The full length 3′UTR sequence of Elavl2 including the endogenous poly(A) site corresponding to 2529 bp of NM_001374696.1 was gene synthesized (Genewiz) and is referred as WT-3′UTR Elavl2. Using NEB Q5 Site directed mutagenesis kit and the gene synthesized as a template, the three 7-mer AREs from Elavl2 3′UTR were deleted to create ΔARE 3′UTR Elavl2 (2508 nt long). The WT-3′UTR Elavl2 and the ΔARE 3′UTR Elavl2 sequences, which differ in size by 21 nt were cloned into pNL3.2.CMV (Promega) using XhoI and NheI restriction sites located downstream of the PEST domain. The pNLD3.2.CMV vector expresses nanoluciferase in frame with a PEST domain that prevents protein accumulation ensuring a short half-life of nanoluciferase. All constructs were verified by Sanger sequencing and restriction digest.
Pnl3.2 cmv
Manufactured by Promega
PNL3.2.CMV is a lab equipment product from Promega. It is designed to perform a specific function, but a detailed description cannot be provided while maintaining an unbiased and factual approach. More information may be available from the manufacturer.
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Lab products found in correlation
In fusion snap assembly master mix, by Takara Bio (1 mentions)
Zero blunt cloning kit, by Thermo Fisher Scientific (1 mentions)
Pgl4.10 luc2, by Promega (1 mentions)
Fugene hd, by Promega (1 mentions)
Vero cell, by American Type Culture Collection (1 mentions)
Nano glo luciferase assay kit, by Promega (1 mentions)
Spectramax m5e plate reader, by Molecular Devices (1 mentions)
Gel extraction, by Qiagen (1 mentions)
5 protocols using pnl3.2 cmv
1
Elavl2 3'UTR Regulation Analysis
2
Luciferase Assay Protocol with Promoter Cloning
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The vectors for the luciferase assay, pGL4.10[luc2] and pNL3.2.CMV, were purchased from Promega. The latter was edited for each promoter assay. For Fig. 3D , the hg19 genome region of chr1:3593076–3594185 was cloned using the Zero Blunt Cloning Kit (Thermo Fisher Scientific) and inserted between the KpnI and XhoI sites of pNL3.2.CMV. For all remaining luciferase assays in this study, the minimal promoter (minP) was removed using the In-Fusion Snap Assembly Master Mix (TaKaRa), and the appropriate sequences (enumerated in Supplementary Table S3) were inserted between the KpnI and XhoI sites of pNL3.2.CMV. Ectopic gene expression in the luciferase study was induced by pME18S or pCAGGS containing the corresponding coding sequence. HEK293 or Jurkat cells were transfected with the indicated plasmids using TransIT (for HEK293) or the Neon transfection system (for Jurkat). After 24 hours of incubation, the transfected cells were subjected to luciferase assay.
3
Generating Luciferase Constructs with 3'UTR Variants
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We gene synthesized the full 3′UTR sequence of SERPINA1 (Twist biosciences) with a mutated proximal polyA site (AUUAAA to AUCAAG) and 60 nucleotides of coding sequence. This coding region and full 3′UTR were cloned into pNL3.2.CMV (Promega) using sequence homology (NEB Builder HiFi). A short 3′UTR isoform was amplified from the gene synthesized construct using PCR with the same coding sequence portion. This short isoform was cloned into the same backbone with sequence homology. The nanoluciferase expressed from this construct contains a PEST domain to prevent protein accumulation. All additional deletion and mutation constructs were created from verified long 3’UTR plasmids. Primers for cloning are listed in S3 Table . The mutated polyA site was reverted back to wildtype for both the long and short 3′UTR constructs (NEB Q5 Site directed mutagenesis kit) for structural studies. All constructs were verified by Sanger sequencing and restriction digest.
4
Measuring Protein Synthesis Inhibition
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Vero cells (ATCC) were transduced with a lentivirus expressing NanoLuc® luciferase (Vero-NLucP cells) as described previously9 . Vero-NLucP cells were plated (5000 cells/well) in a white clear-bottom 96-well plate and treated with toxin the following day. Following overnight incubation (20 h), luminescence signal was developed using the NanoGlo® Luciferase Assay kit (Promega) and read with a Spectramax M5e plate reader (Molecular Devices). Relative luminescence units (RLU) were normalized relative to untreated cells and data was analyzed with GraphPad Prism 7.0. For protein synthesis inhibition assays in HEK293T cells, cells were transiently transfected with the pNL3.2CMV (Promega) rather than by lentiviral transduction. Cells were plated in a 6-well plate at 1 × 106 cells/well and transfected the following day with 3.0 µg of plasmid DNA, in a 3:1 ratio of FuGENE®HD (Promega) transfection reagent to DNA. After 24 h, cells were re-plated at 5 × 103 cells/well, in a white clear-bottom 96-well plate and treated the following day with toxin. After overnight incubation (20 h), luminescence signal was developed using the NanoGlo® Luciferase Assay kit (Promega) similar to above. Each construct was tested 3 times with 2 technical replicates (at a minimum). Wildtype and DPH4-/- HEK293T cells were a gift from Dr. Mikko Taipale at the University of Toronto.
5
Nanoluciferase Reporter Substrate Preparation
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The core substrate vector, generated by gene synthesis (GeneWiz), comprised the C-terminal region of the nanoluciferase gene upstream of a SV40 poly A terminator, followed by a CMV promoter upstream of the N-terminal region of the nanoluciferase gene. The sequences were derived and rearranged from pNL3.2CMV (Promega) with the incorporation of silent nucleotide substitutions introducing an EcoRV restriction site into the nanoluciferase coding region. A region of the vector was excised by restriction digest with EcoRV and the blunt end fragment constituting the reporter substrate was separated from the vector backbone by agarose gel electrophoresis and purified by gel extraction (Qiagen).
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