Construction and screening of plasmid expression vectors encoding short hairpin RNA targeting TWIST gene

Objective To construct a plasmid expression vector coding for the short hairpin RNA(shRNA) targeting Twist mRNA.Methods Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed.The recombinant plasmids were identified by PCR,and then transfected separately into bladder cancer cell line T24.The Twist gene silencing effect was detected by RT-PCR and Western blotting.Results The expected band of 400 bp was amplified from the plasmids coding for shRNA by PCR.By DNA sequencing,it was the same with the insertion element as with the shRNA of synthetic.Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in inhibition of Twist mRNA and protein expressions by 90% and 86%,respectively.The shRNA1 had the most obvious effect in Two types of plasmids interference.Conclusion The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully,of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.