Construction of recombinant lentivirus carrying mouse estrogen receptor α and identification in infected neurons

Abstract

Objective To construct a recombinant lentivirus carrying C57BL/6 mouse estrogen receptor α(Erα) and to infect mouse neurons,so as to pave a way for further studying the relationship of Erα with some nervous system diseases.Methods Erα gene was inserted into the main virus vector LV-GFP-flag to construct recombinant lentiviral vector LV-Erα-flag,which was confirmed by agarose gel electrophoresis(AGE) and DNA sequencing.Recombinant lentivirus V-Erα-flag was produced by 293T cells following the co-transfection of LV-Erα-flag with the packaging plasmids pHelper 1.0 and pHelper 2.0,and the virus titer was examined.The neurons of C57BL/6 mouse were cultured using a serum-free culture medium,and then control lentivirus V-GFP-flag was used to infect the neurons.The infection efficiency and apoptosis rate were examined by flow cytometry to obtain optimal multiplicity of infection(MOI).GFP expression was detected daily under an inverted fluorescent microscope.After that,V-Erα-flag with the optimal MOI was used to infect neurons;the expression of Erα mRNA and protein in the neurons was detected by quantitative real-time PCR and Western blotting analysis.Results AGE and DNA sequencing confirmed that LV-Erα-flag was successfully constructed,and packaged into 293T cells with a titer of 2×10~8 TU/ml.Control lentivirus V-GFP-flag could infect neurons,with the infection efficiency being(89.8±4.03)% and the cell apoptosis rate being only(3.6±0.29)% when MOI=5.Neurons could survive in the culture for at least 8 weeks,during which the GFP was persistently expressed,indicating the lentivirus could efficiently and stably infect the neurons.The expression of Erα mRNA and protein was greatly increased in neurons infected with V-Erα-flag(MOI=5).Conclusion The recombinant lentivirus carrying Erα has been constructed successfully,which can infect neurons and lead to increase the expression of Erα mRNA and protein.