Objectives: To construct a lentiviral vector for RNA interference (RNAi) of the HJURP gene and to identify the silencing efficiency in the human embryo villus cells and to provide a human embryo villus cells multiplication and chromosome segregation.
Materials and methods: In accordance with the study, three specific sequences of siRNA targeting HJURP gene were designed, synthesized, then the complementary DNA containing both sense and antisense oligonucleotides of the targeting sequences were annealed and inserted into the lentiviral vector.The correct clonings were confirmed by PCR and sequencing. The most effective recombinant lentivirus vector was screened, and the recombinant plasmids with the lentivirus packaging mixes were co-transfected into 293T cells to obtain packaged lentivirus particles. Then viral titer was determined. The silencing efficiency of target gene in human embryo villus cells was detected by Real-Time PCR.
Results: DNA sequencing showed that the shRNA sequence was successfully inserted into the lentivirus vector. The recombinant lentiviral vector was successfully transfected into 293T cells. The recombinant lentivirus had a titer of 108 PFU/ml. After silencing HJURP gene in human embryo villus cells, the expression level of HJURP mRNA decreased significantly and the RNAi efficiency was greater than 70%.
Conclusion: A lentiviral shRNA expression vector targeting the HJURP gene was successfully constructed and may effectively silence the target gene at a cellular level, which provides a experimental model for the influence of HJURP gene expressing inhibition on human embryo villus cells multiplication and chromosome segregation.