Psiren shuttle vector

Manufactured by Takara Bio

Sourced in United States

The PSIREN-shuttle vector is a lab equipment product designed for gene expression and protein production. It serves as a tool for the cloning and expression of target genes in various cell lines.

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Lab products found in correlation

Mission shrna lentiviral particles, by Merck Group (1 mentions) Geneticin, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Facsaria 2, by BD (1 mentions) Easysep human cd4 t cell enrichment kit, by STEMCELL (1 mentions)

3 protocols using psiren shuttle vector

Constructing psiRNA Vectors with U6 and H1 Promoters

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The U6 promoter was amplified by PCR using the pSIREN-shuttle vector (Clontech Laboratories, Mountain View, CA, USA) as the template and the following primers:

Forward: 5′-GCGCTATCGATGGAAGAGGCTATTTCCCA-3′

Reverse: 5′-GCGGAGGTACCGTCCTTTCCACAAGATAT-3′.

These amplicons were digested with Acc651 (isoschisomer of KpnI) and ClaI and then ligated into Acc651- and ClaI-digested psiRNA-7SKGFP::Zeo (InvivoGen, San Diego, CA, USA) plasmid to create psiRNA-U6GFP::Zeo. The DNA inserts coding for the shRNAs were generated by annealing complementary oligonucleotides as described previously.¹⁷ (link)^,⁵⁰ (link) These DNA inserts were then ligated into BbsI-digested psiRNA-7SKGFP::Zeo, as well as into BbsI-digested psiRNA-U6GFP::Zeo. Plasmids containing the H1 promoter were constructed in a previous study.¹⁷ (link)

Goguen R.P., Del Corpo O., Malard C.M., Daher A., Alpuche-Lazcano S.P., Chen M.J., Scarborough R.J, & Gatignol A. (2021). Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters. Molecular Therapy. Nucleic Acids, 23, 1020-1034.

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Lentiviral-Mediated CD74 Knockdown

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To knock-down CD74 expression, BJAB and Raji cells were transduced using MISSION shRNA lentiviral particles targeting CD74 or non-target (NT) controls, according to manufacturer’s protocol (Sigma-Aldrich), and kept under selection with 1.9 μg/mL of puromycin.
The shRNA sequences from MISSION shRNA lentiviral particles were inserted between BamHI and EcoRI restriction sites in the multiple cloning cassette of pSIREN-Shuttle vector (Clontech). The U6 promoter-shRNA cassettes were then recloned to KpnI and ApaI sites of the pEPI-1 vector kindly provided by Dr. A. C. Jenke (HELIOS Children's Hospital Wuppertal, Germany) [28 (link)]. The correct sequences of inserted shRNAs were confirmed by sequencing (SeqWright).
Obtained plasmids were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s recommendations. Transfected cells were sorted 48 h post transfection based on green fluorescent protein (GFP) expression, using a flow-activated cell sorter BD FACSAria II (BD Biosciences) followed by limited dilutions and selection with geneticin (1350 μg/mL; Invitrogen) for 2 weeks.

Berkova Z., Wang S., Ao X., Wise J.F., Braun F.K., Rezaeian A.H., Sehgal L., Goldenberg D.M, & Samaniego F. (2014). CD74 interferes with the expression of fas receptor on the surface of lymphoma cells. Journal of Experimental & Clinical Cancer Research : CR, 33(1), 80.

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Silencing Ets1 and Runx1 in P5424 Cells

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Previously reported shRNA target sequences for Ets1 and Runx1 (36 (link), 37 (link)) were cloned into the pSIREN shuttle vector (Clontech), excised as BglII-MluI fragments, and inserted into pcDNA3.1 containing a truncated human CD4 cDNA (38 (link)). P5424 cells were cultured as described (39 (link)) and were transfected by electroporation (Biorad, 250 V/960 μF ) with control (scrambled GFP 5′-AAGCTGGAGTACAACTACA-3′), ETS1-specific (target sequence 5′-GCAGACAGACTACTTTGCCAT-3′), or RUNX1-specific shRNA vectors (target sequence 5′-GCCCTCCTACCATCTATACTA-3′). Transfected cells were purified 48 hours post-transfection with an EasySep Human CD4+ T Cell Enrichment Kit (Stem Cell Technologies #19052).

Zhao J.Y., Osipovich O., Koues O.I., Majumder K, & Oltz E.M. (2017). Activation of Mouse Tcrb: Uncoupling RUNX1 Function from its Cooperative Binding with ETS1. Journal of immunology (Baltimore, Md. : 1950), 199(3), 1131-1141.

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