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Lentiviruses are considered one of the most effective recombinant viruses for gene transfer to mammalian cells and tissues. In this study, the potential of HIV-1-based lentiviral vector to deliver transgenes into avian cells was examined. We co-transfected human embryonic kidney cell line HEK-293T with three lentivirus vectors called transfer, packaging and envelope vectors. We collected the supernatant from transfected cells 24 and 48 hours post-transfection and filtered them immediately. Then we subjected the filtered supernatant to Amicon protein columns for concentration purposes. Centrifugation removed a larger part of the supernatant presumably free of viruses and left behind a small volume of darken solution full of virions. We thereby produced a 500-µl-volume of virus stock. Various dilutions of this stock were added to chicken liver cell line LMH. The initial sign of infection appeared within 48 hours and by 96 hours post-infection 100% the LMH cells positively expressed transgenes. Our results indicated that the human HIV-1-based lentivirus vectors are capable of transducing and transferring foreign genes into chicken cells. Given the need for a high-titer virus stock for successful target cell transduction, our results indicate that the filtration method of virus concentration is able to produce high virus titer and is cost-effective and less time consuming than ultracentrifugation or other traditional methods.

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Lentiviruses are considered one of the most effective recombinant viruses for gene transfer to mammalian cells and tissues. This study comprises of two essential parts: (1) evaluation of efficiency of protein purification columns in concentration of recombinant lentiviruses, and (2) production of recombinant lentiviruses carrying GDNF coding sequences. In part (1) we co-transfected human embryonic kidney cell line HEK-293T with three lentivirus vectors called transfer (carrying either GFP or Jred), packaging and envelope vectors. After a filtration step, we applied the supernatant from transfected cells to Amicon protein columns for concentration purposes. Centrifugation removed 99% of the supernatant and left behind 500-µl-volume of solution full of virions. We thereby produced a of virus stock. Various dilutions of this stock were added to HEK-293T cells that produced up to 100% infected cells positively expressing transgenes. To examine whether the removed supernatant (overflow) has any trace of infective virus by chance, we also used dilutions of the overflow for infection and observed no sign of eGFP or Jred expression. Given the need for a high-titer virus stock for successful target cell transduction, our results indicate that our filtration method of virus concentration is able to produce high virus titer and is cost-effective and less time consuming than previous methods. In part (2), due to the importance of neurotrophic factor GDNF in differentiation and neuroprotection as well as in therapy of neurodegenerative disorders, we ligated GDNF coding sequence into the lentivirus backbone in the second phase of our study. We applied the same method outlined above to produce high-titer recombinant viruses. Following infection of human astrocytoma cells with this virus stock, we detected 3-fold increase in GDNF mRNA expression using RT-PCR. Lentiviruses carrying GDNF can therefore be generated at high titer using the column method and applied for differentiation and neuroprotection studies.

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Objective: The aim of the present study was the production of recombinant lentviruses that express miR-16. After transduction, altered expression levels of miRNA and its target protein were analyzed. Methods: A DNA fragment that contained the miR-16 precursor was cloned in a lentiviral plasmid. Lentiviral vector particles were produced by transient calcium phosphate co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids. Viral supernatants were harvested and concentrated by ultracentrifuge. Virus titration was determined by fluorescent microscopy and flow cytometry. Altered expression levels of miR-16 were evaluated by real-time PCR; its protein target was evaluated by Western blot. Results: The identity of DNA was established by colony-PCR, enzymatic digestion of positive clones, and DNA sequencing. After co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids, viral particles were concentrated and the virus titer determined. Maximum expression of the GFP reporter gene was obtained in more than 80% of the cells transduced with lentivirus at MOI=1. Real-time PCR assay showed that miR-16 expression levels significantly increased in transduced cells compared with the control group. As shown by Western blot analysis, miR-16 overexpression downregulated Bcl-2 expression at the protein level. Conclusion: This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells.

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Objective: microRNAs (miRNAs) are noncoding RNAs that function as key regulators of diverse biological activities such as cellular metabolism, cell proliferation and cell cycle regulation. Recent studies have indicated the high potential of these small molecules to control stem cell differentiation into desired cells. The aim of present study is to investigate the possible effect of let-7f on expression of hepatic nuclear factor 4 alpha (HNF4a) and some hepatic specific factors such as albumin (ALB), alpha fetoprotein (AFP), cytokeratin18 (CK18) and cytokeratin19 (CK19) in human adipose tissue derived stem cells (hADSCs). Methods: ADSCs were isolated from human adipose tissue using collagenase type I and were transduced by recombinant lentiviruses that contained human inhibitor let-7f and Scramble (negative control). Afterward, the expressions of HNF4a, ALB, AFP, CK18 and CK19 were evaluated by Real-time PCR at different time points. Results: Transduction efficiency of lentiviral vectors into ADSCs was more than 80% as judged by the expression of the GFP reporter gene. Real-time PCR analysis revealed that inhibition of let-7f in hADSCs resulted in significant up regulation of hepatic specific genes compared with the negative control. The expression level of HNF4a also increased in experimental cells at day 14, which supported the suppression of HNF4a expression by let-7f. Conclusion: The results of this study identified let-7f as a negative regulator of HNF4a expression in hADSCs and increased the expression of hepatocyte specific factors through silencing of let-7f. Therefore, suppression of let-7f could be a considerable tool for hepatic differentiation of hADSCs.