Functional analysis of gene LRRC8 causing agammaglobulinemia

我们以前分离到一个新的基因，富含亮氨酸的重复序列包含8（LRRC 8），导致无丙种球蛋白血症在人类。在患者中发现的突变的LRRC 8转导到小鼠造血细胞中导致B细胞祖细胞在pro-B阶段的成熟阻滞。为了进一步阐明先天性免疫缺陷的机制，我们尝试制造LRRC 8缺陷小鼠。制备缺失完整LRRC 8的靶向载体，并转染ES细胞。电穿孔进行两次：第一次64个样品，第二次160个样品。挑取约200个克隆，并筛选阳性克隆。通过Southern杂交筛选阳性克隆以确定正确的重组，并分离出一个正确的克隆。将ES克隆（细胞系）注射到胚胎中，获得生殖系嵌合小鼠，但未成功地产生嵌合小鼠。

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.