Functional analysis of gene LRRC8 causing agammaglobulinemia

无丙种球蛋白血症基因LRRC8的功能分析

• 批准号: 17591088
• 负责人: OHTA Hideaki
• 金额: $ 2.18万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591088/
• 关键词: gene; biotechnology; immunology

We previously isolated a novel gene, Leucine-rich repeat-containing 8 (LRRC8) which causes agammaglobulinemia in human. Transduction of mutated LRRC8 found in a patient into mouse hematopoietic cells lead to maturation block of B-cell progenitor at pro-B stage. In order to further elucidate the mechanism of congenital immunodeficiency, we tried making LRRC8-deficient mice. Targeting vector lacking the whole LRRC8 were made, and transfected into ES cells. Electroporation were performed twice: 64 samples at first time, 160 samples at second. Approximately 200 clones were picked up, and screened for positive clones. Positive clones were screened by Southern blot for right recombination, and one right clone was isolated. The ES clone (cell line) was injected into embryos to get germline chimeric mice, but birth of chmeric mice was unsuccessful.

我们以前分离到了一个新的基因，富含亮氨酸重复序列的8(LRRC8)，它会导致人类无丙种球蛋白血症。将患者发现的突变的LRRC8转导入小鼠造血细胞，可导致B细胞前体细胞在PRO-B期成熟受阻。为了进一步阐明先天性免疫缺陷的机制，我们尝试制作LRRC8缺陷小鼠。构建缺失完整LRRC8基因的靶向载体，并将其导入ES细胞。电穿孔进行了两次：第一次是样品，第二次是160个样品。挑选了大约200个克隆，并对阳性克隆进行了筛选。经Southern杂交筛选阳性克隆进行正确重组，分离到1个正确克隆。将ES克隆(细胞系)注射到胚胎中，获得了生殖系嵌合小鼠，但中国仓鼠的出生没有成功。