Gene profiling of v-Src transformed primary chicken embryo fibroblasts (CEFs) under the condition of AP-1 repression. Gallus gallus

To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEF expressing either the c-Jun dominant-negative mutant TAM67 or JunD shRNA. Overall design: CEFs were transfected with B-type RSV viral vectors expressing JunD shRNA, GFP control, or the c-Jun TAM67 dominant negative mutant. Transfected CEFs were superinfected with the temperature sensitive A-type NY72-4 RSV. Infected CEFs were cultured at the non-permissive temperature until such time when temperature shift was performed to induce v-Src activation. All experimental groups consisted of three biological replicates except for NY72-4/RCAS(B)-JunD shRNA permissive which was conducted in sextuplicate. [Type] viral transformation of primary cells, transformation, AP-1 inhibition, temperature sensitive mutant, v-Src, apoptosis

为探究激活蛋白1（AP-1）在Src介导的细胞转化中的作用，本研究通过基因表达谱分析，对分别表达c-Jun显性负性突变体TAM67或JunD短发夹RNA（short hairpin RNA, shRNA）的v-Src转化鸡胚成纤维细胞（chicken embryo fibroblast, CEF）的转录组特征进行表征。实验设计：将携带B型劳斯肉瘤病毒（Rous sarcoma virus, RSV）载体、分别表达JunD shRNA、绿色荧光蛋白（green fluorescent protein, GFP）对照或c-Jun TAM67显性负性突变体的重组载体转染至CEF；随后用温度敏感型A型NY72-4 RSV对转染后的CEF进行超感染。将感染后的CEF置于非允许温度下培养，直至通过温度转换诱导v-Src激活。除NY72-4/RCAS(B)-JunD shRNA允许温度组设置为6次生物学重复外，其余所有实验组均设置3次生物学重复。【研究类型】原代细胞病毒转化、细胞转化、AP-1抑制、温度敏感突变体、v-Src、细胞凋亡