Abstract：
[Abstract] Objective: To construct recombinant adenovirus encoding full-length T cell factor 4(TCF4) and a mutant TCF4 (ΔTCF4), which delete the binding sites of β-catenin, and to investigate the expression and activity of these recombinant adenoviruses in MSCs, thereby providing the foundation for further study about the role of Wnt/β-catenin signaling in MSCs and sheding a light on the usefulness of MSCs in cell transplantation and gene therapies. Methods: The DNA sequences of TCF4 and ΔTCF4 were amplified from cDNA of MSCs by RT-PCR, and were subcloned into the shuttle plasmid pAdtrack-CMV, forming transfer vectors pAdtrack-CMVTCF4 and pAdtrack-CMV-ΔTCF4. The plasmids were recombined in BJ5183 that has transformed by adenoviral backbone plasmid pAdEasy-1. Subsequently, both recombinant plasmids of Ad-TCF4 and Ad-ΔTCF4 were confirmed by RT-PCR, PacⅠ digesting analysis, and DNA sequencing. The identified Ad-TCF4 and Ad-ΔTCF4 were transfected to QBI-293A cells to produce the adenoviruses. To purify and titrate of recombinant adenoviral vectors, and to discuss Ad-TCF4 and Ad-ΔTCF4 vectors infected MSCs. Western blotting was applied to detect the expression of TCF4 in MSCs infected with Ad-TCF4. Luciferase activity assay was used to measure the activity of Ad-ΔTCF4 in MSCs.Results: The adenovirus vectors encoding target genes were proved to be recombined successfully in QBI 293A; and their titer virus were 1.1×107 pfu／mL and 1.3×107 pfu／mL respectively, The optimal virus of Ad-TCF4 and Ad-ΔTCF4 were 150 MOI, in which the efficiency of infection reached more than 90％. Western blot analysed the high expression of TCF4 in Ad-TCF4-infected MSCs, while luciferase activity was effectively suppressed in MSCs infected with Ad-ΔTCF4. MSCs infected with Ad-ΔTCF4 displayed decreased migration as compared with the control, however, infection with Ad-TCF4 showed no obvious differences. Conclusion: The recombinant adenoviruses encoding TCF4 and ΔTCF4 were successfully constructed and could infect MSCs efficiently.
收稿日期: 2013-05-16
基金资助:
国家自然科学基金资助项目（30870642; 31071220）
通讯作者:
张焕相，男，教授，博士生导师，E-mail：hzhang@suda.edu.cn
作者简介: 胡君（1985—），女，硕士研究生
引用本文:
胡君, 叶荣, 王春燕, 等. 大鼠TCF4和ΔTCF4重组腺病毒载体的构建和鉴定[J]. 江苏大学学报:医学版, 2013, 23(4): 277-283.
HU Jun, YE Rong, WANG Chun-yan, et al.
Construction and identification of recombinant adenovirus encoding TCF4 and ΔTCF4. Journal of Jiangsu University(Medicine Edition), 2013, 23(4): 277-283.
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2013, Vol. 23 
