Human GLP1R gene was PCR-amplified from the human brain cDNA library (BioChain, Newark, CA, USA) using DNA polymerase KOD-Plus-Neo (Toyobo) with primers 3 and 4 (Table 1 ). The DNA fragment coding human GLP1R was inserted into pIRES (Clontech) digested with EcoRV and BamHI by using In-Fusion (Clontech), resulting in pIRES-hGLP1R. CHO cells were transfected with pIRES-hGLP1R using Xfect (Clontech), and then, selected with G418 for about 2 weeks to construct a stable cell line producing hGLP1R. Single cell cloning of the resistant cells was conducted by limiting dilution, resulting in GLP1R-CHO.
Pires
Manufactured by Takara Bio
Sourced in United States
PIRES is a versatile plasmid-based expression system developed by Takara Bio. It facilitates the efficient expression of recombinant proteins in a wide range of host cells, including E. coli, yeast, and mammalian cells. The core function of PIRES is to enable the simultaneous expression of two genes of interest from a single promoter.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pad pl dest, by Thermo Fisher Scientific (2 mentions)
Virapower adenoviral expression system, by Thermo Fisher Scientific (2 mentions)
Pdrive, by Qiagen (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Pgl3 basic vector, by Takara Bio (1 mentions)
Pcmv lacz vector, by Takara Bio (1 mentions)
Pcat3 basic vector, by Promega (1 mentions)
Cat elisa kit, by Roche (1 mentions)
Pcdna3, by Takara Bio (1 mentions)
Plasmid mega kit, by Qiagen (1 mentions)
Pgp cmv gcamp6s, by Addgene (1 mentions)
Pmir report luciferase vector, by Thermo Fisher Scientific (1 mentions)
Xfect, by Takara Bio (1 mentions)
In fusion, by Takara Bio (1 mentions)
Kod plus neo, by Toyobo (1 mentions)
Pcr2.1 topo ta cloning vector, by Thermo Fisher Scientific (1 mentions)
Nucleospin plasmid kit, by Macherey-Nagel (1 mentions)
21 protocols using pires
1
Constructing Stable Cell Line Expressing Human GLP1R
2
Construction and Validation of Plasmid Vectors
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All plasmids used as PCR templates in this study are listed in Table 1 . The pIRES-DHFR plasmid is a modified version of the IRES domain of the plasmid pIRES (Clontech, Palo Alto, CA, USA), which was linked to the dihydrofolate reductase (DHFR) gene in house. Oligonucleotides for PCR are listed in Table S1 and oligonucleotides for DNA hybridization of building blocks are listed in Table S2 . Plasmids generated in this study are found in Table S3 .
3
Quantifying ϕX174 DNA Replication
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The ϕX174 dsDNA replicative form was quantified using the Quant-iT PicoGreen dsDNA broad range assay kit (Life technologies), and all extracts were brought to the same concentration (30 ng/µl). DNA from each virus was split into three aliquots, which were treated with XhoI to linearize the genome, with XhoI and DpnI to digest methylated GATCs, or with XhoI and MboI (i.e., DpnII) to digest non-methylated GATCs. Double digestions were performed according to the manufacturer instructions (Thermoscientific). A standard plasmid (pIRES, Clontech) was used as a digestion control (not shown). A monochrome picture of the gel was transformed to an eight-bit image, and the pixel area and intensity of each band were quantified using ImageJ.
4
Cloning and Transfection of Murine HDAC11 and Mmp3
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The full-length CDS of murine HDAC11 (NM_144919) or Mmp3 (NM_010809) was amplified by a PCR with murine colorectal tissue cDNA as template. The primers used were 5'-ATCGATATCATGCCTCACGCAACAC-3', 5'-TATGGATCCTCAAGGCACAGCACAG-3' and 5'-ATCGATATCATGAAAATGAAGGGTC-3', 5'-TATGGATCCTTAACAATTAAACCA-3'. The PCR cycles were as follows: 95 °C for 5 min followed by 30 cycles of 95 °C for 30 s, 56 °C for 30 s, and followed by a final extension at 72 °C for 2 min. After that, the PCR fragment was purified and was subcloned into the mammalian expression vector pIRES (Clontech, CA). The vectors were named pIRESneo3-HDAC11and pIRESneo3-Mmp3 for gene transfection. Plasmids were transfected into CRC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
5
Notch1 and Jagged1 cDNA Overexpression
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Full-length Notch1 and Jagged1 cDNA lacking the 3′-untranslated region (UTR) were purchased from GeneCopoeia (Rockville, MD, USA) and subcloned into the eukaryotic expression vector pIRES (Clontech Laboratories, Inc., Mountain View, CA, USA). The empty pIRES vector was used as a negative control. Rabbit monoclonal anti-Notch1 (cat no. EP1238Y) and anti-Jagged1 (cat no. EPR4290) were purchased from Epitomics (Abcam, Cambridge, UK), mouse monoclonal anti-GAPDH (cat no. MAB374) was purchased from EMD Millipore (Billerica, MA, USA), and mouse monoclonal anti-vimentin (cat no. sc-6260), anti-fibronectin (cat no. C6F10) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat no. sc-2004) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Synthetic miR-34a mimic and negative control (NC) were purchased from GenePharma Co., Ltd. (Shanghai, China). The miR-34a LNA 5′-digoxigenin (DIG)-labeled (cat no. 612537-340) and scrambled LNA 5′-DIG-labeled probes (cat no. 699004-340) were purchased from Exiqon A/S (Vedbaek, Denmark). The probe sequences are as follows: miR-34a, AGG GCA GTA TAC TTG CTG AT; and scramble, GTG TAA CAC GTC TAT ACG CCC A. The probes were detected using an enhanced sensitive ISH detection kit (cat no. MK1030; Wuhan Boster Biological Technology, Ltd., Wuhan, China).
