MCF-7 cell line was transfected with 5μg of vectors expressing JMJD6 WT (pcDNA3-JMJD6WT-V5), JMJD6 mut (pCDNA3-JMJD6MUT-V5) or with empty pCDNA3 (2 X 106 cells in a 100mm dish) using Exgen 500 (Euromedex) according to the manufacturer’s guidelines. Selection was initiated 48h after transfection in medium containing 750μg/ml of G418. After selection, cells were maintained in medium containing 300μg/ml of G418. Three commercially available lentiviral short hairpin RNA (shRNA) constructs targeting human JMJD6 and one negative control construct created in the same vector backbone (pLKO.1-Puro) were purchased from SIGMA. Puromycin selection (1μg/mL) was started 48h after lentiviral infection. The established cell lines have been already described and characterized [23 (link)].
Lentiviral short hairpin rna shrna constructs
Manufactured by Merck Group
Lentiviral short hairpin RNA (shRNA) constructs are gene-silencing tools used in research laboratories. They contain short sequences of RNA that bind to and degrade specific target mRNA molecules, effectively reducing the expression of target genes. These constructs are delivered via lentiviral vectors, which can efficiently transduce a wide range of cell types.
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3 protocols using lentiviral short hairpin rna shrna constructs
1
Overexpression of JMJD6 in MCF-7 Cells
2
Adenoviral and Lentiviral Vector Construction
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NOXA-expressing adenovirus (Ad-NOXA) vector was constructed by inserting Flag-tagged NOXA cDNA (Ref) into pAdTrack-CMV vector (Addgene, Cambridge, MA), while the control adenovirus (Ad-Con) contained the vector alone. Adenoviruses were produced at the VCU shared resource core. Lentiviral short-hairpin RNA (shRNA) constructs were purchased from Sigma-Aldrich (St. Louis, MO). Constructs were transfected with psPAX2 and pMD2.G plasmids (Addgene, Cambridge, MA) into 293T cells with EndoFectin Lenti (GeneCopoeia, Rockville, MD), and the supernatants containing lentivirus were collected. The cell line of interest was infected with the lentiviruses and stable cell lines were established by puromycin (2 μg/ml) selection.
3
Lentiviral shRNA Constructs for LSD1 Knockdown
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Lentiviral short hairpin RNA (shRNA) constructs were obtained from Sigma-Aldrich with the following target sequences: sh non-target (shNT), 5 0 -CCG GGC GCG ATA GCG CTA ATA ATT TCT CGA GAA ATT ATT AGC GCT ATC GCG CTT TTT-3 0 ; shLSD1 #1, 5 0 -CCG GGC TAC ATC TTA CCT TAG TCA TCT CGA GAT GAC TAA GGT AAG ATG TAG CTT TTT G-3 0 ; shLSD1 #2, 5 0 -CCG GGC TAC ATC TTA CCT TAG TCA TCT CGA GAT GAC TAA GGT AAG ATG TAG CTT TTT G-3 0 .
The human Snail-1 ORF with six serine to alanine mutations (30) was fused with a modified estrogen receptor (ER) and cloned into the pBABE-puro retroviral expression vector. The pLV-MFGE8 plasmid was purchased from VectorBuilder. Conditioned medium containing infective lentiviral or retroviral particles was generated by cotransfect-ing 293T human embryonic kidney cells (2 Â 10 6 cells) with 3 mg of lentiviral vector, 1 mg pHCMV-VSV-G, and 3 mg of pCMV q8.91 or Amphotropic using Lipofectamine Ltx (Invitrogen Corporation) according to the manufacturer's instructions. Supernatants were collected 48 hours after transfection and filtered using a 0.45 mmol/L membrane (Millipore. Cells were infected directly using 8 mg/mL polybrene.
The human Snail-1 ORF with six serine to alanine mutations (30) was fused with a modified estrogen receptor (ER) and cloned into the pBABE-puro retroviral expression vector. The pLV-MFGE8 plasmid was purchased from VectorBuilder. Conditioned medium containing infective lentiviral or retroviral particles was generated by cotransfect-ing 293T human embryonic kidney cells (2 Â 10 6 cells) with 3 mg of lentiviral vector, 1 mg pHCMV-VSV-G, and 3 mg of pCMV q8.91 or Amphotropic using Lipofectamine Ltx (Invitrogen Corporation) according to the manufacturer's instructions. Supernatants were collected 48 hours after transfection and filtered using a 0.45 mmol/L membrane (Millipore. Cells were infected directly using 8 mg/mL polybrene.
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