Construction and identification of the lentiviral RNAi vector of rat ICAM-1 gene in vitro

Objective To construct the recombinated lentivirus carrying small interfering RNA(siRNA) of rat intercellular adhesion molecule-1(ICAM-1) gene and identify the most effective lentivirus.Methods Four specific target sequences were selected according to rat ICAM-1 mRNA sequence.The DNA oligonucleotide was subcloned into the plasmid pGCL-GFP.After igated by DNA ligase,the acquired recombinants were subsequently transfected into E coli strain DH5α.Using PCR,the possitively cloned recombinants were obtained and the gene sequencing was made.The recombinanted plasmid was cotransfected along with Helper 1.0 and Helper 2.0 into HEK293T to package lentivirus particles.The recombinated lentivirus was compared with the RNA interference(RNAi) effect by RT-PCR.Results PCR and DNA seuqencing demonstrated that the lenitvirus RNAi vector of ICAM-1 was constructed successfully.The No.3 lentivirus was the most effective one.When MOI=50,the rate of lentivirus inhibiting the gene expression was 88.4%.Conclusion The recombinated lentivirus plasmid carrying siRNA of rat ICAM-1 gene has been constructed successfully