|
|
|
|
Construction and identification of recombinant adenovirus encoding TCF4 and ΔTCF4 |
| HU Jun, YE Rong, WANG Chun-yan, CUI Heng-jin, XU Xiao-jing, ZHANG Huan-xiang |
(Department of Cell Biology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou Jiangsu 215123, China) |
|
|
|
|
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.
|
|
Received: 16 May 2013
|
|
|
|
| [1]Nusse R. Wnt signaling and stem cell control［J］.Cell Res,2008,18(5):523-527.[2]Neth P, Ciccarella M, Egea V, et al. Wnt signaling regulates the invasion capacity of human mesenchymal stem cells［J］.Stem Cells,2006,24(8):1892-1903.[3]Barker N, Huls G, Korinek V, et al. Restricted high level expression of Tcf-4 protein in intestinal and mammary gland epithelium［J］.Am J Pathol,1999,154(1):29-35.[4]Liao Y,Lonnerdal B.Beta-catenin／TCF4 transactivates miR-30e during intestinal cell differentiation［J］. Cell Mol Life Sci, 2010, 67(17): 2969-2978.[5]Jiang Y, Zhou XD, Liu YK, et al. Association of hTcf-4 gene expression and mutation with clinicopathological characteristics of hepatocellular carcinoma［J］. World J Gastroenterol, 2002, 8(5): 804-807.[6]Beildeck ME, Islam M, Shah S, et al. Control of TCF-4 expression by VDR and vitamin D in the mouse mammary gland and colorectal cancer cell lines［J］. PLoS One, 2009, 4(11): e7872.[7]Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages［J］. Blood, 2008, 112(2): 295-307.[8]Wang XJ, Li QP. The roles of mesenchymal stem cells (MSCs) therapy in ischemic heart diseases［J］. Biochem Biophys Res Commun, 2007, 359(2): 189-193.[9]Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow［J］. Nature, 2002, 418(6893): 41-49.[10]Ozawa K, Sato K, Oh I, et al. Cell and gene therapy using mesenchymal stem cells (MSCs)［J］.J Autoimmun,2008,30(3):121-127.[11]Lo WC, Chen WH, Lin TC, et al. Preferential therapy for osteoarthritis by cord blood MSCs through regulation of chondrogenic cytokines［J］. Biomaterials, 2013, 34(20): 4739-4748[12]Zheng B, Wang C, He L, et al. Neural differentiation of mesenchymal stem cells influences chemotactic responses to HGF［J］. J Cell Physiol, 2013, 228(1): 149-162.[13]Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology, and potential applications［J］. Stem Cells, 2001, 19(3): 180-192.[14]Coffelt SB, Marini FC, Watson K, et al. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells［J］. Proc Natl Acad Sci USA, 2009, 106(10): 3806-3811.[15]Logan CY, Nusse R. The Wnt signaling pathway in development and disease［J］. Annu Rev Cell Dev Biol, 2004, 20: 781-810.[16]Daniels DL, Weis WI. Betacatenin directly displaces Groucho／TLE repressors from Tcf／Lef in Wntmediated transcription activation［J］. Nat Struct Mol Biol, 2005, 12(4): 364-371.[17]Graham TA, Weaver C, Mao F, et al. Crystal structure of a beta-catenin／Tcf complex［J］. Cell, 2000, 103(6): 885-896.[18]Lu R, Qu Y, Ge J, et al. Transcription factor TCF4 maintains the properties of human corneal epithelial stem cells［J］. Stem Cells, 2012, 30(4): 753-761. |
|
|
|
2013, Vol. 23 
