Mutations were introduced into human KCNC1 cDNA encoding transcript 1 (NM_001112741; Kv3.1b) or transcript 2 (NM_004976; Kv3.1a) cloned into a pCMV‐Entry vector that were obtained from OriGene Technologies using QuikChange Lightning Site‐Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions. Insertion of mutation was confirmed and additional mutations excluded by Sanger sequencing. cRNA was prepared using the T7 mMessage mMachine kit from Ambion (Austin, TX).
Pcmv entry vector
Manufactured by OriGene
Sourced in United States
The PCMV-Entry vector is a plasmid designed for the expression of recombinant proteins in mammalian cells. It contains a human cytomegalovirus (CMV) promoter, which drives the expression of the target gene. The vector also includes a poly(A) signal for efficient mRNA processing and a multiple cloning site for the insertion of the gene of interest.
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Lab products found in correlation
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (3 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Calcium phosphate, by Takara Bio (1 mentions)
On targetplus smartpool, by Horizon Discovery (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dharmafect 1, by Horizon Discovery (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Xl10 gold ultra competent e coli cells, by Agilent Technologies (1 mentions)
Quickchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Mut express 2 fast mutagenesis kit v2, by Vazyme (1 mentions)
9 protocols using pcmv entry vector
1
Engineered KCNC1 Transcripts for Study
2
MLK4 Silencing and Overexpression
3
Knockdown and Overexpression of METTL3
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For knockdown, either METTL3 small interfering RNA (On-target plus SMART Pool, Dharmacon, Lafayette, CO, USA) or a non-targeting siRNA control (siNT) were transfected using Dharmafect I (Dharmacon) according to the manufacturer’s instructions. The shRNA construct for METTL3 (TRCN0000289814) was taken from the MISSION shRNA TRC whole genome library.
For overexpression, a METTL3 construct in pCMV-Entry vector (#RC200869, Origene, Rockville, MD, USA) was used; transfection of the empty vector acted as a control. Plasmids were transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 48 h, cells were harvested and transfection with the correct construct was confirmed by qRT-PCR. Simultaneously, cells were split into single cell suspension and counted; equal cell numbers were plated.
For overexpression, a METTL3 construct in pCMV-Entry vector (#RC200869, Origene, Rockville, MD, USA) was used; transfection of the empty vector acted as a control. Plasmids were transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 48 h, cells were harvested and transfection with the correct construct was confirmed by qRT-PCR. Simultaneously, cells were split into single cell suspension and counted; equal cell numbers were plated.
4
Engineered KCNC1 Missense Mutation
5
KCNT1 Mutant Protein Expression
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The full-length human KCNT1 cDNA in pCMV-Entry vector (Cat#SC311132) was sourced from Origene, Rockville, MD, USA. The mutagenesis of KCNT1 cDNA was done using Quickchange lightning site-directed mutagenesis kit from Agilent Technologies Inc., Santa Clara, CA, USA (Cat# 210518) to introduce following mutation: G288S (Gly288Ser), T314A (Thr314Ala), R398Q (Arg398Gln), N449S (Asn449Ser), L781V (Leu718Val), E893K (Glu893lys), M896V (Met896Val), L924F (Leu942Phe), R928C (Arg928Cys), F932L (Phe932Leu), S937G (Ser937Gly), R961H (Arg961His) and A965T (Ala965Thr). Primers were designed to introduce specific mutations (www.agilent.com/genomics/qcpd (accessed on 20 April 2018)), and PCR amplification was done using high-fidelity DNA polymerase. Following PCR, Dpn1 endonuclease treatment was performed to digest template DNA, leaving the nicked PCR product with desired mutation. The digested PCR product with EcoR1 and Xho1 restriction enzymes was ligated with pCMV-Entry vector using T4-Ligase (NEB# M0202S) and then transformed in to XL10-Gold ultra-competent E. coli cells (Agilent Technologies # 210518). Mutated sequences were verified by Sanger sequencing. In addition, we have generated YFP-6His tagged versions of wild type and mutant KCNT1 using 3 piece ligation (6His-YFP, KCNT1, pCMV-Entry vector).
6
Functional Analysis of KCNC1 Variant
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Functional analysis of c.949C>A (p.R317S) was performed as previously described (20 (link)). Briefly, the variant was introduced into cDNA encoding KCNC1 transcript variant 1 (NM_001112741; Kv3.1). The cDNA cloned into a pCMV‐Entry vector was obtained from OriGene Technologies (Rockville, MD). Quik Change Lightning Site-directed mutagenesis kit (Agilent, Santa Clara, CA) was used according to the manufacturer’s instructions (20 (link)). Sanger sequencing was performed to verify the insertion of the variant and exclude additional changes. In vitro transcription was performed using T7 mMessage mMachine kit (Ambion, Austin, TX).
7
Tomosyn-1 siRNA Manipulation Protocol
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SNAP25 (clone ID; 4504644) from the RIKEN mouse FANTOM clone was obtained through DANAFORM (Yokohama, Japan). Tomosyn-1 cDNA (clone ID; MC223621) and PCMV-Entry Vector (clone ID; PS100001) were purchased from Origene (Rockville, MD, USA). Tomosyn-1-specific siRNA and scrambled siRNA were procured from Sigma-Aldrich (St. Louis, MO, USA). The following siRNA sequences were used: scrambled, #1 (5'-CAGUCGCGUUUGCGACUGG-3'); Tomosyn-1 #1 (5'-GUACUAUAUUGAGGUUAAATT-3'); and tomosyn-1 #2 (5'-CAGUUGUGCAUAUAAGUGATT-3').
In this manuscript, tomosyn-1 refers to syntaxin binding protein 5, and tomosyn-2 refers to syntaxin binding protein 5-like, whereas tomosyn refers to both paralogs.
In this manuscript, tomosyn-1 refers to syntaxin binding protein 5, and tomosyn-2 refers to syntaxin binding protein 5-like, whereas tomosyn refers to both paralogs.
8
Generating Mutant ZC3HC1 Constructs
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The wild-type ZC3HC1 open reading frame (ORF) with a C-terminal FLAG tag in a pCMV-Entry vector was obtained from Origene (Rockville). A QuikChange site-directed mutagenesis kit (Agilent, Santa Clara, CA) was then used to mutate the wild-type (CAD-risk) version to the mutant (CAD-non-risk) version of the gene. The two versions of the ORF were then subcloned into the pLEICS-10 vector, which contains an N-terminal MBP directly prior to the ORF, by The Protein Expression Laboratory at the University of Leicester (Leicester, UK).
9
Cloning and Mutagenesis of CMPK2 cDNA
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A Flag-tagged human CMPK2 cDNA sequence was cloned into the pCMV-Entry vector (Origene). Based on this template, point mutations of CMPK2 were introduced using a Mut Express® II Fast Mutagenesis Kit V2 (Vazyme), according to the manufacturer’s instructions. The primers for the plasmid constructs are listed in Supplementary Table S2 .
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