Mmu-miR-34c慢病毒表达载体构建及在奶山羊雄性生殖干细胞的表达

doi:10.3864/j.issn.0578-1752.2012.16.022

中国农业科学 ›› 2012, Vol. 45 ›› Issue (16): 3414-3421.doi: 10.3864/j.issn.0578-1752.2012.16.022

Mmu-miR-34c慢病毒表达载体构建及在奶山羊雄性生殖干细胞的表达

刘超, 于萌, 朱海鲸, 李明昭, 华进联

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

收稿日期:2012-01-17 出版日期:2012-08-15 发布日期:2012-06-14
通讯作者: 通信作者华进联，Tel：029-87080068；Fax：029-87080068；E-mail：jlhua2003@ 126.com
作者简介:刘超，Tel：13933065541；E-mail：5595758@qq.com
基金资助:
国家自然科学基金项目（30972097）、教育部新世纪优秀人才支持计划（NCET-09-0654）

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

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

摘要/Abstract

摘要： 【目的】用两种方法构建miR-34c的慢病毒表达载体，并包装成慢病毒颗粒，为经济、高效的进行miR-34c超表达提供方案，为进一步研究miR-34c的作用奠定基础。【方法】从小鼠基因组中扩增miR-34c前体，分别插入pLL3.7的CMV启动子起始的GFP之后或U6启动子后，构建成载体pLL3.7-CMV-34c及pLL3.7-U6-34c。将重组慢病毒载体和包装质粒混合物转染包装细胞293T细胞，包装产生慢病毒颗粒，测定病毒滴度。感染293T细胞与关中奶山羊雄性生殖干细胞，用293T细胞与奶山羊雄性生殖干细胞的GFP表达程度测定转染效率，用定量PCR方法测定miR-34c的表达效率。【结果】重组质粒酶切及PCR分析及转化菌液测序，证明插入序列正确，qPCR检测显示：miR-34c的表达在慢病毒载体感染的293T细胞与关中奶山羊雄性生殖干细胞均显著上调。【结论】用 pLL3.7慢病毒载体中的CMV和U6两种启动子均可经济高效地进行miR-34c超表达，且由U6启动子启动的miR-34c慢病毒表达载体可以成功高效感染关中奶山羊雄性生殖干细胞。

关键词: miR-34c, 慢病毒载体, 雄性生殖干细胞, 奶山羊

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

刘超, 于萌, 朱海鲸, 李明昭, 华进联. Mmu-miR-34c慢病毒表达载体构建及在奶山羊雄性生殖干细胞的表达[J]. 中国农业科学, 2012, 45(16): 3414-3421.

LIU Chao, YU Meng, ZHU Hai-Jing, LI Ming-Zhao, HUA Jin-Lian. Construction of miR-34c Lentiviral Expression Vector and Its Infection in Dairy Goat Germ Line Stem Cells[J]. Scientia Agricultura Sinica, 2012, 45(16): 3414-3421.

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Mmu-miR-34c慢病毒表达载体构建及在奶山羊雄性生殖干细胞的表达

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

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