Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (16): 3414-3421.doi: 10.3864/j.issn.0578-1752.2012.16.022

• VETERINARY SCIENCE • Previous Articles     Next Articles

Construction of miR-34c Lentiviral Expression Vector and Its Infection in Dairy Goat Germ Line Stem Cells

 LIU  Chao, YU  Meng, ZHU  Hai-Jing, LI  Ming-Zhao, HUA  Jin-Lian   

  1. 西北农林科技大学动物医学院/陕西省干细胞工程技术研究中心/农业部动物生物技术重点开放性实验室, 陕西杨凌 712100
  • Received:2012-01-17 Online:2012-08-15 Published:2012-06-14

Abstract: 【Objective】In order to over-express mouse miR-34c economically and efficiently, mouse miR-34c lentiviral expression vector was constructed using two different methods.【Methods】The pri-miR-34c was amplified and inserted into downstream of CMV and U6 promotors in pLL3.7, respectively. The recombinant lentiviral vector together with lentivirus package plasmid mixtures were transfected into 293T cells to package virus. The titres of the viruses were examined and then the viruses were infected with 293T cells and dairy goat male germ line stem cells and 293T cells. The transfection efficiency was measured by the percentage of GFP expression cells, and the expression level of miR-34c was determined by qPCR.【Results】The recombinant pLL3.7-CMV-34c vector was successfully constructed, confirmed by endonuclease digestion analysis and DNA sequencing. The expression level of miR-34c in cells infected with the packaged pseudoviriius was distinctively increased. 【Conclusion】Using both CMV and U6 promotor can over-express miR-34c vector. And the virus packaged with recombined vector using U6 promoter can infect dairy goat male germ line stem cells more efficiently.

Key words: miR-34c, lentivirus vector, male germ line stem cells, dairy goat

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