Generation of the HAI‐2/SPINT2 expression plasmid was described previously.13 Briefly, the full‐length coding region for HAI‐2/SPINT2 was cloned into XbaI‐SalI sites of a pCI‐neo expression vector (Promega, Madison, WI, USA). An empty vector (mock) or SPINT2 expression plasmid was linearized and transfected into U87, U251, and T98G to establish clones stably expressing HAI‐2/SPINT2. To determine growth curves of the mock‐ or SPINT2‐transfected subline, triplicate 35‐mm dishes were seeded at 1 × 104 cells/3 mL growth medium and the number of viable cells was counted at the indicated time period.
Pci neo expression vector
Manufactured by Promega
Sourced in United States
The PCI-neo expression vector is a plasmid designed for the expression of recombinant proteins in mammalian cell lines. It contains a neomycin resistance gene for selection of transfected cells and a multiple cloning site for the insertion of the gene of interest.
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Lab products found in correlation
Pci neo vector, by Promega (2 mentions)
Dcas9 vp64 p65 rta construct19, by Addgene (2 mentions)
Sc 423402 act, by Santa Cruz Biotechnology (2 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Topo ta vector system, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Endofree plasmid mega kit, by Qiagen (1 mentions)
17 protocols using pci neo expression vector
1
Stable Expression of HAI-2/SPINT2 in Glioma Cells
2
Cloning and Mutagenesis of HAZV N and Claudins
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pcDNA3.1 vector (Invitrogen, Carlsbad, CA, United States) carrying HAZV N cDNA was previously described (Matsumoto et al., 2019 (link)). Construction of N mutants was performed by standard polymerase chain reaction (PCR) mutagenesis methods. HAZV N cDNA was also cloned into the episomal Epstein–Barr virus-based expression plasmid, pEBS-PL (Bontron et al., 1997 (link)). pcDNA3.1 vector carrying CLDN1 cDNA was previously described (Yumine et al., 2019 (link)). cDNA of CLDN7 was obtained from total RNA of HeLa cells by reverse transcription polymerase chain reaction (RT-PCR) as previously described (Ohta et al., 2018 (link)). CLDN mutants were constructed by a standard PCR mutagenesis method. cDNAs of CLDN1, CLDN7 and their mutants with 3xFLAG tag at their N-termini were cloned into the pCI neo expression vector (Promega, Madison, WI). These constructs were all confirmed by DNA sequencing.
3
Synthetic Transcriptional Activators for CRISPR Modulation
4
Overexpressing SOCS3 in Cell Lines
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The construct pCIneo-SOCS3 was made by subcloning the human SOCS3 cDNA amplified from 786-O cells into the pCIneo expression vector (Promega, WI) as described previously [24 (link)]. The empty vector was used as a negative control. For pCIneo-SOCS3 vector and empty vector transfection, Lipofectamine LTX (Invitrogen, CA) was used according to the manufacturer's protocol. The transfected cells were kept at 37°C in a 5% CO2 incubator for 24 h until treatment.
5
Jurkat Cell Transfection Protocol
6
SNF2L and SNF2H cDNA Cloning
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Human SNF2L cDNA comprising the entire open reading frame (+exon1/−exon13/+NLS) was amplified from a pcDNA3 expression plasmid64 (link) in a two-step process. The 5′ end of SNF2L cDNA was amplified with SNF2L-EcoRI-Fwd (5′-TAAGGAATTCATGGAGCA-3′) and SNF2L-XhoI-Rev-internal (5′-ACCTCTCGAGCTATGT-3′) primers, followed by purification of the PCR products, restriction digest by EcoRI and XhoI, and directional subcloning into the pBRIT-LoxP-NTAP and pBRIT-LoxP-CTAP retroviral plasmid vectors, which have been previously described65 (link). The remaining 3′ sequence of SNF2L was then subcloned non-directionally following amplification with SNF2L-XhoI-Fwd-internal (5′-ATAGCTCGAGAGGTAG-3′) and SNF2L-XhoI-Rev (5′-ATGCTCGAGGGATTTCACCTTCTTG-3′) primers and a restriction digest with XhoI. Similarly, SNF2H cDNA comprising the entire open reading frame was cloned into the pCI-neo expression vector (Promega Corp. Madison, WI, USA) then amplified with the SNF2H-BamHI-Fwd (5′-AAAAGGATCCATGTCGTCCGCGGCCGAGCC-3′) and SNF2H-XhoI-Rev (5′-AAAACTCGAGTAGTTTCAGCTTCTTTTTTCTTCC-3′) primers and directionally subcloned into the pBRIT-LoxP-NTAP and pBRIT-LoxP-CTAP vectors, following restriction digest with BamHI and XhoI. All clones were verified by sequencing at McGill University and Génome Québec Innovation Centre (Montreal, QC, Canada).
7
Cloning and Transfection of GRM3 Constructs
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The open reading frames for GRM3, GRM3Δ4, and GRM3Δ4 with a C-terminal V5 tag (GRM3Δ4-V5) were cloned into the pCI-neo expression vector (Promega E1841) using EcoR1 and XbaI restriction sites. This vector uses the human cytomegalovirus promoter to drive constitutive expression in mammalian cells. Constructs were sequenced and corrected by site-directed mutagenesis (Stratagene 200523) prior to use for transfection of human embryonic kidney (HEK293T/17) cells. This cell line was chosen since it does not express endogenous mGlu3, confirmed by reverse transcription polymerase chain reaction (data not shown).
8
Cloning and Expression of Human and Rat Gcom15
9
Expression vector construction: BTG1 cDNA
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cDNA encoding human BTG1 was generated by RT-PCR and subcloned into the EcoRI and MluI restriction sites of the pCI-neo expression vector (Promega Corporation, Madison, Wisconsin, USA). The plasmid sequence was confirmed using sequencing.
10
HIV-1 Gag Fusion Protein Expression
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The pGag-EGFP plasmid used, codes for a Rev independent HIV-1 Gag protein fused in frame to the enhanced GFP (Hermida-Matsumoto and Resh, 2000). The plasmid from the NIH AIDS Reagent Program (Cat 11468) was constructed by cloning the Gag sequence from pCMV55M1–10 (Schwartz et al, 1992) into the pEGFP-N1 plasmid (Clonthec, Mountain View, CA).
The coding sequence for the GH-RVG fusion protein was constructed inserting the G-H loop 63 bp sequence of FMDV A/Arg/2001 strain, between the RV signal peptide and the N-term extreme of the glycoprotein. This 1650 bp fragment was then cloned in pCiNeo expression vector (Promega, Madison, WI, USA), obtaining the pCiNeo- GH-RVG fusion protein plasmid.
The coding sequence for the GH-RVG fusion protein was constructed inserting the G-H loop 63 bp sequence of FMDV A/Arg/2001 strain, between the RV signal peptide and the N-term extreme of the glycoprotein. This 1650 bp fragment was then cloned in pCiNeo expression vector (Promega, Madison, WI, USA), obtaining the pCiNeo- GH-RVG fusion protein plasmid.
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