To generate a RhoGDI2 expression plasmid, the full-length coding region of RhoGDI2 cDNA was amplified using the primers RhoGDI2-forward CATACTCGAGCGG ACA GAG ACG TGAA GCAC and RhoGDI2-reverse CACTGGATCCGAGT GAC AGG GTG GGA AAAG (the restriction sites of XhoI and BamHI were italic) and inserted into pIRES2-EGFP (Clontech Laboratories, Mountain View, CA) at XhoI and BamHI sites. MKN-45 cells were transfected with pIRES2-EGFP-RhoGDI2 or pIRES2-EGFP using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Stable plasmid-transfected clones were selected using 800 μg/ml G418 (Invitrogen) for 2 weeks, isolated colonies were picked up with tips, and the cells were further cultured in the presence of 400 μg/ml G418. MKN-45 cells transfected with pIRES2-EGFP-RhoGDI2 were named MKN-45/RhoGDI2 cells. MKN-45 cells transfected with pIRES2-EGFP were named MKN-45/GFP cells.
Pires2 egfp
Manufactured by Thermo Fisher Scientific
Sourced in United States
PIRES2-EGFP is a fluorescent protein construct designed for use in laboratory experiments. It consists of the enhanced green fluorescent protein (EGFP) fused to the PIRES2 sequence, which allows for bicistronic expression. The core function of this product is to enable the simultaneous expression and visualization of two different proteins within the same cell.
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Lab products found in correlation
Pires2 egfp, by Takara Bio (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Facsvantage se, by BD (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Pcr xl topo, by Thermo Fisher Scientific (1 mentions)
Ix71 inverted epi fluorescent microscope, by Olympus (1 mentions)
7 protocols using pires2 egfp
1
Generation of RhoGDI2 Expression Plasmid
2
Functional Analysis of IGFBP3 in H1299 Cells
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IGFBP3 cDNA was generated by RT-PCR as described above. The PCR product was subcloned using the TOPO TA Cloning kit (Invitrogen). To express IGFBP3 cDNA insert of pCR®2.1-TOPO® was digested with EcoRI and cloned to the expression vector pIRES2-EGFP (Clontech). The DNA sequence of the inserts were analyzed using DNA Sequencing (ABI PRISM® 377).
H1299 cells were transfected with pIRES2-EGFP (Invitrogen) with or without IGFBP3 cDNA using EffecteneTM transfection reagent. After 72 h, cells were harvested and sorted by fluorescence cell sorting system (FACSVantage SE, Becton Dickinson). Then, 72 h after sorting the rate of EGFP positive cells was redetermined with flow cytometry. Lysates and supernatants of these cells were tested for IGFP3 with western blot and enzyme-linked immunosorbent assay (ELISA).
To determine the functionality of IGFBP3, supernatants of IGFBP3 positive cells were incubated (37 °C) with IGF-1 (2.5 ng/mL). After 2 h, ELISA was used to analyze these supernatants for free IGF-1.
H1299 cells were transfected with pIRES2-EGFP (Invitrogen) with or without IGFBP3 cDNA using EffecteneTM transfection reagent. After 72 h, cells were harvested and sorted by fluorescence cell sorting system (FACSVantage SE, Becton Dickinson). Then, 72 h after sorting the rate of EGFP positive cells was redetermined with flow cytometry. Lysates and supernatants of these cells were tested for IGFP3 with western blot and enzyme-linked immunosorbent assay (ELISA).
To determine the functionality of IGFBP3, supernatants of IGFBP3 positive cells were incubated (37 °C) with IGF-1 (2.5 ng/mL). After 2 h, ELISA was used to analyze these supernatants for free IGF-1.
3
Overexpression and Knockdown of TMEM196
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The full-length human TMEM196 gene cDNA was confirmed by sequencing and subcloned into the mammalian expression vector pIRES2-EGFP (Invitrogen, Carlsbad, CA, USA). Cells were transfected with the TMEM196 vector or empty vector using the X-treme Gene HP DNA transfection reagent (Roche, Mannheim, Germany). Overexpression of TMEM196 was confirmed by RT-PCR.
