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Pcdh cmv mcs ef1 puro lentiviral vector

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The PCDH-CMV-MCS-EF1-Puro lentiviral vector is a lentiviral expression vector designed for the stable transduction and expression of genes of interest in mammalian cells. The vector includes a CMV promoter for strong and constitutive expression, a multiple cloning site for insertion of the gene of interest, and a puromycin resistance gene for selection of transduced cells.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions) Polybrene, by Merck Group (4 mentions) Plko 1 puro vector, by Addgene (3 mentions) Egfp c2, by Takara Bio (3 mentions) Puromycin, by Merck Group (3 mentions) Ad cre ires gfp, by Vector Biolabs (3 mentions) Ad gfp, by Vector Biolabs (3 mentions)

12 protocols using pcdh cmv mcs ef1 puro lentiviral vector

1

Lentiviral Myc-SYK and NLyn-SYK Constructs

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The Myc-SYK construct was generated as previously described [28 (link)]. Myc-SYK and Myc-epitope tag cDNAs were subcloned into the pLL-1 vector (Active Motif, Carlsbad, CA, USA) to generate Myc-SYK-eDHFR and Myc-eDHFR constructs. These were then further subcloned into the pCDH-CMV-MCS-EF1-Puro lentiviral vector (System Biosciences, Palo Alto, CA, USA). NLyn-SYK-eDHFR was generated by cloning SYK with a forward primer coding for the first five N-terminal residues of LYN and inserting the construct into the pLL-1 vector.
The following antibodies were used for Western blotting and/or immunofluorescence: SYK (D3Z1E XP, Cell Signaling Technology, Danvers, MA, USA), Myc (9B11, Cell Signaling Technology, Danvers, MA, USA), G3BP (611126, BD Biosciences, San Jose, CA, USA) and GAPDH (AM4300, Ambion, Austin, TX, USA). HRP-coupled secondary antibodies were purchased from Pierce and AlexaFluor 405- and 594-coupled secondary antibodies from Invitrogen (Carlsbad, CA, USA).
Westbroek M.L, & Geahlen R.L. (2017). Modulation of BCR Signaling by the Induced Dimerization of Receptor-Associated SYK. Antibodies, 6(4), 23.
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2

Cloning and Tagging of Spastin and NA14

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Complementary DNAs comprising the coding sequences of human M1 and M87 spastin (GenBank NM_014946, including the exon 4 splice cassette) were cloned into the mammalian expression vector pGW1-Myc, which contains an N-terminal Myc-epitope tag [11] (link), [28] (link). Human full-length NA14 (GenBank NP_003722.1) and a Δ1–32 amino acid deletion form were cloned into the EcoRI site of pGW1-HA, which harbors an N-terminal hemagglutinin (HA)-epitope tag. For producing lentivirus, wild-type NA14 cDNA was cloned into the pCDH-CMV-MCS-EF1-Puro lentiviral vector (Systems Biosciences, Mountain View, CA, USA); sequences encoding HA and EGFP epitopes were cloned into XbaI and EcoRI sites. To establish NA14-depleted cell lines, shRNA sequences specific for NA14 or else a control sequence (CTL) were cloned into the pLKO.1 Puro vector (Addgene, Cambridge, MA, USA): shCTL, 5′-GCAATCGAAGCTCGGCTAC-3′; shNA14-1, 5′-ACAACAACGAGCTGGTCAA-3′; shNA14-2, 5′-AGGAGGAGGAGGACGAGAA-3′; and shNA14-3, 5′-AGGAGGAGGACGAGAAGCA-3′. All constructs were confirmed by DNA sequencing.
Goyal U., Renvoisé B., Chang J, & Blackstone C. (2014). Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development. PLoS ONE, 9(11), e112428.
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3

