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Pmscvhyg

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The PMSCVhyg is a plasmid vector used for gene expression in mammalian cell lines. It contains a hygromycin resistance gene for selection of positive transformants.

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Lab products found in correlation

Pmscvpuro, by Takara Bio (2 mentions) Pegfp c1, by Takara Bio (1 mentions) Western lightning chemiluminescence ecl reagents, by PerkinElmer (1 mentions) Psiren luc luciferase shrna, by Takara Bio (1 mentions) Nucleobond pc 500 kit, by Macherey-Nagel (1 mentions) Pqcxip, by Takara Bio (1 mentions)

4 protocols using pmscvhyg

1

Retroviral Vector Construction for SERINC and CD4

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The human SERINC3 and SERINC5 genes preceded by a Kozak sequence were inserted into pMSCVhyg and pMSCVpuro, respectively (Clontech). The human CD4 gene was inserted into the retroviral vector pCXbsr53 (link).
Usami Y., Wu Y, & Göttlinger H.G. (2015). SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. Nature, 526(7572), 218-223.
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2

Isoform-specific p53 Protein Constructs

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The human p53 isoform constructs used in this report were previously described (Bourdon et al., 2005 (link)). They were sub-cloned into the EcoRI and BamHI sites of pEGFPC1 (Clonetech) to obtain GFP-tagged proteins or in the BamHI and EcoRI sites of pLPCmyc to obtain myc-tagged proteins. Constructs were amplified using the Nucleobond PC 500 kit (Macherey-Nagel) according to the manufacturer’s instructions. Human Δ133p53 isoforms (α, β and γ) were cloned into pMSCVhyg (Clontech Laboratories) plasmid for retrovirus production. pSIREN-Luc (luciferase)-shRNA was purchased from Clontech. The mouse anti-E-Cadherin (clone 36) and the mouse anti-Beta1-Integrin, were purchased from BD-transduction laboratories and were diluted at 1/400° and 1/250°, respectively. The mouse N-Cadherin (clone 32/BD Transduction laboratories), and the rabbit polyclonal anti-GFP (Invitrogen, Life Technologies, A-6455) were used at 1/400°, 1/250°, 1/300° and 1/2000°, respectively.
The sheep pantropic p53 antibody Sapu was diluted at 1/5000°. The rabbit polyclonal KJC8 antibody specific of the β p53 isoforms was diluted at 1/4000° (Bourdon et al., 2005 (link)). The Horse-Radish Peroxidase (HRP)-conjugated anti-IgG antibodies were purchased from GE-Healthcare and were diluted at 1/5000°. The Western Lightning Chemiluminescence (ECL) reagents were purchased from PerkinElmer.
Gadea G., Arsic N., Fernandes K., Diot A., Joruiz S.M., Abdallah S., Meuray V., Vinot S., Anguille C., Remenyi J., Khoury M.P., Quinlan P.R., Purdie C.A., Jordan L.B., Fuller-Pace F.V., de Toledo M., Cren M., Thompson A.M., Bourdon J.C, & Roux P. (2016). TP53 drives invasion through expression of its Δ133p53β variant. eLife, 5, e14734.
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3

Mitochondrial Dynamics Regulation Protocols

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All cell lines were cultured in DMEM containing 10% fetal bovine serum, 100 IU/ml streptomycin, and 100 μg/ml penicillin. Mff-null cell lines were previously published (Loson et al., 2013 (link)). Drp1-null cell lines were a generous gift from Katsuyoshi Mihara (Kyushu University, Fukuoka, Japan). Drp1-null cells lines stably expressing Drp1 and Drp1 mutants were generated by retroviral transduction of pQCXIP (Clontech) vectors with Kozak-Drp1 cloned into the BamHI/EcoRI sites and maintained in 0.5 μg/ml puromycin.
To generate the LAMP1 constructs, the Kozak sequence and FLAG-tag-Strep-tagII was fused to the rat Lamp1 sequence to generate a Kozak-LAMP1-FLAG construct, which was then fused to the N-terminus of Mff, MiD51, and MiD49. LAMP1-FLAG-Mff isoform 4 ΔTM (Δ272–291), LAMP1-FLAG-MiD51 (134–463), and LAMP1-FLAG-MiD49 (124–454) were cloned into the XhoI/HpaI sites of pMSCVhyg (Clontech), retrovirally transduced into Drp1-null cell lines expressing Drp1 or Drp1 mutants, and maintained in 0.5 μg/ml ­puromycin and 100 μg/ml hygromycin.
Liu R, & Chan D.C. (2015). The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1. Molecular Biology of the Cell, 26(24), 4466-4477.
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4

Retroviral Vector Construction for SERINC and CD4

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The human SERINC3 and SERINC5 genes preceded by a Kozak sequence were inserted into pMSCVhyg and pMSCVpuro, respectively (Clontech). The human CD4 gene was inserted into the retroviral vector pCXbsr53 (link).
Usami Y., Wu Y, & Göttlinger H.G. (2015). SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. Nature, 526(7572), 218-223.
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