pig (Sus scrofa) Fat1 Gene and Transgenic Study In vitro[J]. 农业生物技术学报, 2019, 27(8): 1369-1381. |
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