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Studies on the regulatory mechanisms of V (D) J rearrangement using gene-inserted mice.

使用基因插入小鼠研究 V(D)J 重排的调节机制。

基本信息

批准号：
08839005
负责人：
TAKI Shinsuke
金额：
$ 1.54万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

In order to understand the regulatory mechanisms of immunogoloulin heavy chain rearrangement, studies have been performed using a mutant mouse strain which carries a functionally rearranged V_H gene (V_HT15) inserted in the V_H locus on one chromosome (T15i/+ mice). I have shown followings. 1. In T15i/+ mice, B cells bearing the V_HT15 product occupies around 40% in the peripheral B cell compartment. In contrast, V_HT15^+B cells comply of around 80% of the B cell pool in T15i/+ mice carrying additional mutation in the IL-7 receptor loci (T15i/+IL-7R^<-/->), indicating that IL-7 is required for the survival of pro-B cells which undergo V(D)J rearrangement, so that in the absence of IL-7, pro-B cells cannot survive long enough to perform secondary rearrangements to disrupt the inserted V_H gene and generate functional V_H gene on the wild-type chromosome. 2. A kappa-chain transgene was additionally introduced into the T15i/+IL-7R^<-/-> mice. Even in these mice, the percentage of VHT15+B cells are still as high as that in non-transgenic T15i/+IL-7R^<-/-> mice. Moreover, the reduction of the B cell number due to the lack of IL-7 signals could not be restored by introducing both heavy and light cahins. These results indicate that the IL-7 signals function independently of surface Ig signals. 3. Secondary rearrangements on the mutant chromosome which destroy the inserted VH gene in T15i/+ mice might be due to the presence of the neomycin resistant cassette (neo) present upstream of the inserted VH gene which had been used as a selection marker in gene targeting. However, deletion of the ned gene using FLIP-FRT system did not alter the frequency of such secondary rearrangement. Thus, the secondary rearrangements occur as a result of the unique structure of VHT15 gene itself (for example, the sequence or the transcriptional activity of the accompanying promotor).

为了了解免疫goloulin重链重排的调控机制，研究人员使用一种突变小鼠（T15i/+小鼠）进行了研究，该突变小鼠在一条染色体的V_H位点上插入了一个功能重排的V_H基因（V_HT15）。我展示了以下内容。1. 在T15i/+小鼠中，携带V_HT15产物的B细胞约占外周B细胞区室的40%。相比之下，在携带IL-7受体位点额外突变（T15i/+IL-7R^<-/->）的T15i/+小鼠中，V_HT15^+B细胞符合约80%的B细胞库，这表明IL-7是进行V(D)J重排的pro-B细胞存活所必需的，因此在没有IL-7的情况下，pro-B细胞无法存活足够长的时间来进行二次重排，从而破坏插入的V_H基因并在野生型染色体上产生功能性的V_H基因。2. 在T15i/+IL-7R^<-/->小鼠中引入kappa链转基因。即使在这些小鼠中，VHT15+B细胞的百分比仍然与非转基因T15i/+IL-7R^<-/->小鼠一样高。此外，由于缺乏IL-7信号而导致的B细胞数量减少不能通过引入重蛋白和轻蛋白来恢复。这些结果表明IL-7信号独立于表面Ig信号起作用。3. T15i/+小鼠突变染色体上的二次重排破坏了插入的VH基因，这可能是由于插入的VH基因上游存在新霉素耐药盒（neo），该盒被用作基因靶向的选择标记。然而，使用FLIP-FRT系统删除ned基因并没有改变这种二次重排的频率。因此，二次重排的发生是由于VHT15基因本身的独特结构（例如，伴随启动子的序列或转录活性）。