Ferrara Luciana, Engstrom Julia U, Schwartz Timothy, Parekh-Olmedo Hetal, Kmiec Eric B
Department of Biological Sciences, University of Delaware, Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA.
DNA Repair (Amst). 2007 Oct 1;6(10):1529-35. doi: 10.1016/j.dnarep.2007.04.007. Epub 2007 Jun 11.
We have previously shown that activation of the homologous recombinational repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long-term impact of this stalling on the growth of cells that have enabled gene repair events. Using a mutated eGFP gene as an episomal reporter, we show that corrected (eGFP-positive) cells contain only a few active replication templates 2 weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By 6 weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP-negative) population. These results indicate that the initial stalling is transient and eGFP-positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.
我们之前已经表明，同源重组修复途径的激活会导致校正后的细胞出现细胞分裂阻滞，这可能是通过检查点蛋白Chk1和Chk2的活性实现的。在本研究中，我们研究了这种停滞对已发生基因修复事件的细胞生长的长期影响。使用突变的eGFP基因作为游离型报告基因，我们发现校正后的（eGFP阳性）细胞在电穿孔后2周仅含有少数活跃的复制模板，但未表现出凋亡或衰老表型。到电穿孔后6周，细胞恢复活跃复制，其细胞周期谱与未校正的（eGFP阴性）群体相当。这些结果表明，最初的停滞是短暂的，eGFP阳性细胞最终恢复正常的表型生长模式，从而允许在体外传代和扩增。
