RHBDF2-shRNA慢病毒载体的构建和Neuro-2a-RHBDF2-shRNA稳转细胞系的建立

doi:10.13481/j.1671-587x.20190606

Abstract

Abstract: Objective: To construct the RHBDF2-shRNA lentiviral vector and to establish a stably transfected Neuro-2a-RHBDF2-shRNA cell line,and to detect the expression levels of RHBDF2 mRNA and protein in the stably transfected cell line. Methods: According to the RHBDF2 sequence in NCBI,a shRNA-oligo targeting RHBDF2 gene was designed and synthesized.It was cloned into the lentiviral vector FV067-RNAi-EGFP-Puro digested by EcoRⅠand AgeⅠrestriction endonuclease to construct the FV067-RHBDF2-shRNA lentiviral plasmid.The positive clones were screen by PCR and verified by sequencing.The HEK293T cells and Neuro-2a cells were used as the experimental materials.The Neuro-2a-FV067 Vector was used as control group,and the Neuro-2a-FV067-RHBDF2-shRNA was used as experiment group.The FV067 Vector and FV067-RHBDF2-shRNA plasmids were co-transfected into the HEK293T cells with lentiviral-assisted plasmids psPAX2 and pMD2G using Lipofectamine 2000,respectively.The lentivirus was harvested and purified 48 h after transfection.The FV067 Vector lentivirus in control group and the FV067-RHBDF2-shRNA lentivirus in experiment group were used to infect the Neuro-2a cells when the multiplicity of infection(MOI) was 50,respectively,and the expression of green fluorescence was detected under fluorescence microscope.Then 2 mg·L^-1 puromycin was used to screen the cell line stably silencing RHBDF2.Real-time fluorescence quantitative PCR was used to detect the expression levels of RHBDF2 mRNA in the cells in two groups and Western blotting method was used to detect the expression levels of RHBDF2 protein in the cells in two groups. Results: The PCR and the sequencing analysis results indicated that the interference sequence of RHBDF2-shRNA lentiviral vector was identical with the sequence of designed and synthesized RHBDF2-shRNA-oligo.The titer of FV067 Vector lentivirus was 5×10⁸ TU·mL^-1 and the titer of FV067-RHBDF2-shRNA lentivirus was 3×10⁸ TU·mL^-1.The real-time fluorescence quantitative PCR results revealed that the expression level of RHBDF2 mRNA in the stably transfencted cell line in experiment group was significantly decreased by 52.26% compared with control group(t=11.44,P=0.007 6).The Western blotting results indicated that the expression level of RHBDF2 protein in the stably transfected cell line was significantly decreased by 47% compared with control group(t=6.374,P=0.007 8). Conclusion: The FV067-RHBDF2-shRNA lentiviral vector is constructed successfully and the Neuro-2a-RHBDF2-shRNA cell line is established,and the expression levels of RHBDF2 mRNA and protein in the Neuro-2a-RHBDF2-shRNA stably transfected cell line is decreased significantly.

Key words: RHBDF2, RNA interference, lentivirus, Neuro-2a cells, stable cell line

CLC Number:

Q343.37

LI Shengnan, CHEN Wuhai, CHEN Shaofeng, DENG Fu, ZHU Peiyi, LI You. Construction of RHBDF2-shRNA lentiviral vector and establishment of stably transfected Neuro-2a-RHBDF2-shRNAcell line[J].Journal of Jilin University(Medicine Edition), 2019, 45(06): 1224-1230.

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Construction of RHBDF2-shRNA lentiviral vector and establishment of stably transfected Neuro-2a-RHBDF2-shRNAcell line

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