Myostatin基因沉默绵羊成纤维细胞系的建立

畜牧兽医学报 ›› 2011, Vol. 42 ›› Issue (10): 1368-1373.

Myostatin基因沉默绵羊成纤维细胞系的建立

唐大运1,2,3，朱化彬2，吴健敏3，杜卫华2，王栋2，赵学明2，陈汉忠1*，林秀坤2*

1.广西大学动物科技学院，南宁 530004； 2.中国农业科学院北京畜牧兽医研究所,北京100193； 3.广西兽医研究所，南宁 530001

收稿日期:2010-10-14 修回日期:1900-01-01 出版日期:2011-10-26 发布日期:2011-10-26
通讯作者: 陈汉忠1*，林秀坤2*

Establishment of Sheep Fibroblast Cell Line with Myostatin Gene Silencing

TANG Dayun1,2,3, ZHU Huabin2, WU Jianmin3, DU Weihua2,
WANG Dong2, ZHAO Xueming2, CHEN Hanzhong1*, LIN Xiukun2*

1.College of Animal Science and Technology, Guangxi University, Nanning 530004, China; 2.Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193 China; 3. Guangxi Veterinary Research Institute, Nanning 530001, China

Received:2010-10-14 Revised:1900-01-01 Online:2011-10-26 Published:2011-10-26
Contact: CHEN Hanzhong1*, LIN Xiukun2*

摘要/Abstract

摘要： 旨在获得肌肉生长抑制素（Myostatin，MSTN）基因沉默的成纤维细胞系，为进一步探索Myostatin的调控机理，获得 Myostatin的基因沉默的转基因羊奠定基础。针对小尾寒羊Myostatin的基因序列，设计4条siRNA干扰序列，构建pSilencer干扰载体，转染成纤维细胞，采用RTPCR进行筛选；构建慢病毒干扰载体，包装病毒，感染成纤维细胞，采用流式细胞技术分选阳性细胞，阳性细胞予以测序鉴定并检测沉默效果。结果，获得 Myostatin基因沉默效率约90%的RNA干扰片段PSL1，包装病毒获得滴度为1×108的病毒颗粒，流式细胞技术分选得到纯度达99%的 Myostatin基因沉默成纤维细胞系。采用慢病毒感染接合流式细胞术分选，能较快获得无抗性标记的转基因阳性细胞系，为培育 Myostatin基因沉默的转基因羊及探索Myostatin的调控机理奠定基础。

关键词: 肌肉生长抑制素, RNA干扰, 慢病毒, 绵羊成纤维细胞

Abstract: The aim of this study were to obtain fibroblast cell line with Myostatin silencing, to further understand the regulation mechanism of Myostatin and to get Myostatin silencing transgenic sheep. Four siRNAs were designed according to Myostatin gene sequences and Myostatin silencing pSilencer vectors were constructed, and then transfected into fibroblasts, the silencing effects were analysised by RTPCR; A lentiviral vector with Myostatin gene silencing was constructed and then lentivirus particles was packaged. Fibroblasts were infected by lentivirus particles and positive cells were isolated by flow cytometry. Positive cells were identified by sequencing and the silencing effect was analyzed by RTPCR. An effective siRNA sequence(PSL1) was obtained, and it inhibited the Myostatin expression significantly with a rate of 90%. The virus particles which were 108 titer determined by fluorescence microscope were obtained. Positive cell line with the purity of 99% were isolated by flow cytometry. The result indicate that positive transgene cell line without resistance marker can be quickly obtained by using lentivirus and flow cytometry. This study provides a useful cell line for further research the regulation mechanism of Myostatin and get transgenic sheep.

Key words: Myostatin, RNA interference, lentivirus, sheep fibroblast

唐大运;;朱化彬;吴健敏;杜卫华;王栋;赵学明;陈汉忠;林秀坤. Myostatin基因沉默绵羊成纤维细胞系的建立[J]. 畜牧兽医学报, 2011, 42(10): 1368-1373.

TANG Dayun;;ZHU Huabin;WU Jianmin;DU Weihua;WANG Dong;ZHAO Xueming;CHEN Hanzhong;LIN Xiukun. Establishment of Sheep Fibroblast Cell Line with Myostatin Gene Silencing[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2011, 42(10): 1368-1373.

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