Construction and verification of RNA interfering lentiviral vector targeting Suv39h1 gene / 中国职业医学

ABSTRACT

OBJECTIVE: To construct the RNA interference(RNAi) lentiviral vector of suppressor of variegation 3-9 homolog 1(Suv39 h1) and verify its interfering efficiency by transfecting it to the bone marrow-derived mesenchymal stem cells(BMSCs). METHODS: The oligonucleotides of RNA plasmid were designed and synthesized according to the gene sequence of Suv39 h1 and short hairpin RNA design principles. Three kinds of LV-Suv39 h1-RNAi recombinant plasmids with different lentivirus knockdown targets(KD1, KD2 and KD3) were constructed. After identification by restriction analysis and sequencing, the packaged lentivirus vectors with the three kinds of Suv39 h1 gene were transfected into rat BMSCs at logarithmic growth stage, and were named KD1, KD2 and KD3 transfection groups. The control group was transfected with the negative control virus. After 72 hours transfection, the transfection efficiency was evaluated, and the relative mRNA levels of Suv39 h1 were determined by quantitative real-time polymerase chain reaction(qPCR). RESULTS: Sequencing analysis demonstrated that three kinds of LV-Suv39 h1-RNAi recombinant plasmids were constructed correctly. The results of transfection efficiency evaluation showed that more than 80.00% green fluorescence was expressed in the BMSCs transfected with the three lentiviral vectors with a multiplicity of infection of 20. These results indicated that lentivirus was successfully constructed and transfection efficiency was high. The results of qPCR showed that the relative expression of Suv39 h1 mRNA in BMSCs of KD1, KD2 and KD3 transfection groups was lower than that in the control group(all P<0.05), and the relative expression of Suv39 h1 mRNA in KD1 and KD3 transfection groups was lower than that in KD2 transfection group(both P<0.05). However, there was no significant difference in the relative expression of Suv39 h1 mRNA between KD1 and KD3 transfection groups(P>0.05). CONCLUSION: The constructed lentiviral vector with low expression of Suv39 h1 was constructed successfully. This vector can be expressed in rat BMSCs, which lays a foundation to study the effect of Suv39 h1 gene in acute myeloid leukemia.