ABSTRACT
We have optimized lentiviral vector constructs and cassettes for expression of short hairpin RNAs (shRNAs) in order to create genome-wide library capable of inhibition of full variety of human mRNAs. The vector optimization has resulted in 15-20-fold improvement in virus stock titers. We found that in the context of lentiviral vector the most effective structure for the shRNA is simple hairpin with 21 nucleotide stem. The shRNA-expressing lentiviral constructs contain choice of puro(R), copGFP or H-2K(k) selective markers. The efficiency of the optimized library was evaluated in experiments on screening of shRNAs that reactivate oncosuppressor p53 in HeLa cells. The cells contained reporter construct with p53-dependent expression of a fluorescent protein, which allows cytofluorimetric isolation of cell population with reactivated p53.
Subject(s)
Gene Library , Genome, Human/genetics , Lentivirus , MicroRNAs/genetics , RNA Interference , Gene Expression Regulation , Genes, p53/genetics , Genetic Markers , Genetic Vectors , HeLa Cells , Humans , Nucleic Acid ConformationABSTRACT
Structure and dynamics of actin cytoskeleton play a role ih regulation of cell adhesion, spreading and migration. TRIP6 is a LIM domain-containing protein interacting with many actin-associated proteins and in addition modulating activity of certain transcription factors. To study functions of TRIP6 we inhibited its expression in A549 and A431 cells by short interfering RNAs (siRNAs). The TRIP6 knock-down lead to the increased number and length of stress fibers and to the induction of locomotive phenotype. There was observed decreased number and reorganization of focal adhesions revealed by staining for paxillin, and loss of cell to cell adhesions revealed by staining for E-cadherin. The above changes in cell morphology were accompanied by 2-fold increase in the cell motility rate assessed by the wound healing assay. Thus, down-regulation of TRIP6 in the cell lines used results in increase in the features characteristic to malignant transformation of epithelial cells. Possible mechanisms for the observed effects are discussed.
