Laboratory Animal Science of Gene Therapy Using Mutant Mice for T-cell Apoptosis

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Vectors from retroviruses are known to transfer gene effectively into cells. However, the therapeutic use of viruses entails a problem as gene delivery vehicles, because retroviruses might disrupt DNA of the host cells when they infect, and would cause potentially harmful results. On the other hand , nonviral methods of gene transfer would be suitable for repeated use, since they do not result in the immune responses. Liposome is one of nonviral vector.It is considered that a gene therapy using Fas antigen in vivo is an important way to eleminate or regress tumors through apoptosis. A mouse lymphoproliferation (lpr) , which is a nonfunctional mutation of Fas antigen, has been assigned to chromosome 19. Transcript of the Fas antigen in MRL-lpr mice is nearly expressed in the thymus and liver compared to that in normal mice because an insertion by an early transposable elment is occured. The clinical syndrome of MRL-lpr mice is characterized as lymphoadenopathy, hypergammaglobulinaemia a … More nd arthritis, which resembles human systemic lupus erythematosus. In this study the expression vectors carrying FAS antigen cDNA are constructed in the correct orientation named pEF-Fas and pCMV-Fas. First, it became apparent in vitro experiment and Western blot analysis that Fas antigen protein is expressed in COS-l cells transfected by lipofectin and pEF-Fas or pCMV-Fas complexes. Then MRL-lpr mice were used for investigating the effect of Fas antigen expression by means of transfection of the expression vectors enclosed in cationic liposomes. After injected through tail vein, the vectors carrying Fas antigen cDNA-liposome complex were successfully transferred in mainly lung, liver, spleen, kidney and pancreas as detected by Southern blot analysis. Furthermore, the RNA transcripts were found in lung and liver by Norhtern blot analysis, but not observed in any other organ. Fas antigen protein, however, was not observed even in lung and liver by Western blot analysis. Finally 66% partial hepatectomy was attempted to find Fas antigen protein effectively, but could not detected. Future study is nessesary to investigate the reason why transferred DNA-liposome complex was not expressed as protein, and to search the way to express Fas antigen in vivo. Less