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：

甲狀腺轉錄因子-2; 表達載體; 骨髓間質干細胞; 轉染; 基因; 小鼠

DOI：

10.7507/1002-0179.20130522

Video：

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目的　構建小鼠甲狀腺轉錄因子-2（TTF-2）轉基因動物表達載體（pBROAD3-TTF-2），觀察其在小鼠骨髓間充質干細胞（BMSC）中的表達。方法　從C57BL/6J小鼠肝臟組織中提取基因組DNA，利用聚合酶鏈式反應方法擴增出TTF-2基因1 113 bp開放閱讀框，通過DNA重組技術將TTF-2基因片段插入克隆載體pMD18-T中，經測序正確后，再重組于pBROAD3-mcs中，構建轉基因動物表達載體pBROAD3-TTF-2，用酶切電泳分析對其進行鑒定。運用脂質體轉染試劑將其轉染BMSC后，蛋白質印跡法檢測TTF-2基因的表達。結果　① DNA測序證實目的基因序列正確無突變，酶切電泳分析得到相應的目的片段，大小與理論計算值一致，成功構建轉基因動物表達載體pBROAD3-TTF-2。② 蛋白質印跡法顯示轉染的BMSC高表達TTF-2蛋白。結論　成功構建了pBROAD3- TTF-2轉基因動物表達載體，顯示其轉染BMSC后TTF-2基因的表達，為下一步建立TTF-2轉基因小鼠模型奠定了基礎。

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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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. 　黃磊, 石冰, 宋慶高, 等. TTF-2 和TGFβ3 在C57BL/6J 小鼠腭突發育過程中的表達變化[J]. 河北醫藥, 2011, 33(14): 2085-2087.
7. 　黃培堂, 俞諱源, 陳天彌, 等. PCR技術實驗指南[M]. 北京: 科學出版社, 1998: 23-33.
8. 　王素梅, 李力, 張瑋, 等. 組織蛋白酶L基因真核表達質粒的構建與鑒定[J]. 廣西醫科大學學報, 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薩姆布魯克,拉塞爾DW. 分子克隆實驗指南[M]. 3版. 黃培堂, 等, 譯. 北京: 科學出版社, 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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