Expression of Dopaminergic Transcription Factor Nurr1 in Human Cells Via Recombinant Lentiviruses
Author(s):
Abstract:
Material and
Methods
The IRES-EGFP fragment was isolated from the pIRES2-EGFP vector using restriction enzymes BglII/NotI and made blunt-ended using Klenow. The transfer vector PNL-EGFP/CMV/WPREdU3 was digested with NheI/XhoI and made blunt-ended. Finally, the isolated IRES-EGFP fragment was inserted into this lentivirus vector to generate lentivirus construct (I). The human Nurr1 gene was then isolated from the PCMX-NOT vector using BamHI and XhoI and inserted into construct (I) pre-digested with BamHI and SalI. At this step lentivirus construct (II) as our final transfer construct was generated. In order to generate recombinant lentiviruses, we then transfected the HEK-293T cell line with transfer vector plus packaging and envelope vectors. Cell medium full of virus particles was collected and passed through Amicon filters to produce a concentrated virus stock. The stock was ultimately used for transduction of fresh HEK-293T cells. EGFP expression was shown under fluorescent microscope and Nurr1 expression was analyzed using RT-PCR.Results
Enzymatic tests confirmed the correct cloning of the hNurr1gene into the lentivirus backbone. Observation by fluorescent microscopy showed EGFP expression post-transfection and post-transduction. RT-PCR demonstrated Nurr1 expression at both stages.Conclusion
In this study, lentiviruses carrying the human Nurr1 gene were produced and used for transduction of human cell line HEK-293T. The transduced cells successfully expressed the Nurr1 gene.Keywords:
Nurr1 , Dopaminergic , Transcription factor , Lentivirus , HEK , 293T
Language:
Persian
Published:
Journal of Cell &Tissue, Volume:7 Issue: 1, 2016
Page:
1
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