Lentiviral-mediated BCL2 gene knockdown using comparative microRNA adaptive shRNAs
| Autor: | Leila Nasehi, Ali Fallah, Baharak Abdolhossein Zadeh, Gholamreza Tavoosidana, Mehdi Banan, Kamal Yavari, Moloud Absalan |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Gene knockdown
Expression vector Genetic enhancement Blotting Western Lentivirus HEK 293 cells General Medicine Biology Viral vector Cell biology MicroRNAs HEK293 Cells Proto-Oncogene Proteins c-bcl-2 RNA interference Gene Knockdown Techniques microRNA Gene expression Humans RNA Interference RNA Small Interfering |
| Zdroj: | Cellular and Molecular Biology. 64:25-30 |
| ISSN: | 1165-158X 0145-5680 |
| DOI: | 10.14715/cmb/2018.64.11.5 |
| Popis: | B-cell lymphoma 2 (BCL2) family proteins play a critical role in tuning cell death processes. Almost in half of all human cancers, a dysregulation in BCL2 family gene expression has been shown which made it an impressive target for human gene therapy as a novel approach in cancers. In this study we will optimize lentiviral-mediated RNA interference (RNAi), recombinant lentiviruses accommodating anti-BCL2 micro adaptive short hairpin RNAs (shRNAs), to downregulate BCL2 in human embryonic kidney 293T (HEK293T) cells to produce stable cell lines. We tested 4 different Dharmaconâ„¢ GIPZâ„¢ shRNAmir lentiviral vectors targeting BCL2 in different positions and a pGIPZ non-silencing shRNAmir lentiviral vector (as a negative control). Lentivirus packaging was performed by the calcium phosphate precipitation method. HEK293T cells were transduced by each type of recombinant lentiviruses individually and selected by puromycin within 10 days. The relative mRNA level and protein expression were assayed by using real-time polymerase chain reaction (PCR) technic and western blotting, respectively. Lentivirus (LV) packaging was performed in high efficiency (transfection rate was > 90%). Recombinant viruses of 4 expression vector addition to a control vector were produced then transduced to HEK293T cells successfully. All the 4 cell groups showed a significant down regulation of BCL2 gene (~90-95%) at mRNA level compared to the control group (p 0.05). We showed that the lentivirus-mediated RNAi technique is an efficient method to establish HEK293 cell lines with stable down-regulation of BCL2 gene. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|