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

已知逆转录病毒载体能有效地将基因转移到细胞中。然而，病毒作为基因传递载体的治疗用途带来了一个问题，因为逆转录病毒在感染宿主细胞时可能会破坏DNA，并可能造成有害的结果。另一方面，非病毒的基因转移方法适合重复使用，因为它们不会导致免疫反应。脂质体是一种非病毒载体。在体内利用Fas抗原进行基因治疗是通过细胞凋亡消除或逆转肿瘤的重要途径。小鼠淋巴增生（lpr）是一种Fas抗原的无功能突变，已被分配到19号染色体上。与正常小鼠相比，MRL-lpr小鼠的Fas抗原转录本在胸腺和肝脏中几乎表达，这是因为早期转座元件的插入发生了。MRL-lpr小鼠的临床症状表现为淋巴腺病、高γ -球蛋白血症和关节炎，类似于人的系统性红斑狼疮。本研究构建了携带FAS抗原cDNA的正确定向表达载体，命名为pEF-Fas和pCMV-Fas。首先，体外实验和Western blot分析表明，Fas抗原蛋白在脂质体和pEF-Fas或pCMV-Fas复合物转染的cos - 1细胞中表达。用阳离子脂质体包裹的表达载体转染MRL-lpr小鼠，观察其对Fas抗原表达的影响。携带Fas抗原cdna -脂质体复合物的载体经尾静脉注射后，经Southern blot检测成功转移至肺、肝、脾、肾和胰腺。此外，通过northern blot分析，在肺和肝脏中发现了RNA转录本，但在其他任何器官中均未观察到。而在肺和肝脏中均未观察到Fas抗原蛋白。最后66%的肝部分切除术试图有效地寻找Fas抗原蛋白，但未能检测到。未来的研究需要进一步研究转移的dna -脂质体复合物不以蛋白形式表达的原因，并寻找在体内表达Fas抗原的途径。少

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