Generation of Insulin Receptor Cell Lines

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Human wild type and S323L low-ligand binding mutant insulin receptor (IR) expression vectors were kindly provided by Jonathan Whittaker PhD (Case Western Reserve University, Cleveland, OH). Wild type and S323L IR open reading frames were cloned into pCDH-CMV-MCS-EF1-Puro lentiviral vector (System Biosciences). Lentivirus particles were generated by following the manufacturer’s recommendation (Open Biosystems). Lentiviral particles for murine CENP-A short hairpin RNA (shRNA) (sc-37556-v), murine PLK1 shRNA (sc-36278-v), and control scramble shRNA (sc-108080) were purchased from Santa Cruz. Cells were infected by adding the lentiviral particles to the culture with polybrene (sc-134220). For generating stable cell lines, cells were treated with 4 mg/mL of puromycin 48 hours after the transduction and were maintained in selection media for more than 14 days. We generated two separate stable cell lines in each group.
Shirakawa J., Fernandez M., Takatani T., El Ouaamari A., Jungtrakoon P., Okawa E.R., Zhang W., Yi P., Doria A, & Kulkarni R.N. (2017). Insulin signaling regulates the FoxM1/PLK1/CENP-A pathway to promote adaptive pancreatic β-cell proliferation. Cell metabolism, 25(4), 868-882.e5.
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4

Lentiviral Expression of LANA Mutants

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The pCDHCMV-MCS-EF1-puro lentiviral vector (System Biosciences) was used to express WT and mutant forms of LANA. The LANA mutants were based on studies of Dr. Kenneth Kaye at the Harvard Medical School [28 (link)]. Virus-containing supernatants from 293T cells transfected by the lentiviral vector and packaging vectors were collected 60 hours post-transfection, concentrated by centrifugation (24000 rpm, 1.5 hr, 4°C) and used for infection of cells in the presence of 8 μg/ml polybrene. The next day the infected cells were split, and then infected with KSHV the following day.
Toth Z., Papp B., Brulois K., Choi Y.J., Gao S.J, & Jung J.U. (2016). LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection. PLoS Pathogens, 12(9), e1005878.
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5

Lentiviral Transduction of Rat SLC1A3

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The coding sequence of rat SLC1A3 (GenBank accession number NM_019225.2) was cloned into the XbaI/EcoRI sites of pCDHCMV-MCS-EF1-puro lentiviral vector (System Biosciences, Palo Alto, CA) by PCR amplification to generate expression vectors named pCDH-puro-SLC1A3. The primer sequences used for cloning are listed in Table 1. The construct was confirmed by direct DNA sequencing. Lentivirus-containing supernatants were collected and used for transduction as previously described (24 (link)). MM-vector, KMM-vector, MM-SLC1A3, and KMM-SLC1A3 cells were selected with 2 μg/ml puromycin after transduction.
Zhu Y., Li T., Ramos da Silva S., Lee J.J., Lu C., Eoh H., Jung J.U, & Gao S.J. (2017). A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus. mBio, 8(4), e01179-17.
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6

Lentiviral Overexpression of dCK in Pancreatic Cancer Cells

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To overexpress dCK in PANC‐1 and MIA PaCa‐2 cells, a lentivirus‐mediated transfection method was used. The dCK coding sequence was constructed into the pCDH‐CMV‐MCS‐EF1‐Puro lentiviral vector (System Biosciences, Palo Alto, CA, USA). Lentivirus was produced by co‐transfecting dCK‐overexpressing constructs with psPAX2 and pMD2.G vectors at a ratio of 4:3:1 into HEK293T cells. Stable dCK‐overexpressing cell lines were obtained by infecting PANC‐1 and MIA PaCa‐2 cells and subsequent puromycin selection.
Hu Q., Qin Y., Xiang J., Liu W., Xu W., Sun Q., Ji S., Liu J., Zhang Z., Ni Q., Xu J., Yu X, & Zhang B. (2018). dCK negatively regulates the NRF2/ARE axis and ROS production in pancreatic cancer. Cell Proliferation, 51(4), e12456.
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7

