SELECTIVE GENE ABLATION IN MATURE B LYMPHOCYTES

拟议项目的目标是开发一种方法， 只在成熟的B细胞中表达。 这将使研究 在体内成熟B细胞中基因的功能，即使在 动物生存能力和/或成熟 B细胞。此外，由于其他细胞类型不会受到影响， 观察到的表型应该更简单。 因此，我们认为， 第一个具体目标是仅在成熟的大肠杆菌中表达Cre重组酶， B细胞。 这样做的主要策略是插入Cre和 内部核糖体进入位点的3'非翻译区的 δ重链基因座。 由于该位点仅在VDJ后表达， IgH基因座的重组，表达应严格限制 到B细胞。 此外，δ重链在B期间不表达 细胞发育，但在每个成熟的表达水平很高， 静息B细胞，抗原刺激前。 因此，Cre将是 通过同源重组插入胚胎的δ基因座， 干细胞和小鼠将由这些细胞制成。 这些老鼠将 与β-半乳糖苷酶报告小鼠杂交，直接评估 Cre重组酶组织和细胞型表达模式 功能测定。 在第二个目标中， 表达的信号传导组分Shc将通过引入loxP而改变 位于必需外显子两侧的位点。 这应该不会影响Shc 除了在表达Cre的细胞中表达之外，这将删除DNA 在两个loxP位点之间，使Shc基因失活。 因此，Shc应该 在成熟B细胞中丢失，但在所有其他细胞中应正常表达。 细胞类型。这种缺陷对成熟B细胞功能的影响将 在融资期结束后进行评估。

The goal of the proposed project is to develop a method of inactivating genes only in mature B cells. This will make it possible to study the function of genes in mature B cells in vivo, even if the gene in question is required for animal viability and/or development of mature B cells. Also, as other cell types will not be affected, interpretations of the phenotypes observed should be more straightforward. Therefore, the first Specific Aim is to express the Cre recombinase only in mature B cells. The primary strategy for doing this is to insert Cre and an internal ribosome entry site into the 3' untranslated region of the delta heavy chain locus. As this locus is only expressed following VDJ recombination of the IgH locus, expression should be tightly restricted to B cells. Moreover, the delta heavy chain is not expressed during B cell development, but is expressed at high levels in every mature resting B cell, prior to antigenic Stimulation. Thus, Cre will be inserted by homologous recombination into the delta locus of embryonic stem cells and mice will be made from these cells. These mice will be crossed to beta-galactosidase reporter mice to directly assess the tissue and cell-type expression pattern of Cre recombinase by a functional assay. In the second Aim, the gene for the ubiquitously expressed signaling component Shc will be altered by introducing loxP sites on either side of essential exons. This should not affect Shc expression except in cells expressing Cre, which will delete the DNA between the two loxP sites, inactivating the Shc gene. Thus, Shc should be lost in mature B cells, but should be normally expressed in all other cell types. The effects of this defect on mature B cell function will be assessed after the funding period.