MUTATIONS OF THE INTRONIC IGH ENHANCER AND ITS FLANKING SEQUENCES DIFFERENTIALLY AFFECT ACCESSIBILITY OF THE J(H)-LOCUS
MUTATIONS OF THE INTRONIC IGH ENHANCER AND ITS FLANKING SEQUENCES DIFFERENTIALLY AFFECT ACCESSIBILITY OF THE J(H)-LOCUS
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DOI：
10.1002/j.1460-2075.1993.tb06152.x
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发表时间：
1993-12-01
期刊：
影响因子：
11.4
通讯作者：
ALT, FW
中科院分区：
文献类型：
--
作者：
CHEN, JZ;YOUNG, F;ALT, FW
为了研究内含子免疫球蛋白重链（IgH）增强子（Emu）在产生J（H）位点对VDJ重组的可及性中的作用，我们产生了ES细胞，其中Eu或其侧翼序列通过用表达的neo（r）基因替换或插入而突变。杂合突变ES细胞用于产生嵌合小鼠，通过用Abelson鼠白血病病毒（A-MuLV）转化骨髓细胞从所述嵌合小鼠衍生前B细胞分支。在个别前B细胞系中正常和突变等位基因的重排状态的比较允许我们测定突变的顺式作用效应。替换核心Emu下游的700 bp区域[包括部分3'基质相关区（MAR）和Imu外显子]对靶向等位基因的重排没有明显影响，表明将转录的neo（r）基因插入J（H）-Cmu内含子不影响J（H）可及性。相反，替换包含鸸鹋的重叠的1 kb DNA片段导致相关J（H）基因座的重排、去甲基化和生殖系转录的显著顺式作用抑制。令人惊讶的是，将neo（r）基因插入核心鸸鹋上游约100 bp的5' MAR序列中也显著降低了连锁J（H）基因座的重组;但是，在许多品系中，并不能阻止该基因座的去甲基化。我们得出结论，鸸鹋和上游侧翼序列的完整性是需要有效重排的J（H）基因座和该基因座的去甲基化，本身，并不一定使其成为一个良好的基板VDJ重组。
To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (Emu) in generating accessibility of the J(H) locus for VDJ recombination, we generated ES cells in which Eu or its flanking sequences were mutated by replacement with or insertion of an expressed neo(r) gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell tines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core Emu [which includes part of the 3' matrix associated region (MAR) and the Imu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neo(r) gene into the J(H)-Cmu intron does not affect J(H) accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the Emu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated J(H) locus. Surprisingly, insertion of the neo(r) gene into the 5' MAR sequence approximately 100 bp upstream of the core Emu also dramatically decreased recombination of the linked J(H) locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the Emu and upstream flanking sequences is required for efficient rearrangement of the J(H) locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.