Generating Of Tiam1 Transgenic Mice And Exploring The Function Of Tiam1 Gene

BACKGROUND & OBJECTIVETiam1 is one of guanine nucleotide exchange factors(GEFs),which activate GTPases by promoting the exchange of the GDP-bound forms from inactive to active. Although Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro,it specifically activates Rac in vivo.Recent evidence suggests that Tiam1 could also influence Rac GTPases signaling specificity by promoting their activation.Tiam1 has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins,which couples Tiam1-Rac activity to specific signaling pathways.Tiam1 was originally identified as the invasion-and metastasis-inducing gene in T lymphoma cells.The role of Tiam1 in cellular migration,invasion and metastasis may not be limited to T lymphoma.It was reported that Tiam1 plays an important role in promoting the tumor progression in a variety of cancers such as breast cancer,lung cancer and Ras-induced skin tumors.However,the possible role of Tiam1 in the metastasis of colorectal cancer is still not very clear.The transgenic animal is the one that carries a foreign gene which has been deliberately inserted into its genome.The transgenic animal could express exogenous genes and make it an indispensable tool for modern biologists.Transgenic mice are currently generated by pronuclear injection;another approach is the use of retroviruses as gene delivery vehicles.Retroviruses are able to stably integrate into the genome of cells.Lentiviruses are a class of retroviruses which can cause host organisms chronic illnesses.Among retroviruses,lentiviruses have the distinguished property of being able to infect both split-phase cells and non-split-phase cells.The lentiviral vector used in these experiments is a self-inactivating vector,which carry an internal promoter driving the GFP reporter gene.The lentiviral vector we used not only promoted the safety of the vector,but also increased the detection ratio of the exogenous gene it carried.After established the pCDF1-Tiam1-copGFP lentivirus vector,we successfully transfected the HT29 colorectal cancer cell line which do not express Tiam1,also we investigated Tiam1 biological functions in vitro.Meanwhile,by means of lentivirus-mediated perivitelline space microinjection of Tiam1 gene into zygotes,we generated the Tiam1 transgenic mice.Based on the Tiam1 transgenic mice model, this study will further investigate the functions of Tiam1 gene in vivo.METHODS1.Construction of lentiviral vector pCDF1-Tiam1-copGFPLentiviral vector pCDF1-Tiam1-copGFP was constructed by gene cloning.The recombinant plasmid of pCDF1-Tiam1-copGFP was identified by restriction endonuclease analysis and DNA sequencing.Recombinant lentivirus was harvested from 293FT cells cotransfected with the pPACKF1 Packaging Plasmid Mix.2.Effect of Tiam1 gene on the biological behaviors of human CRCTiam1/C1199HA cDNA was transfected into HT29.The expression level of Tiam1 gene was determined by RT-PCR,immunohistochemistry and Western blot. The biological behaviors of transfected HT29 were investigated by flow cytometry, MTT assay and plate colony formation assay in vitro.Cell migration and invasion were assessed by Transwell chamber and Boydon chamber. 3.Generation of Tiam1 transgenic mice by lentivirus-mediated gene transferLentiviruses harboring Tiam1 were subzonally injected into single-cell fertilized eggs of ICR mice,and subsequently embryos were transplanted into the pseudopregnant mice to attain F₀ mice.4.Screening Tiam1 transgenic mice(Founder,F₀)PCR,sequence analysis and Southern Blot were used to explore the Tiam1 gene combination in transgenic mice.5.Mouse propagation and transgene transmissionF₁ generation was produced after Tiam1 transgenic mice mated with wild-type ICR mouse.Detect GFP expression(F₁)under fluorescent stereo microscope. Immunohistochemistry analysis were used to explore the expressions of Tiam1 and GFP in F₁ mice,confirming that Tiam1 and GFP transgene actually integrated into genome of transgenic mice and could be transmitted to the next generation.6.Detection of positive phenotype of Tiam1 transgenic micePathological morphology of transgenic mice was detected.Corresponded cell proliferation activities were analysised by immunohistochemistry.7.Statistical analysisStatistical analysis was done using SPSS 13.0.Cell cycle,plate colony formation efficiency assay,cell migration and invasion assay were analyzed by two sample t-test. In vitro proliferation assay and subcutaneous tumor growth assay were analyzed by repeated measure square analyzeRESULTS1.Recombinant pCDF1-Tiam1-copGFP lentivirus vector was constructed and identified by restriction endonuclease analysis and DNA sequencing.6771bp vector segment and 3800bpTiam1 were got after EcoRI digested.After 48 hours of transferring pCDF1-Tiam1-copGFP and packaging plasmid into 293FT cell,the green fluorescence could be observed in most of the transferring cells.2.Single clone HT29 cells infected with recombinant lentivirus were selected and observed in inversion fluorescence microscope after 48 hours.HT29/Tiam1 cells showed a significantly enhanced proliferation features compared with the HT29/mock cells as determined by in vitro MTT assay.In addition,HT29/Tiam1 cells had a significant promotion in their ability to form colonies in plate compared with HT29/mock cells.Next,the effect of Tiam1 on tumor growth was assessed by subcutaneous injection of HT29/Tiam1 and HT29/mock cells for thirty days in vivo. Compared with HT29/mock cells,the expression of Tiam1 led to a pronounced increase in HT29/Tiam1 cell growth starting from day 15,up to 2.3-fold increase in mice of tumor volume at day 30 after cell injection.The results of migration and invasion assays showed that the HT29/Tiam1 cells posses a remarkable increasement in migration and invasiveness compared with HT29/mock cells in vitro.3.Transgenic mice were generated by subzonal injection of lentivirus harboring Tiam1 gene into single-cell fertilized eggs of ICR mice.We transplanted 588 virus-injected eggs into 33 pseudopregnant mice and attained 76 F0 mice(potential transgenic founders).Founder mice were selected from 76 potential transgenic founders(F0)by PCR assay,sequence analysis and Southern Blot.A total of 5 PCR-positive founders were attained,all of them transmitted Tiam1 gene to their offspring.The ratio of founders to born mice was 7%.4.Green fluorescence was detected in tissue samples.Those tissues including colon, stomach,lung,kidney and testis,which isolated from the Tiam1 transgene-positive mice but not from wild-type mice.Immunohistochemistry showed that the localization of GFP is consistent with that of Tiam1.Tiam1 can stablely transfer from one generation to the next generation. 5.Comparing to the control mice,significantly over-expressed Tiam1 was detected in multiple organs(colon,stomach,lung,kidney and testis)of Tiam1 transgenic mice. Correspondingly,cell proliferation activity was significantly enhanced in those organs,especially in the colon.CONCLUSION1.Successfully constructed lentiviral expression vector pCDF1-Tiam1-copGFP;2.Tiam1 gene plays an important role in proliferation,metastasis and invasion of CRC in vitro;3.Tiam1 transgenic mice were established by lentivirus-mediated gene transfer. Tiam1 gene could be transmitted from founders to subsequent generation;4.Significantly over-expressed Tiam1 was detected in multiple organs(colon, stomach,lung,kidney and testis)of Tiam1 transgenic mice.Correspondingly,cell proliferation activity was significantly enhanced in those organs,especially in the colon.The results of these experiments in vivo are consistent with that in vitro, which we have mentioned before.