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小鼠前B细胞中逆转录病毒载体内免疫球蛋白重链开关区重组

Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells.

作者信息

Ott D E, Alt F W, Marcu K B

出版信息

EMBO J. 1987 Mar;6(3):577-84. doi: 10.1002/j.1460-2075.1987.tb04793.x.

DOI:10.1002/j.1460-2075.1987.tb04793.x
PMID:3034595
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC553436/
Abstract

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a substrate for detecting the presence of immunoglobulin heavy chain constant region (CH) gene switch (S) recombination activity in murine pre-B cells. ZN(Smu/S gamma 2b)tk1 contains a neomycin (neo) resistance gene in addition to the herpes simplex virus thymidine kinase (Htk) gene which is positioned between murine Smu and S gamma 2b sequences. Stable acquisition of the ZN(Smu/S gamma 2b)tk1 vector was selected in G-418 and switch region recombination within these proviruses was selected by resistance to the drug bromodeoxyuridine (BUdR). Fluctuation analyses of ZN(Smu/S gamma 2b)tk1 infected 18-8tk- and 38B9tk- pre-B lines revealed Htk gene inactivations with apparent frequencies of 5 X 10(-5) and 1 X 10(-5) events/cell/generation, respectively, while G-418 resistant Ltk- fibroblasts lost the HTK phenotype at an apparent rate of 4 X 10(-8). Southern blot analysis demonstrated that switch recombination caused the deletion of the Htk gene in all pre-B clones examined while the loss of Htk in Ltk- clones was not mediated by S region recombination. In 21 out of 24 pre-B clones, the recombinations involved the tandemly repetitive portions of the Smu and S gamma 2b sequences. These results demonstrate that the CH gene S region segments inserted into ZN(Smu/S gamma 2b)tk1 are sufficient for B-cell-specific recombination/deletion within the S region tandem repeats.

摘要

我们使用了一种逆转录病毒载体ZN(Smu/Sγ2b)tk1，作为检测小鼠前B细胞中免疫球蛋白重链恒定区(CH)基因开关(S)重组活性的底物。ZN(Smu/Sγ2b)tk1除了含有单纯疱疹病毒胸苷激酶(Htk)基因外，还含有一个新霉素(neo)抗性基因，该基因位于小鼠Smu和Sγ2b序列之间。通过在G-418中筛选稳定获得ZN(Smu/Sγ2b)tk1载体，并通过对药物溴脱氧尿苷(BUdR)的抗性筛选这些原病毒中的开关区域重组。对感染ZN(Smu/Sγ2b)tk1的18-8tk-和38B9tk-前B细胞系进行波动分析，发现Htk基因失活的明显频率分别为5×10(-5)和1×l0(-5)事件/细胞/代，而对G-418有抗性的Ltk-成纤维细胞以4×10(-8)的明显速率丧失HTK表型。Southern印迹分析表明，开关重组导致所有检测的前B细胞克隆中Htk基因缺失，而Ltk-克隆中Htk的缺失不是由S区域重组介导。在24个前B细胞克隆中的21个中，重组涉及Smu和Sγ2b序列的串联重复部分。这些结果表明，插入ZN(Smu/Sγ2b)tk1中的CH基因S区域片段足以在S区域串联重复内进行B细胞特异性重组/缺失。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/741e12b46bad/emboj00243-0040-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/90fd90e60857/emboj00243-0038-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/e83404971db9/emboj00243-0039-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/974e231abd63/emboj00243-0040-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/741e12b46bad/emboj00243-0040-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/90fd90e60857/emboj00243-0038-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/e83404971db9/emboj00243-0039-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/974e231abd63/emboj00243-0040-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb98/553436/741e12b46bad/emboj00243-0040-b.jpg

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