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个不同的基因座被报道，称为IDD。然而，除了Idd1与MHC II类分子连锁外，它们的实质性基因还不清楚。我们以前的研究表明，IL-2基因是Idd3的一个负责基因。通过建立除IL-2基因外的NOD表型的同源小鼠，这一结论是明显的，该基因应该被MSM IL-2取代。为了获得这样的小鼠，我们计划了三个步骤：1)将NOD小鼠与B6来源的IL-2KO小鼠回交，获得IL-2KO 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T细胞系。3)IL-2 KO NOD小鼠与同源小鼠回交，NOD/Shi.Idd3msm/MSm。在本学年，我们建立了一个逆转录病毒感染系统，用于高频率地将基因导入发育中的胸腺细胞。病毒感染率或基因整合到T细胞的比率一直被认为是非常低和困难的，但我们通过以下进展克服了这一问题。1)新型大鼠逆转录病毒载体的构建，便于包装和感染细胞的检测和浓缩。2)胸腺细胞与包装细胞共培养，而不是含病毒的培养液。3)未成熟胸腺细胞与包装细胞的复合培养。除了我们的项目，我们开发的这个系统还有助于将某些基因引入发育中的T细胞。我们也开始建立IL-2 KO NOD小鼠，现已获得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.