| T1DM is an autoimmune disease. Treatments of T1DM mainly include exogenous insulin injection and pancreas ( islet ) transplant to maintain normal blood glucose level. However, there are unavoidable defects in these two methods. With the development of molecular biology ,transgene technique will be the best choice for T1DM after we overcome the technical problem, moral ethics problem and artificial operational problem of the regulation mechanism. Now transgene treatment for T1DM is still on the stage of experiment. Its basic process includes six steps : ( 1 ) Obtain the human proinsulin gene. ( 2 ) Connect the regulate elements and conjunct them with proper vector. ( 3 ) Introduce the recombinant DNA into retroviral packaging cell line. (4) Screen the positive cell clone whose genome was integrated with the recombinant gene. ( 5 ) Hie expression examination and purification in the recombinant gene in the clone. ( 6 ) Animal experiment. Basing on recombinant piasmid has been constructed successfully, we introduced the recombinant piasmid into the genome of PA317 packaging cell line. Then we determined the virus liter of every positive clone to screen one cell clone producing high liter virus. We paved our experimental way for the further research in cellular and animal level. Then we could explore a mature technical route for the same kinds of experiments.MethodsAccording to the feature of the retroviral vector carrying human proinsulin gene that has been constructed, we chose an amphotropic retrovirus packaging cell line-PA317 and adopted liposome to introduce the recombinant DNA. After screening one cell clone producing high liter virus, we amplified and took its supernatant virus. At last, we abstracted the DNAs from PA317 cells and PA317/ pLXSN cell to check the objective gene fragment.Result1. We didnt find obvious band in the plasmid we obtained by AGE. Ultraviolet spectrophotometer showed that its concentration was 50 g/ml.2. We could see dead cells after 4 days when the PAS 17 cell had been introduced the recombinant gene in its genome. Clone formed at the 13th day, meanwhile the cells were almost dead and no clone was found in control group.3. NIH3T3 cell infected by the virus supernatant began to die on the 3rd day and formed clones at the 10th day.4. One 970bp band was amplificated from the cell genome in experimental group while no band was seen in control group. So we could certify that the exogenous gene had been stably integrated in the PA317 cells genome.ConclusionAccording to the retrovirus vector's characteristics, we adopted liposome and the packaging cell lines PAS 17 to tran duct the recombinant gene. Then we chose NIH3T3 cell to screen one clone in G418-medium that can produce high titer virus. This cell clone could keep on producing the recombinant gene as we expect, which would pave the way for future research of cellular and animal level experiments. |
