Hartl M, Vogt P K, Bister K
Institute of Biochemistry, University of Innsbruck, Austria.
Virology. 1995 Feb 20;207(1):321-6. doi: 10.1006/viro.1995.1086.
A chimeric construct (VCD) consisting of parts from viral jun, chicken c-jun, and chicken junD was cloned into the replication-competent retroviral RCAS vector. This construct, RCAS-VCD, was found to have a higher focus forming potential in quail fibroblasts than the equivalent construct RCAS-VJ-1, expressing viral jun. DNAs from RCAS-VCD and RCAS-VJ-1 were transfected into primary quail embryo fibroblasts. Cells derived from one RCAS-VCD-induced focus survived cell crisis, which became manifest after 15 passages, and could be expanded into a long-term culture. This cell line, termed VCD, has been passaged for over 100 times so far. The cells grow to very high densities and then pile up into clumps of rounded cells. The culture releases a transforming virus with a titer of 10(5) FFU/ml, as tested on primary quail embryo fibroblasts. The transformed phenotype of VCD cells was verified by agar colony formation. VCD cells are capable of anchorage-independent growth with a cloning efficiency of 10%. Southern blot analysis of genomic DNA from VCD cells showed proviral integration of the RCAS construct without detectable rearrangements. Northern and Western blot analyses confirmed correct expression from integrated RCAS-VCD of predicted RNAs and of the chimeric Jun(VCD) protein. jun(VCD)-transformed cells provide a constant source of homogeneous cellular material for the investigation of the molecular mechanisms of jun-induced cell transformation and for the identification of direct and indirect targets of Jun protein function.
一种由病毒jun、鸡c-jun和鸡junD的部分组成的嵌合构建体（VCD）被克隆到具有复制能力的逆转录病毒RCAS载体中。发现这种构建体RCAS-VCD在鹌鹑成纤维细胞中比表达病毒jun的等效构建体RCAS-VJ-1具有更高的集落形成潜力。将来自RCAS-VCD和RCAS-VJ-1的DNA转染到原代鹌鹑胚胎成纤维细胞中。来自一个RCAS-VCD诱导集落的细胞在15代后出现细胞危机并存活下来，并可扩展为长期培养物。这个细胞系被称为VCD，到目前为止已经传代超过100次。细胞生长到非常高的密度，然后堆积成圆形细胞团。如在原代鹌鹑胚胎成纤维细胞上测试的那样，培养物释放出滴度为10(5) FFU/ml的转化病毒。通过琼脂集落形成验证了VCD细胞的转化表型。VCD细胞能够进行不依赖贴壁的生长，克隆效率为10%。对VCD细胞基因组DNA的Southern印迹分析显示RCAS构建体的前病毒整合，没有可检测到的重排。Northern和Western印迹分析证实了整合的RCAS-VCD对预测RNA和嵌合Jun（VCD）蛋白的正确表达。jun（VCD）转化的细胞为研究jun诱导的细胞转化的分子机制以及鉴定Jun蛋白功能的直接和间接靶点提供了恒定的均匀细胞材料来源。
