Chromosome movements that regulate tissue-specific gene expression

在2016财年，我们完成了以下工作： 1.开发试剂和细胞系，进行RAG1/2在前B细胞抗原受体位点的活体细胞成像。这些包括用HaloTag或Suntag标记RAG1和RAG2，以及突变的RAG2蛋白。这些蛋白在Abelson病毒转化的Pro-B细胞系6312中得到表达。 2.检测Suntag需要与Suntag结合的单链FV蛋白的共表达，以放大荧光信号，从而增加在体内检测到单分子的可能性。我们建立了稳定的细胞系，表达这种单链FV蛋白。 3.为了测试酶失活的Cas9-Suntag(dCas9-Suntag)在基因组特定位置的招募，我们产生了表达针对端粒重复序列的引导RNA(GRNAs)的Pro-B细胞。 4.我们在Janelia Farm的活体Pro-B细胞中进行了RAG1-Halo和RAG2-Halo的初步可视化研究。观察到每个荧光衍生物的离散斑点。下一步是确定由这些荧光蛋白产生的RAG复合体的化学计量比。 5.我们开发了针对IgH基因可变区(VH)的gRNA的表达载体。如果在上述实验中很容易看到端粒重复序列，我们将把dCas9-Suntag招募到VH结构域来研究IgH基因座的动力学。

During FY16 we accomplished the following: 1. Developed reagents and cell lines to carry out live cell imaging of RAG1/2 recruitment to antigen receptor loci in pro-B cells. These included tagging RAG1 and RAG2, and mutant RAG2 proteins, with a HaloTag or a SunTag. These proteins were expressed in Abelson virus-transformed pro-B cell line 6312. 2. Detection of SunTag requires co-expression of a single-chain Fv protein that binds to the SunTag to amplify the fluorescence signal that increases the probability of detecting single molecules in vivo. We generated stable cell lines that express this single chain Fv protein. 3. To test recruitment of enzymatically inactive Cas9-SunTag (dCas9-SunTag) to specific sites in the genome, we generated pro-B cells that express guide RNAs (gRNAs) targeted to telomere repeats. 4. We carried out preliminary visualization studies of RAG1-Halo and RAG2-Halo in live pro-B cells at Janelia Farms. Discrete puncta of each fluorescent derivative were observed. The next step is to determine the stoichiometry of RAG complexes generated by these fluorescent proteins. 5. We developed vectors to express gRNAs that target the variable (VH) domain of the IgH locus. If telomeric repeats are readily visualized in the experiments described above, we will recruit dCas9-SunTag to the VH domain to study dynamics of the IgH locus.