TMEM196 mRNA siRNAs and the negative control sequence were designed, synthesised, and subcloned into pcDNA6.2™ GW/EmGFP siRNA vectors (Invitrogen). The HBE cell line with TMEM196 expression was transfected with vectors carrying siRNA-TMEM196 or the siRNA negative control. RNA was extracted 48 h after transfection and the siRNA producing the greatest TMEM196 knockdown was used to assess cell function.
TMEM196 mRNA siRNAs and the negative control sequence were designed, synthesised, and subcloned into pcDNA6.2™ GW/EmGFP siRNA vectors (Invitrogen). The HBE cell line with TMEM196 expression was transfected with vectors carrying siRNA-TMEM196 or the siRNA negative control. RNA was extracted 48 h after transfection and the siRNA producing the greatest TMEM196 knockdown was used to assess cell function.
4
Cloning and Expression of Hes-1 Gene
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The PIRES2-EGFP and PCR-XL-TOPO vectors (containing Hes-1, which was assembled with chemically synthesized oligos through PCR) were purchased from Invitrogen Corporation. The fragment of EcoRI-Hes-1-IRESEGFP-XhoI was amplified using the templates of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. EcoRI-Hes-1-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested with EcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent Escherichia coli DH5a cells for further amplification and use. The recombinant pcDNA3.1-Hes-1 plasmids were verified by sequencing and transfected using Lipofectamine 3000 transfection reagent, according to the manufacturer’s instructions. Stable transformants were selected for 4 weeks and isolated by a single cell manipulation technique.
5
Overexpression of Foxa2 and LXR-alpha
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The PIRES2-EGFP and PCR-XL-TOPO vectors (containing Foxa2 or LXRα which was assembled by the chemically synthesized oligos through PCR) were purchased from Invitrogen. Segments of EcoRI-Foxa2 or LXRα and IRES-EGFP-XhoI were amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. EcoRI-Foxa2-IRES-EGFP-XhoI or EcoRI- LXRα-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested by EcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent E.coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-Foxa2 and pcDNA3.1-LXRα. The plasmid transfection process was performed using Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.
6
Generation of Chimeric Antigen Receptors
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The human genes encoding CD7 and hIL-2 were obtained from RIKEN gene bank. The CAR genes were cloned into pcDNA3.1(−) or pIRES2-EGFP, the mammalian expression vector (Thermo Fisher Scientific, Waltham, MA, USA; Figure 1A ). The series of CAR genes with CD3ζ and with or without CD7/CD8α as a spacer/hinge, or CD28 as a secondary signal, were designated mCR-0, mCR-1, mCR-2, and mCR-3, respectively, as shown in Figure 1B . The cloning of mCR-2 and hCR-2 genes into pcDNA3.1(−) has been described previously (F39scFv/CIR-2 and L45scFv-CIR, respectively).23 (link),26 (link) The hCR-2 in pIRES-EGFP was generated by digesting mCR-2/pcDNA3.1(−) with BglII/BamHI. The purified fragment was then cloned into the pIRES-EGFP vector. The antibody-cytokine fusion protein, scFv-IL2, was constructed by splice-overlap extension (SOE)-polymerase chain reaction (PCR) using appropriate primers (Figure 2A and B and Table 1 ). The purified fragment was digested with NheI/HindIII and cloned into the pBAD/gIII Escherichia coli expression vector. The integrity of all plasmid constructs was confirmed with restriction digestion and DNA sequencing.
7
Generation of Myc-BirA*-Txnip cell line
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Myc-BirA∗-Txnip was generated by first generating Myc-BirA∗ by PCR from the pcDNA3.1 mycBioID plasmid [20 (link)]. PCR products were digested and ligated into pIRES2-EGFP (Clontech). Next, pCR4-TOPO-Txnip (Thermo Fisher Scientific) was used as template to amplify Txnip which was ligated in frame with Myc-BirA∗ in pIRES2-EGFP. Subsequently, Myc-BirA∗-TxnipC247S was generated by site-directed mutagenesis (Agilent Technologies). Plasmids were linearized and transfected into HEK293 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were selected in 200 μg/mL hygromycin and stable clones were selected based on EGFP fluorescence visualized with an Olympus IX71 inverted epifluorescent microscope.
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