Regulatory mechanism of T cell activation in NOD mice

NOD小鼠已被用作研究多基因疾病的IDDM模型，因为至少有16个不同的基因座，命名为Idd。但是，除了与MHC Ⅱ类分子连锁的Idd 1基因外，其实质基因尚不清楚。这一提示性结论可以通过产生NOD表型的同源小鼠而得到证实，但IL-2基因除外，其应被MSM IL-2取代。为了产生这样的小鼠，我们计划了三个步骤：1）通过将NOD与B6衍生的IL-2 KO小鼠回交来产生IL-2 KO NOD小鼠。2)利用逆转录病毒感染系统将MSM IL-2基因导入IL-2 KO NOD小鼠的T细胞谱系。3)IL-2 KO NOD小鼠与同源小鼠NOD/Shi.Idd3msm/msm的回交。在本学年中，我们建立了一个逆转录病毒感染系统，用于将基因以高频率导入发育中的胸腺细胞。病毒感染或基因整合到T细胞中的比例被认为是非常低和困难的，但我们通过以下发展克服了这个问题。1)新构建的逆转录病毒载体与大鼠cDNA易于检测和富集包装和感染细胞。2)胸腺细胞与包装细胞共培养代替含病毒培养基。3)未成熟胸腺细胞与包装细胞的重组培养。除了我们的项目，我们开发的这个系统对于将某些基因引入发育中的T细胞是有用的。我们还开始建立IL-2 KO NOD小鼠，现在获得了F2（IL-2 KO B6 x NOD）x NOD。

The NOD mice have been used as a IDDM model for studying multigenic diseases because at least 16 different loci, named Idd, have been reported. However, their substantial genes are remained unclear except Idd1 linked to MHC class II.Our previous studies suggested that IL-2 gene is a responsible gene in Idd3. This suggestive conclusion is able to be evident by generating congenic mice with NOD phenotypes except IL-2 gene, which should be replaced by MSM IL-2. To produce such a mouse, we planed three steps : 1) Production of IL-2 KO NOD mice by back-crossing of NOD to B6 derived IL-2 KO mice. 2) Introduction of MSM IL-2 gene into T cell lineage of IL-2 KO NOD mice using retrovirus infection system. 3) Back-crossing of IL-2 KO NOD mice with congenic mice, NOD/Shi.Idd3msm/msm. In this academic year, we established a retroviral infectin system for gene introduction into developing thymocytes in a high frequency The ratio of the viral infection or gene integration into T cells has been thought to be very low and difficult but we overcome this problem by following developments. 1) New construction of retroviral vector with rat cDNA for easy detection and enrichment of packaging and infected cells. 2) Coculture of thymocytes with packaging cells instead of virus containing media. 3) Reaggrigation culture of immature thymocytes with packaging cells. In addition to our project, this system we developed is useful for introducing certain genes into developing T cells. We also started to establish IL-2 KO NOD mice and now obtained F2 (IL-2 KO B6 x NOD) x NOD.