A Method For Constructing GLP-1 Overexpression Intestinal Organoids

ObjectivesTo optimize the method of lentivirus infection of organoids and construct a lentiviral eukaryotic expression vector with the glucagon-like peptide-1(GLP-1)gene overexpression and transfect small intestinal organoids,and to provide a new strategy for clinical treatment of type II diabetes.MethodsThe primers of GLP-1/Fc gene were designed and PCR extended to obtain the full-length sequence of mouse GLP-1/Fc.The recombinant lentivirus plasmid pLenti-CMV-GLP-1/Fc-IRES2-ZsGreen was constructed by using restriction enzyme digestion to obtain GLP-1/Fc gene sequence and cloned into the lentivirus vector pLenti-CMV-IRES2-ZsGreen.The recombinant lentivirus plasmid was transformed into DH5 alpha bacteria.Positive clones were selected and amplificated after overnight culture,then sequenced after extraction of plasmid DNA.The correct recombinant plasmids were obtained.293T cells were transfected with the recombinant plasmid for 48h and the expression of green fluorescence was observed under a fluorescence microscope.Furthermore,The recombinant lentivirus plasmid was co-transfected into the 293T cells with lentivirus assisted packaging plasmid.The lentivirus was packaged and the titer of lentivirus was determined.Used the above-mentioned lentivirus to infect small intestinal organoids,and optimized the infection conditions to test the best infection effect.ELISA method was used to detect the activity of GLP-1/Fc,and the glucose tolerance test was performed on wild-type and diabetic mice respectively.The crypts of GFP mice were cultured with green fluorescence in vitro and transplanted into the intestinal of wild type mice in situ to observe the integration of intestinal organoids.ResultsSequencing verified that the recombinant plasmid was constructed successfully.Then it was found that when the multiplicity of infection=1000,the expression of green fluorescence could be observed in most of the intestinal organoids and the infection rate of the lentivirus to intestinal organoids was the best.It was found that after two passages,the intestinal organoids could still express strong green fluorescence.The ELISA results showed that compared with the wild-type mice,the activity of GLP-1/Fc protein in the experimental group was significantly enhanced.Besides,it was found that supernatants secreted by the GLP-1 overexpression organoids effectively enhanced glucose tolerance in wild-type and diabetic mouse.Orthotopic transplantation experiments found that one week after transplantation,the expression of green fluorescent protein could be detected and the intestinal organoids cultured in vitro in GFP mice could be successfully integrated into the recipient mice.ConclusionIn summary,this study successfully constructed mouse intestinal organoids that overexpress GLP-1 by optimizing the GLP-1 lentivirus infection method and obtained intestinal organoids capable of stably expressing GLP-1/Fc,laying a certain experimenal foundation for the treatment of type Ⅱ diabetes in the future.Figure 10 table 3 reference 51...