Gene activation by Rag-mediated DNA double strand breaks. Mus musculus

The objective is to identify genes that are differentially expressed following the introduction of DNA double strand breaks (DSBs) by the Rag proteins in murine pre-B cells. Cells lacking Artemis are used since the Rag-induced DSBs will not be repaired and, thus, will provide a continuous stimulus to the cell. Cells lacking Artemis and Atm are used to determine which gene expression changes depend on Atm and cells lacking Artemis that express an I kappa B alpha dominant negative are used to determine which gene expression changes depend on NFkB. Overall design: Murine v-abl-transformed pre-B cells were treated with 3 uM STI571 for 48 hours. Cell types included Wild type (3 biological replicates), RAG-2-deficient (3 biological replicates), Artemis-deficient (3 biological replicates), Artemis and ATM-deficient (2 biological replicates, each with a technical replicate), and Artemis-deficient expressing dominant negative IkB (3 biological replicates). Each sample was hybridized once using Affymetrix Mouse Genome 2.0 GeneChip arrays (Mouse 430 v2, Affymetrix, Santa Clara, CA). Data were analyzed using the RAG-2-deficient samples as the controls. Additionally, purified bone marrow pre-B cells were harvested from RAG-1-deficient (2 biological replicates) and Artemis-deficient (2 biological replicates) mice with an IgH transgene. Each sample was hybridized once using Affymetrix Mouse Genome 2.0 GeneChip arrays (Mouse 430 v2, Affymetrix, Santa Clara, CA). Data were analyzed using the RAG-1-deficient:IgH samples as the controls.

本研究旨在鉴定小鼠前B细胞中经Rag蛋白诱导DNA双链断裂（DNA double strand breaks, DSBs）后差异表达的基因。本研究选用Artemis缺陷细胞，因Rag诱导的DNA双链断裂无法在此类细胞中得到修复，从而可对细胞产生持续的刺激信号。同时选用Artemis与ATM联合缺陷细胞，以明确哪些基因的表达变化依赖于ATM；并选用表达IκBα显性负突变体的Artemis缺陷细胞，以确定哪些基因的表达变化依赖于NF-κB。

实验整体设计：将经v-abl转化的小鼠前B细胞用3 μM STI571处理48小时。所用细胞类型包括：野生型（3次生物学重复）、RAG-2缺陷型（3次生物学重复）、Artemis缺陷型（3次生物学重复）、Artemis与ATM联合缺陷型（2次生物学重复，各含1次技术重复），以及表达显性负突变IkB的Artemis缺陷型（3次生物学重复）。所有样本均单次采用Affymetrix Mouse Genome 2.0 GeneChip arrays（Mouse 430 v2, Affymetrix, 美国加利福尼亚州圣克拉拉市）进行杂交。本研究以RAG-2缺陷型样本作为对照进行数据分析。

此外，从携带IgH转基因的RAG-1缺陷型（2次生物学重复）和Artemis缺陷型（2次生物学重复）小鼠中分离纯化骨髓前B细胞。所有样本均单次采用Affymetrix Mouse Genome 2.0 GeneChip arrays（Mouse 430 v2, Affymetrix, 美国加利福尼亚州圣克拉拉市）进行杂交。本研究以RAG-1缺陷型:IgH样本作为对照进行数据分析。