Construction of Lentiviral Vector of DPC4 RNA Interference and Its Silencing Effects

Objective :To construct and design lentiviral shRNA targeting DPC4 gene and gene silencing. Methods :Web-based program was used to analyze the DPC4 sequence for the shRNA target sites and to design RNA interference sequence which fits the designing principles. Based on the designing software evaluation,the sequences with the best dynamic parameters were selected for synthesis of DNA oligonucleotide with sense-loop-antisense structure and restriction enzyme sticky ends. DNA oligos were PAGE purified and inserted into the shRNA lentiviral vector cutted with restriction enzymes. The product was transformed into the competent cells. The positive constructs were further confirmed by sequencing. In addition,the Western blot was prepared from the infected 293T cells for analysis of the knowdown effect of the shRNA lentivirus. Results :LVshDPC4 vectors were confirmed by restriction analysis and DNA sequencing. The lentiviral particles were prepared and showed effective infection capacity and knockdown effects. Conclusion :Lentiviral shRNA targeting DPC4 gene is successfully constructed and can effectively knock down DPC4 expression.