Constructing pGFAP-CreERT2 Lentiviral Vector

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To construct pCDH-pGFAP-MCS-EF1-puro, the CMV promoter sequence of the pCDH-CMV-MCS-EF1-puro lentiviral vector (System Biosciences, USA) was removed by using two restriction enzymes, SnaBI and NheI. The pGFAP promoter sequence (−1,817 to −17 upstream from the pGFAP transcription start site +1) in the pGL3-basic-pGFAP promoter plasmid [8 (link)] was digested with EcoRI (blunted by the Klenow fragment) and NheI. Subsequently, the pGFAP promoter sequence was ligated into the aforementioned CMV promoter-deleted lentiviral vector. Finally, to construct a lentiviral vector containing the pGFAP-CreERT2 gene construct, the CreERT2 gene digested by EcoRI from the pCre-ERT2 plasmid [19 (link)] was inserted into the identical restriction enzyme sites of pCDH-pGFAP-MCS-EF1-puro.
To generate the CMV-LoxP-EGFP-pA-LoxP construct, LCMV:EGFP(LoxP)MCS (No. 31377; Addgene, USA) was modified by removing the EGFP sequence by using NcoI and BsrGI. Then, the EGFP sequence from EGFP-C2 (No. 6083-1; Clontech Laboratories, USA) that had been digested with the same restriction enzymes was inserted into the aforementioned EGFP-deleted vector.
Hwang S.U., Eun K., Yoon J.D., Kim H, & Hyun S.H. (2018). Production of transgenic pigs using a pGFAP-CreERT2/EGFPLoxP inducible system for central nervous system disease models. Journal of Veterinary Science, 19(3), 434-445.
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8

ITGB1 Gene Overexpression in TCs

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The full-length cDNA of the ITGB1 gene was PCR-amplified from TCs and cloned into the pCDH-CMV-MCS-EF1-Puro lentiviral vector (System Biosciences, Mountain View, CA, USA). The plasmids were confirmed by sequencing. Lentiviruses carrying ITGB1 were produced in HEK293T cells co-transfected with pCDH-ITGB1, pMD2. G, and pSPAX.2 plasmids using Lipofectamine 3000 (Invitrogen). Viral supernatant was collected after 48 h and transduced into TCs with 4 μg/ml polybrene (Sigma-Aldrich). TCs were selected using 2 μg/ml puromycin (Sigma-Aldrich) for 48 h.
Qi R., Wang Y., Yan F, & Zhong J. (2024). Exosomes derived from ITGB1 modified Telocytes alleviates LPS-induced inflammation and oxidative stress through YAP1/ROS axis. Heliyon, 10(5), e27086.
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9

Lentiviral Transduction of SPINT2 cDNA

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A plasmid containing full-length human SPINT2 cDNA was purchase from Open Biosystems (USA). The cDNA was subcloned into the pCDH-CMV-MCS-EF1-Puro lentiviral vector (System Biosciences, USA). Empty vector was used as a negative control. Once sequence-verified, the lentiviral expression vectors were cotransfected with the lenti-packaging plasmids (Addgene, USA) into HEK293FT cells, and the pseudovirus-containing supernatant was collected 48 h post-transfection. Virus supernatant was used to infect U87G cells cultured in 6-well plate. Transduced cells were then selected with puromycin (1 μg/mL) to establish a stable cell line.
Lee E.J., Rath P., Liu J., Ryu D., Pei L., Noonepalle S.K., Shull A.Y., Feng Q., Litofsky N.S., Miller D.C., Anthony D.C., Kirk M.D., Laterra J., Deng L., Xin H.B., Wang X., Choi J.H, & Shi H. (2015). Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells. Journal of genetics and genomics = Yi chuan xue bao, 42(7), 355-371.
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10

Adenovirus-Mediated Cre Recombinase Delivery

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Pre-packaged adenovirus containing an internal ribosome entry site (IRES) and expressing Cre recombinase and green fluorescent protein (GFP; Ad-Cre-IRES-GFP and Ad-GFP) were purchased from Vector Biolabs (Malvern, PA, USA). Human wild-type IR open reading frames (kindly provided by J. Whittaker, Case Western Reserve University, Cleveland, OH, USA) were cloned into the pCDH-CMV-MCS-EF1-Puro lentiviral vector (System Biosciences, Palo Alto, CA, USA). See ESM Methods for further details, and ESM Table 2 for primers.
Kawamori D., Shirakawa J., Liew C.W., Hu J., Morioka T., Duttaroy A., Burkey B, & Kulkarni R.N. (2017). GLP-1 signalling compensates for impaired insulin signalling in regulating beta cell proliferation in βIRKO mice. Diabetologia, 60(8), 1442-1453.
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