Di Fulvio Mauricio, Henkels Karen M, Gomez-Cambronero Julian
Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA.
Biochem Biophys Res Commun. 2007 Jun 8;357(3):737-42. doi: 10.1016/j.bbrc.2007.04.013. Epub 2007 Apr 10.
Grb2 is an SH2-SH3 protein adaptor responsible for linking growth factor receptors with intracellular signaling cascades. To study the role of Grb2 in cell growth, we have generated a new COS7 cell line (COS7(shGrb2)), based on RNAi technology, as null mutations in mammalian Grb2 genes are lethal in early development. This novel cell line continuously expresses a short hairpin RNA that targets endogenous Grb2. Stable COS7(shGrb2) cells had the shGrb2 integrated into the genomic DNA and carried on <10% of normal levels of Grb2. Silencing Grb2 expression reduced, but did not eliminate, basal cell growth rate. This could be reversed by either the addition of neomycin to the cell cultures or by rescuing with an Xpress-Grb2(SiL) construct (made refractory to the shRNA-mediated interference), but not with an SH2-deficient mutant (R86K). Thus, a viable knock-down and rescue protocol has demonstrated that Grb2 is crucial for cell proliferation.
Grb2是一种SH2-SH3蛋白衔接子，负责将生长因子受体与细胞内信号级联反应相连。为了研究Grb2在细胞生长中的作用，我们基于RNA干扰技术构建了一种新的COS7细胞系（COS7(shGrb2)），因为哺乳动物Grb2基因的无效突变在早期发育阶段是致死性的。这种新型细胞系持续表达靶向内源性Grb2的短发夹RNA。稳定的COS7(shGrb2)细胞将shGrb2整合到基因组DNA中，其Grb2水平维持在正常水平的10%以下。沉默Grb2表达会降低但不会消除基础细胞生长速率。这可以通过向细胞培养物中添加新霉素或用Xpress-Grb2(SiL)构建体（对shRNA介导的干扰具有抗性）进行挽救来逆转，但不能用SH2缺陷型突变体（R86K）来逆转。因此，一种可行的敲低和挽救方案证明Grb2对细胞增殖至关重要。
