Construction Of TGF-?1 Gene Overexpression Lentivirus Vector And Identification Of Transfected Beagle Bone Marrow Mesenchymal Stem Cells

Objectives:It is necessary to use broad-spectrum immunosuppressant to treat the rejection after organ transplantation,so as to find a way to replace or reduce the use of immunosuppressant or increase the immune targeting.Combined with the immunosuppression and immunoregulation of transforming growth factor ?1(TGF-?1)and the characteristics of low immunogenicity and immunosuppression of mesenchymal stem cells,we constructed a lentivirus vector of TGF-?1 gene overexpression in beagle dogs,and transfected it into beagle dog bone marrow mesenchymal stem cells(CMSCs)for identification.Methods:1.The whole gene was obtained by oligomix(?)oligonucleotide synthesis.The target gene of TGF-?1 was purified by PCR,and the target gene fragment was recovered and identified by agarose electrophoresis.In order to obtain the target gene(canine)TGF-?1 and lentivirus vector,the two vectors were connected by clonexpress(?)entry one step cloning kit to construct LV5 vector carrying the target gene.The sensitive cells mediated by E.coli were cultured and transfected with bacterial suspension by calcium chloride method.The positive clones were sequenced and compared with the target gene(canine)TGF-?1 for further experiments.2.First,purchased 293T cells were used for cell recovery,counting,passage,and cryopreservation.Packaging culture dishes are prepared with passaged 293T cells.Adherent cells can reach 80%-90%.Serum-free DMEM+helper plasmid V9120+lentiviral packaging plasmid was added to RNAi-Mate at room temperature for fusion,and then inoculated in a packaging petri dish to start cultivation.The cultured cell suspension was collected by centrifugation and the virus solution was concentrated,and the virus titer was detected.3.Bone marrow was obtained by bone marrow puncture under general anesthesia of beagle dogs.The cells were separated and cultured by density gradient centrifugation and adherent culture.When the adherent rate was more than 90%,subculture began.The third generation cells were cultured and identified according to the standard of conventional MSC,and frozen for future use4.The cell infection solution was mixed with canine TGF-?1-containing virus solution and cMSCs and cultured in a 24-well plate.Microscope and fluorescence microscopy were observed,photographed,and puromycin(Puromycin,PM)resistance virus screening to determine the best infection rate.CMSCs-TGF-?1 cells were maintained and cultured with the best resistant virus screened,and quantitative real-time polymerase chain reaction(Q-PCR)was used for detection and Western Blot(WB))Detect the transcription and expression of TGF-?1 as the target gene after transfecting cMSCs with TGF-?1-LV5 lentiviral vector.Results:The target gene of TGF-?1 was successfully extracted and connected with LV5 vector by recombinant clone.After transfection of the receptor cells,the plasmid was collected.After agarose detection and sequencing with the target gene,the positive plasmid of the target gene was successfully obtained,and the TGF-?1-lv5 lentivirus vector suspension was successfully constructed.After cell morphology observation,single adherent cells appeared 24 hours later,with little amount and no aggregation;after 48 hours,adherent cells with different sizes and irregular morphology were observed,with aggregation growing gradually;5-7 days later,the growth of cells gradually accelerated,from the initial appearance of long fusiform cells gradually fused into a vortex arrangement,reaching the conditions of passage.The results of antibody test showed that CD90,CD 105(+),CD45(-),and adipogenic and osteogenic tissues could be induced successfully.The expression of fluorescence and protein were observed in 48 hours and 144 hours after transfection of CMSCs cells with TGF-?1-lv5 lentivirus vector;puromycin resistant virus screening:4ug/ml was the best concentration for puromycin screening,and more than 80%of the infection rate could be obtained under the fluorescence microscope on the ninth day of culture.Quantitative real-time polymerase chain reaction(Q-PCR)and Western blot showed that TGF-?1 was highly expressed.Conclusions:TGF-?1-lv5 lentivirus vector was constructed successfully;Beagle dog bone marrow tissue was isolated,cultured and identified as dog bone marrow mesenchymal stem cells,and the concentration was up to expectations;after beagle dog bone marrow mesenchymal stem cells were transfected,the average transfection rate was more than 80%,and high expression TGF-?1 cell line was obtained.Prospect:Canine bone marrow mesenchymal stem cells were obtained through isolation,culture and identification of bone marrow tissues of organisms.After being transfected with TGF-?1 lentivirus,cMSCs-TGF-?1 cells were successfully obtained.The research and comparison of Tregs cells for subsequent T cell co-culture detection Induction of autoimmune tolerance induced by combined pancreas-kidney transplantation in Beagle dogs provides preliminary experimental basis.