Construction Of PTet-on And PTRE2-H-Ins Double-plasmid Stably Integrated Embryonic Stem Cell Line Of Mouse

ObjectiveDiabetes is a common endocrine disease caused by relative or absolute defect of insulin. Nowadays, diabetes has already been the third disease that damaged human health after cardiovascular diseases and cancer. The pathologic mechanism is still unclear, but it is known that this related with βcell defect, lack of insulin production and genetic factors. Present therapy mainly supplies insulin with various methods, but excessive insulin can cause severe hypoglyce-mia. Though the therapy of pancreas or islets transplantation to substitute the islets without function has achieved some effect, because of the influence of the defect of donor sources, immunologic rejection and long - term immunosuppres-sion therapy, the normal serum glucose regulation cant be rebuild and this method causes severe complications and be restricted in clinical application. Recently , with the rapid development of gene recombination technique and trans-genic technique, reforger of insulin secretory clonal cell line or specific suppression of the autoimmne process aimed at islets should substitute present insulin injection therapy and insulin transplantation therapy. The gene therapy for diabetes has achieved great progress during recent 20 years and gained a great deal of precious information, but still is in the stage of animal experiments, and there are two cardinal problem to be solved: â‘  the regulation of insulin genes â‘¡ the gene transfection method is the key of gene therapy, present transfection method need improve in security and efficiency.Adopting transgenic method to establish animal models of human diseases, compared with other methods, has many advantages. For instance, the geneticbackground is clear, the genetic substance can be changed simply, the models are more natural and closer to the patients'symptoms; the establishing progress is easy to carry on and with short period, the transgenic models need no special feeding condition to keep the disease symptoms, which pass from generation to generation according to Mendelian rules, thus establishing human insulin transgenic mouse model is the preferred animal model for genetic therapy of diabetes. Several laboratories have established the transgenic mice model with human insulin expression since the 1980s, but because of the persistent high - expression of human insulin gene in the animals, the high level of insulin secretion caused hypoglycemia in mice, even death of the animals, and the expression products in gene therapy cant be regulated according to the serum glucose concentration, which causes the stop of gene therapy for diabetes.Tetracycline (tet) inducing expression system, which was established by Gossen et al. in 1992, was an advanced regulating system, and was rebuilt in 1995, then formed Tet - off/Tet - on gene inducting expression regulation system. It can effectively regulate the expression of exogenous genes of eukaryocyte and transgenic animals; the inducing expression is of high efficiency, no cyto-toxicity, perfect on/off function and no leaking expression. Thereinto, tet - on system has been successfully applied into mammalian animal cell culture, transgenic mice, and gene therapy.Embryonic stem cells (ES) are the embryonic cells with various differentiating potential, theoretically can be induced to differentiate into all kinds of cells of the organism. In the condition of in vitro differentiating inhibition culture, they have the ability of keeping undifferentiated and infinite proliferation. After injecting the mouse ES cells into normal blastula, ES cells participate into the development and establishment of embryo, forming Chimera. In Chimera, ES cells take a part in the formation of various tissues, including germ cells. So, ES cells can be used to make genelocation and transgenosis through Chimera path. The best advantage of ES cells - mediated transgenosis is that it can screen at the level of cell; can randomly integrate and homogenous recombination integrate; can introduce, eliminate or replace certain gene, carry on one or several nucleotides decoration. It is impossible to control the expression and reg-ulation of transported exogenous genes by the use of genetic decoration of ES cells in vitro.Thus, we introduced pTet - on and pTRE2 - H - Ins plasmids into ES cells orderly by transfection to establish pTet - on and pTRE2 - H - Ins double -plasmid stably integrated ES cell line, further used RT - PCR method and insulin measure method to quantificationally investigate the expression of human insulin gene secretion at the level of RNA and protein, and grounded for the establishment of inducing expression of human insulin transgenic mice, then discussed the relationship between the expression level of insulin genes and the serum glucose concentration of diabetes mice model, provided