Hale M A, Garrard W T
Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, Dallas 75235-9140, USA.
Mol Immunol. 1998 Jul;35(10):609-20. doi: 10.1016/s0161-5890(98)00060-1.
Previous studies employing ectopic integration of reporter genes have shown that the nuclear matrix association region (MAR) adjacent to the intronic enhancer of the mouse kappa immunoglobulin (Ig) gene is required for high level transcription of rearranged genes, demethylation, reduction of position effects and maximal somatic hypermutation in B cells. To test for the function of this MAR in its natural chromosomal environment, we pursued the 'HIT-and-RUN' procedure with the mouse pre-B cell line 103 to create a targeted MAR deletion. We observed a 'HIT' targeting frequency of 1/684 but 0/2100 'RUN' clones maintained the MAR-deleted germline locus because of an unexpected hyper-recombination for Vkappa-Jkappa joining, specifically to the MAR-deleted allele, and primarily at Jkappa4 and Jkappa5. This hyper-recombination was correlated with undermethylation of the Jkappa-Ckappa region but not with the level of local transcription. These results are consistent with the possibility that the MAR and/or DNA methylation negatively regulate(s) Vkappa-Jkappa joining during the pre-B cell stage of development.
先前利用报告基因异位整合的研究表明，小鼠κ免疫球蛋白（Ig）基因内含子增强子附近的核基质结合区（MAR）对于重排基因的高水平转录、去甲基化、位置效应的降低以及B细胞中最大程度的体细胞超突变是必需的。为了在其自然染色体环境中测试该MAR的功能，我们对小鼠前B细胞系103采用“敲入并敲除”程序来创建靶向MAR缺失。我们观察到“敲入”靶向频率为1/684，但2100个“敲除”克隆中有0个维持了MAR缺失的种系位点，因为Vκ-Jκ连接出现了意外的高频率重组，特别是与MAR缺失的等位基因发生重组，且主要发生在Jκ4和Jκ5处。这种高频率重组与Jκ-Cκ区域的低甲基化相关，但与局部转录水平无关。这些结果与MAR和/或DNA甲基化在发育的前B细胞阶段对Vκ-Jκ连接进行负调控的可能性一致。
