Construction and Identification of Lentiviral Vector for RNA Interference of USE1 Gene

doi:10.13560/j.cnki.biotech.bull.1985.2018-0659

Abstract

Abstract: This work aims to construct a lentiviral vector for RNA interference of ubiquitin-conjugating enzyme USE1 gene and to detect the inhibition level of this gene in cells for identifying the downstream substrates corresponding to the specific ubiquitination pathway of Uba6-USE1. The specific shRNA sequence targeting USE1 gene were selected,synthesized,and cloned into pLL3.7 lentiviral vector,and the recombinant lentiviral plasmids inhibiting the expression of USE1 gene were constructed and identified. The identified recombinant plasmids were co-transfected into HEK-293 cell with psPAX2 and VSVG lentiviral packaging vectors. Then viral supernatants were collected and the titers as well as the optimal dilution factors of the virus were determined. The inhibition of the USE1 gene in viral infected HEK-293 cells was detected by qPCR and Western Blot. Results showed that 3 RNA-interfered lentiviral vectors targeting USE1 gene were constructed successfully,the supernatants of 3 lentiviral packaging vectors with satisfied titers were collected. Number 2 and 3 shRNA sequences presented obvious inhibitions to the expression of USE1 gene with an inhibition rate at 50%（^*P<0.05）. In sum,lentiviral vectors specifically inhibiting the expression of ubiquitin-conjugating enzyme USE1 gene are successfully constructed by RNA interference,and supernatants and shRNA sequences significantly inhibiting the expression of USE1 are obtained after mRNA and protein verification.

Key words: ubiquitin-conjugating enzyme USE1, RNA interference, lentiviral vector, ubiquitination

WANG Jia-yue, LIU Xiang-nan, PENG Kang-li, ZHAO Bo. Construction and Identification of Lentiviral Vector for RNA Interference of USE1 Gene[J]. Biotechnology Bulletin, 2019, 35(3): 117-122.

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