theory base for the gene therapy of type 1 diabetes mellitus.Materials and methods1 Reagenthigh - sugar DMEM, leukemia inhibition factor (LIF) ( ESGRO 10⁶units/ ml) , Gibco/BRL company, USA; fetal calf serum , Hyclone company, USA; β - mercaptoethanol and trypsin , Sigma company, USA; HEPES , Amersco company, USA; Lipofectin and G418, Invitrogen company, USA; hygromycin B, DIG marker and measure kits and mitemycin C , Roche company, USA; plasmid purifying kits , Promege company, USA; luciferase reporting gene measure kits, Biyuntian company; gel recovery kits, RT - PCR kits, PCR relative reagents, various enzymes (BamH I, EcoR I, HindIII, Sca I etc. ) were all from TaKaRa company.2. MaterialsCompetence DH5αE. coli were stored by our laboratory, pTet - on, pTRE2 - H - Ins, pTRE - luc, pWL/neo and pHyg plasmids were donated by professor Jin Zhuang of Institute of Medicine Harward University, NIH3T3 cells were donated by Department of Cell Biology China Medical University, ES - D₃ cells were donated by professor Cong Xiaoqian of Institute of Biochemistry and Cell Biology, Shanghai, Chinese Academy of Sciences.3. Methods(1) By the method of lipofectin, introduced the pWL/neo plasmid and pHyg plasmid orderly into NIH3T3 cells, to build G418 and hygromycin B dual fastness NIH3T3 strain, used PCR and Southern blot method to examine the cell line. Took these cells as feeding cells for the screening of ES - D₃ cell transfect-ed with target genes.(2) By means of culturing ES - D₃ cells in feeding - layer, and took G418 and hygromycin B dual fastness NIH3T3 cells as feeding - layer, introduced pTet - on plasmid into ES - D₃ cells by electroporation, gained the clones screened by G418, and examined whether Tet - on gene were stably integrated into ES - D₃ cell genome by PCR and Southern blot.(3) Introduced pTRE - luc genes into the Tet - on gene positive ES - D₃ cell clones by electroporation, and used luciferase reporting gene measure kits to examine and screen out the Tet - on gene positive ES - D₃ cell clones with high expression and low background.(4) By electrotransfection, co - transfected Tet - on gene positive ES - D₃ cells with pTRE2 - H - Ins plasmid and pHyg plasmid in the proportion of 20: 1, used hygromycin B to screen, used the PCR and Southern blot methods to examine whether human insulin genes were well - integrated in the screened out clones.(5) In the condition of inducible drug dox and without inducible drug dox, examined the the stable integration of pTet - on and pTRE2 - H - Ins double -plasmid into ES - D₃ cells at the level of RNA and protein by RT - PCR and ra-dioimmunity human insulin measuring method, observed the change of human insulin gene expression in ES - D₃ cell.Results1 By PCR and Southern blot, the results indicated that lipofectin could introduce neo gene and hygromycin gene into NIH3T3 cell genome successfully, built G418 and higromycin B dual fastness mouse embryonic stem cell NIH3T3 feeding - layer cells.2 By PCR and Southern blot, the results indicated that electroporationmethod introduced Tet - on gene into ES - D₃ cell genome, and integrated stab-ly.3 Used luciferase reporting gene measure kits to examine the function of acquired ES - D₃ clones, the results proved that we gained one strain of Tet - on gene positive ES - D₃ cell line (No. 1) with high expression and low background, the inducing expression multiple was 21. 31 times.4 Introduced exogenous human insulin target genes into Tet - on gene positive ES - D₃ cell line ( No. 1) by electrotransfection, examined by PCR and Southern blot respectively, proved that human insulin gene has already stably integrated into Tet - on gene positive ES - D₃ cell line (No. 1) genome DNA, gained pTet - on and pTRE2 - H - Ins double - plasmid stably integrated mouse embryonic stem cell line.5 In the condition of inducing expression drug dox and without dox, cultured the pTet - on and pTRE2 - H - Ins double - plasmid stably integrated mouse embryonic stem cell line, estracted total RNA, examined with PCR and proved, 72h after adding dox, the high level transcription of human insulin genes was induced.6 In the condition of inducing expression drug dox and without dox, cultured the pTet - on and pTRE2 - H - Ins double — plasmid stably integrated mouse embryonic stem cell line, abstracted the supernatant fluid at 24h, 48h, 72h, put into dox, at 72h, the average human insulin secretion expression a-mount reached the highest in the supernatant fluid, was 1. 1uU/ml, 4. 78 times as in normal ES - D₃ cells.Conclusions1 Took ES - D₃ cells cultured in MEF cells and NIH3T3 cells as control observation , proved that NIH3T3 cells can be used as feeding - layer to culture ES cells.2 Building G418 and hygromycin B dual fastness NIH3T3 cell line successfully grounded for the screening of ES - D₃ cell target gene transfection.3 Successfully gained one strain of Tet - on gene positive ES - D₃ cell in...