Targeted DNA cleavage at switch regions in immunoglobulin class switch recombinat

免疫球蛋白(Ig)类开关重组(CSR)是由 哪些B细胞用Ig重链的恒定域来交换最佳的 清除病原体。这种独特的DNA重组是由千碱基- 长开关区域和需要B细胞特异性因子激活诱导的胞苷 脱氨酶(AID)以及其他普遍表达的DNA修复因子。它 已知CSR是由AID催化的胞苷脱氨反应启动的，导致 在开关区域内有尿囊虫。然而，切换区域的机制 指导AID在-cis中的作用以及尿嘧啶修复途径的相互作用 最终导致DNA双链断裂仍然没有明确的定义。这个 这项应用的目标是鉴定顺式作用的DNA序列 负责靶向的转换区和反式作用蛋白因子 在类转换重组过程中特定基因组位置的DNA裂解。我们 我开发了一种基于细胞的类转换实验，用于研究 内源染色体位置的切换区序列。这个化验 是基于我们最近在CH12F3高效基因打靶方面的成功 细胞，一种能够在体外强大的细胞因子诱导CSR的小鼠B细胞系。我们 我设计了一种高效的敲门策略，以允许评估大型 开关区域突变的数量。CH12F3的高效基因打靶 细胞还可以通过反向遗传方法研究基因功能 企业社会责任的蜂窝模型。我们现在可以处理几个重要的问题 以前很难在动物模型中解决的未回答问题。我们 提出三个具体目标：(1)确定课堂所需的短序列基序 开关重组；(2)确定班级所需的长序列组织 开关重组；(3)鉴定开关区的DNA切割活性。 这一项目的完成将使人们对 开关区序列的功能与核酸酶的鉴定 参与了企业社会责任中的DNA切割。这些发现将提供机械论 洞察涉及班级切换重组的各种人类疾病。

Immunoglobulin (Ig) class switch recombination (CSR) is a process by which B cells exchange the constant domain of the Ig heavy chain for the optimal clearance of pathogens. This unique DNA recombination is directed by kilobase- long switch regions and requires B cell-specific factor activation-induced cytidine deaminase (AID) as well as other ubiquitously expressed DNA repair factors. It is known that CSR is initiated by AID-catalyzed cytidine deamination resulting in uracils in the switch regions. However, the mechanism by which switch region directs AID actions in-cis and the interplays of uracil repair pathways that ultimately lead to DNA double strand breaks remain poorly defined. The objectives of this application are to identify cis-acting DNA sequences in the switch region and trans-acting protein factors that are responsible for targeted DNA cleavage at defined genomic loci during class switch recombination. We have developed a cell-based class switch assay for studying the function of switch region sequences at the endogenous chromosomal locus. This assay was based on our recent success in highly efficient gene targeting in CH12F3 cells, a mouse B cell line capable of robust cytokine-induced CSR in vitro. We have designed an efficient knock-in strategy to allow assessment of a large number of switch region mutations. Highly efficient gene targeting in CH12F3 cells also allows study of gene function by the reverse genetic approaches in a cellular model for CSR. We are now in position to address several important yet unanswered questions previously difficult to address in animal models. We propose three specific aims: (1) Identify short sequence motifs required for class switch recombination; (2) Identify long sequence organizations required for class switch recombination; (3) Identify the DNA cleavage activity at switch regions. The completion of this project will lead to a more complete understanding of the function of switch region sequences and the identification of the nucleases involved in DNA cleavage in CSR. These findings will provide mechanistic insight to a variety of human diseases involving class switch recombination.