| Type 1 diabetes mellitus is caused by severe insulin deficiency secondary to autoimmune destruction of pancreatic cells. We can transfer the modified human proinsulin gene into diabetes mellitus patients, and it will produce long - term N high - efficient and physiologically controlled insulin. Then we will have a permanent therapy method of type 1 diabetes mellitus.We use retrovirus as the vector of transfection in this experiment. We recombine the retroviral vector with the modified human proinsulin gene by using the experimental technique of molecular biology. The retroviral vector is reconstructed from the Moloney murine leukemia virus ,the gag,env,pol parts of the necessary gene that can encode wild virus granula was removed during the reconstruction, so it can not encode the structure protein of virus. The packaging cells are the cell lines that have been transducted by the gene expressing the structure protein of virus artificially. The vector can form virus granula through the contrary compensation of the packaging cells. The packaging cellline we use in this experiment is PAS 17 , which is the second generation of packaging cell. The retrovirus produced by it can infect mouse, rat,cat,dog and human. PA317 cell line has a large range of hosts and can produce high - titer virus. Moreover, the feature that it will not produce auxiliary virus make it suitable for the experimental research in the genetic therapy of human beings.Materials and Methods1. Plasmid and Cell LinePLXSN was purchased from Clontech, PLXSN - MpINS plasmid was constructed by a researcher of the former part of the experiment, PA317 packaging cell line was purchased from Shanghai Biology Engineer Research Center of Chinese Academic Institute and NIH3T3 cell was presented by our department.2. Purified the plasmid carrying modified human proinsulin gene by means of alkali cleavage.3. The PLXSN - MpINS plasmid was transfected into PAS 17 cells by lipofectamine.4. Determined the virus tilers and selected the cell clones producing high - liter virus.5. Integration of goal gene with genome DNA from PAS 17 cells was assayed by genomic DNA PCR.Results1. Obtain the PAS 17 resistant clones and infect NIH3T3 cells. (1)The PLXSN - MpINS plasmid was transfected into PAS 17 cellsby lipofectamine.(1) After the selection of medium with G418,many cell clones were detected. While in the control group, we could not find any clone and most cells were dead,which indicated that the genetic transfection had been successful.(2)The supernatants from PLXSN - MpINS - PAS 17 were obtained and used to infect NIH3T3 cells.2. Determine the virus liters.Choose seven clones and determine the virus liter by the formula: Virus liter = ( Average clone numbers of each well x Passage times) / Volume of retroviral supernalant.Table 1 The virus liter of PAS 17 ( cfu/ml)3. Identify ihe integration of goal gene.Integration of MpINScDNA gene with genome DNA from PA317 was assayed by genomic DNA PCR. The results showed that MpINScD-NA gene had been integrated wilh ihe genome DNA of resislanl Irans-fected PAS 17 cells.DisscussionTo establish an effective gene transfer system by transfecting MpINS gene into PAS 17 cells is the premise to study the application of the system in genetic therapy of type 1 diabetes. The principal of lipo-some - mediated transfection is: the interaction between liposome and DNA of samples produces liposome - DNA complex. Liposome can bring the exogenous DNA into target cells because it will fuse with the cellular membrane easily. Liposome - mediated transfection is convenient for operation, and its reliable efficiency make it fit for both instantaneous transfection and steady transfection. However, the method has a main defect that the transferred gene can not express for a long period of time steadily. In order to overcome the shortcoming, we use the retrovirus vector to carry the modified human proinsulin gene,and transduct it into the target cells through liposome - mediated tran...
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