ChIP-seq of MOZ (Flag-V5-BIO tagged) in mouse mouse craniofacial development

Carboxy-terminally tagged MOZ (Flag-V5-BIO tagged) was detected by ChIP-seq using anti-V5 antibody (Sigma, A7345) to precipitate chromatin associated with MOZ A Flag-V5-BIO triple tag was added in frame to the carboxy-terminus of the Moz locus by BAC by recombineering in E. coli. The construct was used to generate homologously recombined mouse embryonic stem cells, chimeras and mice that carry the Flag-V5-BIO tag in the Moz locus. Mice homozygous for a null mutation of Moz die between embryonic day 14.5 (E14.5) and birth. Mice homozygous fo the Moz-Flag-V5-BIO allele are viable, healthy and fertile showing that the MOZ-Flag-V5-BIO protein functions normally. The first the second pharyngeal arches of E10.5 embryos homozygous for the Moz-Flag-V5-BIO allele as well as wild-type control pharyngeal arches (negative control) were used for anti-V5 chromatin immunoprecipitation to detect the distribution of tagged MOZ in the mouse genome.

本研究采用抗V5抗体（Sigma，货号A7345）沉淀与MOZ结合的染色质，通过染色质免疫共沉淀测序（ChIP-seq）检测经羧基端标记的MOZ（Flag-V5-BIO标记）蛋白。我们通过大肠杆菌（E. coli）内的重组工程（recombineering）技术，借助细菌人工染色体（BAC）在Moz基因座的羧基端原位插入了Flag-V5-BIO三联标记。利用该构建体，成功制备了携带Moz基因座Flag-V5-BIO标记的同源重组小鼠胚胎干细胞、嵌合体小鼠以及转基因小鼠。携带Moz纯合无效突变的小鼠会在胚胎发育第14.5天（E14.5）至出生前死亡。携带Moz-Flag-V5-BIO等位基因的纯合小鼠存活状态良好、健康且可育，证明MOZ-Flag-V5-BIO蛋白功能正常。本研究选取Moz-Flag-V5-BIO等位基因纯合的E10.5期胚胎的第一、第二鳃弓，以及野生型对照鳃弓（阴性对照），通过抗V5染色质免疫共沉淀实验，检测标记型MOZ蛋白在小鼠基因组中的分布情况。