To generate PB-TRE3G-OKS and PB-TRE3G-c-Myc, pTRE3G-IRES (Clontech) was amplified by PCR and cloned into PB-hCMV*1-cHApA [27] (link), [28] (link) to yield PB-TRE3G-cHApA, and then inserts from PB-TET-OKS [8] (link), [29] (link) and PB-TET-c-Myc were introduced into PB-TRE3G-cHApA. To generate PB-(CAG-Tet3G; EOS-C(3+)-EGFP-IRES-puro), inserts from pCMV-Tet3G (Clontech) and PB-EOS-C(3+)-EiP [8] (link), [9] (link) were cloned into PB-CAG-rtTA_Adv [8] (link), [29] (link). To generate PB-(CAG-TagRFP-IRES-hyg; Acr-EGFP), inserts from pAcr3-EGFP [11] (link) was introduced into empty PB vector, and the resultant vector (PB-Acr-EGFP) was amplified by PCR and introduced into PB-CAG-cHA-IRES-hyg to yield PB-(CAG-cHA-IRES-hyg; Acr-EGFP); next, an insert from pTagRFP-N1 (Evrogen) was introduced into PB-(CAG-cHA-IRES-hyg; Acr-EGFP). To generate pCAG-hyPBase, pCMV-hyPBase [18] (link) was cloned into blunt-ended pCAGGS [10] (link). Primer sequences are shown in Table S1 .
Ptre3g ires
Manufactured by Takara Bio
Sourced in United States
The PTRE3G-IRES is a plasmid vector designed for protein expression in mammalian cells. It contains an internal ribosome entry site (IRES) sequence that allows for the translation of two open reading frames from a single mRNA transcript. The vector also includes a selection marker for antibiotic resistance.
Automatically generated - may contain errors
2 protocols using ptre3g ires
1
Generating Plasmid Constructs for Inducible Gene Expression
2
Analyzing RUNX3 and β-Catenin Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
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To examine the intracellular RUNX3 and β-catenin levels, permeabilized cells were incubated with the primary antibodies for total β-catenin (Sigma) or RUNX3,(5 (link)) followed by the secondary antibodies for rabbit IgG-conjugated with Alexa 488 (Molecular Probes, Grand Island, NY, USA) or mouse IgG-conjugated with Alexa 633 (Invitrogen), and examined using FACS Canto II (BD Biosciences, San Jose, CA, USA). Cells were transfected with a pcDNA-RUNX3-IRES-mGFP expression vector, in which internal ribosome entry site (IRES) fragment from pTRE3G-IRES (Clontech Laboratories, Mountain View, CA, USA) and maxGFP cDNA from pmaxGFP (LONZA, Allendale, NJ, USA) were subcloned to pcDNA-Flag-RUNX3, and RUNX3-expressing cells were isolated using the FACS Aria cell sorter (BD Biosciences, San Jose, CA, USA) to collect GFP-expressing cells.
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