An HIV-based transfer vector encoding CMV-GFP44 (link) was modified to encode TA-FLuc (plenti-TA-FLuc) by exchanging the CMV-GFP cassette with TA-FLuc from the Panomics translucent control vector (Panomics, Madison, WI) using NheI and XbaI restriction enzymes. This construct was further modified to create a library of lentivirus-producing transfer vector constructs with TF-responsive binding elements. Sequences derived from Panomics constructs were digested out of the constructs also using NheI and XbaI and ligated into the plenti-TA-FLuc backbone. For non-Panomics constructs, such as TCF/LEF45 (link), CMYC14 (link), NOTCH146 (link), and PTTG47 (link), custom oligonucleotides were synthesized (Sigma Aldrich), annealed, and inserted into the plenti-TA-FLuc backbone using NheI and BglII. A vector encoding Gaussia luciferase (GLuc), TA-GLuc, was also constructed by transferring the GLuc gene from pCMV-GLuc (New England Biolabs, Ipswich, MA) into the Panomics vector using HindIII and XbaI, and subsequently following the same procedure as the other Panomics-derived constructs.
Pcmv gluc
Manufactured by New England Biolabs
Sourced in United Kingdom
PCMV-GLuc is a plasmid that contains the secreted Gaussia luciferase (GLuc) reporter gene under the control of the cytomegalovirus (CMV) promoter. GLuc is a small, naturally secreted luciferase that can be easily detected in cell culture media or body fluids.
Automatically generated - may contain errors
2 protocols using pcmv gluc
1
Generation and Modification of TA-Luciferase Lentiviral Vectors
2
Plasmid DNA Purification and Transfection
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The expansion, isolation and purification of cell secreted Gaussia-luciferase plasmid (pCMV-GLuc, obtained from New England Biolabs UK) was performed using the Giga-Prep (Qiagen) kit as per protocol and its purity was confirmed by UV spectroscopy (NanoDrop™ ND1000 Spectrophotometer, Thermo Scientific) and gel electrophoresis. The polyplex formed via the electrostatic attraction between the DNA and cationic polymer. P1-3 were firstly diluted in 25 mM aqueous NaOAc (pH = 5.2) and then mixed with DNA (60 μg/mL in 25 mM aqueous NaOAc) at a 1:1 volume ratio with polymer/DNA w/w ratio ranged from 15:1, 30:1 to 45:1. For PEI, N/P ratio 10:1 was used. To prepare the polyplexes, PEI and DNA were dissolved in 40 μl of Opti-MEM media, respectively, and then vortexed for 15 s and allowed to stand for 10 min. SuperFect was used according to manufacturer's instructions, briefly, DNA was diluted in serum free cell culture media, SuperFect was then added, mixed by vortexing for 10 s and incubated for 10 min. DNA binding efficiencies were confirmed by PicoGreen.
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