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Plko 1 puro cmv turbogfp lentivirus plasmid

Manufactured by Merck Group

The PLKO.1-puro-CMV-TurboGFP lentivirus plasmid is a molecular biology tool designed for gene expression studies. It contains a Puromycin resistance gene and a Turbo Green Fluorescent Protein (TurboGFP) reporter gene, both driven by a Cytomegalovirus (CMV) promoter. This plasmid can be used to generate lentiviral particles for the purpose of transducing target cells and monitoring gene expression through fluorescence detection.

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3 protocols using plko 1 puro cmv turbogfp lentivirus plasmid

1

Generating LNK and shRNA Constructs

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The pLKO.1-puro-CMV-TurboGFP lentivirus plasmid (SHC003) was obtained from Sigma. The entire coding region of huLNK, including the HIS tag and V5 tag, was amplified from pcDNA3 LNK using primers TGAGCTAGCATGAACGGGCCTGCCCTGCAGCC (Nhe I) and AAACACGTGCTCGAGCGGCCGCCACTGT (Pml I). The PCR product was ligated to pGEM-T vector and validated by Sanger sequencing. This construct was digested with Nhe I and Pml I, the LNK containing fragment was gel purified, and the GFP coding fragment of pLKO-CMV-GFP vector (SHC003) was replaced with the LNK open reading frame (pLKO-CMV-LNK). For gene silencing, shRNA plasmids targeted to LNK [TRCN0000265715 (shRNA15), TRCN0000265716 (shRNA16), TRCN0000256095 (shRNA95) and Scramble shRNA SHC002 were purchased from Sigma. shRNA plasmids targeted to AKT1 (TRCN0000010174), 14-3-3 Q (YWHAQ TRCN0000078169), 14-3-3 Z (YWHAZ TRCN0000029404) and 14-3-3 G (YWHAG TRCN0000078158) were generated according to the protocol described by Addgene (http://www.addgene.org/tools/protocols/plko/). The sequences of all the shRNA constructs were confirmed by Sanger sequencing using the U6 primer.
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2

Lentiviral Gene Silencing in Myeloid Cells

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The pLKO.1-puro-CMV-TurboGFP lentivirus plasmid (SHC003) was obtained from Sigma. For gene silencing, shRNA plasmids targeted to RBP-jk, TRCN0000016207 (shRBP-jk), Scramble shRNA SHC016 were purchased from Sigma, as well asLentiviral Packaging Mix (SHP001). Plasmids were co-transfected into 293T cells usingMACSfectin transfection reagent (MiltenyiBiotec), at a ratio of 1:1(plasmid of shRNA:Packaging Mix). Media were replaced with DMEM + 10% FBS at 16 hours after transfection; viruses were collected at 48 and 72 hours after transfection and filtered through a 0.45 μm filter. HL-60 cells and THP1 cells were transduced by the lentivirus in the presence of the polycation Polybrene, and the stably transduced cells were then selected by puromycin (1μg/ml) for 4 weeks.
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3

Generating LNK and shRNA Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols
The pLKO.1-puro-CMV-TurboGFP lentivirus plasmid (SHC003) was obtained from Sigma. The entire coding region of huLNK, including the HIS tag and V5 tag, was amplified from pcDNA3 LNK using primers TGAGCTAGCATGAACGGGCCTGCCCTGCAGCC (Nhe I) and AAACACGTGCTCGAGCGGCCGCCACTGT (Pml I). The PCR product was ligated to pGEM-T vector and validated by Sanger sequencing. This construct was digested with Nhe I and Pml I, the LNK containing fragment was gel purified, and the GFP coding fragment of pLKO-CMV-GFP vector (SHC003) was replaced with the LNK open reading frame (pLKO-CMV-LNK). For gene silencing, shRNA plasmids targeted to LNK [TRCN0000265715 (shRNA15), TRCN0000265716 (shRNA16), TRCN0000256095 (shRNA95) and Scramble shRNA SHC002 were purchased from Sigma. shRNA plasmids targeted to AKT1 (TRCN0000010174), 14-3-3 Q (YWHAQ TRCN0000078169), 14-3-3 Z (YWHAZ TRCN0000029404) and 14-3-3 G (YWHAG TRCN0000078158) were generated according to the protocol described by Addgene (http://www.addgene.org/tools/protocols/plko/). The sequences of all the shRNA constructs were confirmed by Sanger sequencing using the U6 primer.
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