Construction of Transgenic Animal Expression Vector of Mouse Thyroid Transcription Factor-2 and Its Expression in Bone Marrow-derived Mesenchymal Stem Cells_West China Medical Journal

Authors：

HUANG Lei ¹ ,  SHI Bing ^2,3 , ZHENG Qian ^2,3 , MENG Tian ^2,3 , WANG Yan ^2,3

1. Department of Stomatology, Guangzhou First People’s Hospital, Guangzhou, Guangdong 510016, P. R. China; 2. State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, Sichuan 610041, P. R. China; 3. Department of Cleft Lip and Palate Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

SHI Bing, Email: huangleikmmc@21cn.com

Keywords：

甲状腺转录因子-2; 表达载体; 骨髓间质干细胞; 转染; 基因; 小鼠

DOI：

10.7507/1002-0179.20130522

Video：

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目的　构建小鼠甲状腺转录因子-2（TTF-2）转基因动物表达载体（pBROAD3-TTF-2），观察其在小鼠骨髓间充质干细胞（BMSC）中的表达。方法　从C57BL/6J小鼠肝脏组织中提取基因组DNA，利用聚合酶链式反应方法扩增出TTF-2基因1 113 bp开放阅读框，通过DNA重组技术将TTF-2基因片段插入克隆载体pMD18-T中，经测序正确后，再重组于pBROAD3-mcs中，构建转基因动物表达载体pBROAD3-TTF-2，用酶切电泳分析对其进行鉴定。运用脂质体转染试剂将其转染BMSC后，蛋白质印迹法检测TTF-2基因的表达。结果　① DNA测序证实目的基因序列正确无突变，酶切电泳分析得到相应的目的片段，大小与理论计算值一致，成功构建转基因动物表达载体pBROAD3-TTF-2。② 蛋白质印迹法显示转染的BMSC高表达TTF-2蛋白。结论　成功构建了pBROAD3- TTF-2转基因动物表达载体，显示其转染BMSC后TTF-2基因的表达，为下一步建立TTF-2转基因小鼠模型奠定了基础。

Citation： HUANG Lei,SHI Bing,ZHENG Qian,MENG Tian,WANG Yan,.. Construction of Transgenic Animal Expression Vector of Mouse Thyroid Transcription Factor-2 and Its Expression in Bone Marrow-derived Mesenchymal Stem Cells. West China Medical Journal, 2013, 28(11): 1647-1650. doi: 10.7507/1002-0179.20130522 Copy

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12.	J萨姆布鲁克,拉塞尔DW. 分子克隆实验指南[M]. 3版. 黄培堂, 等, 译. 北京: 科学出版社, 2002: 331-332.

1. 　Zannini M, Avantaggiato V, Biffali E, et al. TTF-2, a new forkhead protein, shows a temporal expression in the developing thyroid which is consistent with a role in controlling the onset of differentiation[J]. EMBO J, 1997, 16(11): 3185-3197.
2. 　Macchia PE, Mattei MG, Lapi P, et al. Cloning, chromosomal localization and identification of polymorphisms in the human thyroid transcription factor 2 gene (TITF2)[J]. Biochimie, 1999, 81(5): 433-440.
3. 　De Felice M, Ovitt C, Biffali E, et al. A mouse model for hereditary thyroid dysgenesis and cleft palate[J]. Nat Genet, 1998, 19(4): 395-398.
4. 　Clifton-Bligh RJ, Wentworth JM, Heinz P, et al. Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia[J]. Nat Genet, 1998, 19(4): 399-401.
5. 　Castanet M, Park SM, Smith A, et al. A novel loss-of-function mutation in TTF-2 is associated with congenital hypothyroidism, thyroid agenesis and cleft palate[J]. Hum Mol Genet, 2002, 11(17): 2051-2059.
6. 　黄磊, 石冰, 宋庆高, 等. TTF-2 和TGFβ3 在C57BL/6J 小鼠腭突发育过程中的表达变化[J]. 河北医药, 2011, 33(14): 2085-2087.
7. 　黄培堂, 俞讳源, 陈天弥, 等. PCR技术实验指南[M]. 北京: 科学出版社, 1998: 23-33.
8. 　王素梅, 李力, 张玮, 等. 组织蛋白酶L基因真核表达质粒的构建与鉴定[J]. 广西医科大学学报, 2008, 25(2): 219-221.
9. 　Liu ZJ, Gao X, Cai Y, et al. Construction of a full-length iASPP expression plasmid pcDNA3.1+/iASPP and its biological activity[J]. Plasmid, 2009, 62(1): 10-15.
10. 　Kisseberth WC, Brettingen NT, Lohse JK, et al. Ubiquitous expression of marker transgenes in mice and rats[J]. Dev Biol, 1999, 214(1): 128-138.
11. 　Farley FW, Soriano P, Steffen LS, et al. Widespread recombinase expression using FLPeR (flipper) mice[J]. Genesis, 2000, 28(3-4): 106-110.
12. 　J萨姆布鲁克,拉塞尔DW. 分子克隆实验指南[M]. 3版. 黄培堂, 等, 译. 北京: 科学出版社, 2002: 331-332.

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Construction of Transgenic Animal Expression Vector of Mouse Thyroid Transcription Factor-2 and Its Expression in Bone Marrow-derived Mesenchymal Stem Cells

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