6
Shh Expression Construct Generation
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Shh expression constructs were generated from murine cDNA (NM_009170 ; nucleotides 1–1314, corresponding to amino acids 1–438; for ShhN, nucleotides 1–594 were used, corresponding to amino acids 1–198) and human hedgehog acyltransferase (Hhat) cDNA (NM_018194 ). Both cDNAs were cloned into pIRES (Clontech) for their coupled expression from bicistronic mRNA to achieve near-quantitative Shh palmitoylation. Unlipidated C25SShhN cDNA and non-palmitoylated C25AShh cDNA (amino acids 1–438, the alanine modification completely blocks Shh N-palmitoylation, while the serine modification reduces it; Hardy and Resh, 2012 (link)) were generated by site-directed mutagenesis (Stratagene) and inserted into pcDNA3.1 (Invitrogen). Primer sequences are provided in Table S3 . Human Scube2 constructs were a kind gift from Ruey-Bing Yang (Academia Sinica, Taiwan). Murine V5- and C-terminal HA-tagged Disptg inserted into pcDNA3.1 was a kind gift from Stacey Ogden (St. Jude Children's Research Hospital, Memphis, USA). Murine DispΔL2 was generated from Disptg by deletion of the second extracellular loop (L2) between TM domain 7 and 8 (amino acids 752–972). Murine PtcΔL2 was generated from Ptc full length (pcDNA-h-mmPtch1-FL; Addgene 120889 ) by deletion of the second extracellular loop (L2) between TM domain 7 and 8 (amino acids 794–997).
7
Generating Stable Luc-pIRES-KLK3 Cell Line
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Eukaryotic expression vector pIRES and pGL3-basic vector were purchased from Clontech Laboratories, Inc. (Mountain View, USA). PCR primers were designed according to the GenBank expression of human PSA protein (KLK3) sequences (GI:22208990). Luciferase gene sequences (GI:13195703) were obtained from pGL3-basic vector by endonuclease digestion. Using reverse transcriptase PCR (RT-PCR) and PCR amplification technologies, a Luc-pIRES-KLK3 plasmid was constructed (Figure 1A ) for transfectioninto RM9 cells for stable expression in monoclonal strains; G418 was successfully used to filter out the RM9-Luc-pIRES-KLK3 monoclonal cell line. The Cell Proliferation assay showed that potential proliferation was the same between the RM9-Luc-pIRES-KLK3 and normal RM9 subsets by The Cell Proliferation Kit II (XTT, Roche, Basel, Switzerland).
8
Bicistronic Vector Construction and Validation
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The EMCV-IRES-containing bicistronic vector, pβGal-IRES-CAT, was constructed by inserting the β-galactosidase (β-gal) gene from pCMV-LacZ Vector (Clontech, Mountain View, CA, USA) and the chloramphenicol acetyltransferase (CAT) gene from pCAT3-Basic Vector (Promega) into multi-cloning sites A and B of pIRES (Clontech), respectively. The control vector, pβGal-CAT, was obtained by removing the IRES region from the pβGal-IRES-CAT by inverse PCR. The FLAG repeat-containing vector, pβGal-4× FLAG-CAT, was constructed by inserting the 4× FLAG fragment (obtained from a vector which was synthesised at Integrated DNA Technologies Inc.) into the linker region of pβGal-CAT. The expression of the bicistronic mRNAs was driven by the cytomegalovirus (CMV) promoter. Nucleic acid sequences of vector inserts were confirmed by sequencing. HeLa cells were plated in a 6-well plate (4 × 105 cells/well, 2 mL) one day before transfection. The cells were transfected with these plasmids (2.5 μg each/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection the cells were harvested and assessed for expression of β-gal and CAT by enzyme-linked immunosorbent assays (ELISA) using β-Gal ELISA and CAT ELISA kits (Roche Applied Science, Mannheim, Germany), respectively.
9
Protein Substrate Tagging and Expression
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Protein substrates were derived from human Rb, ODC, and E. coli DHFR, and fluorescence proteins used in this study were RFP (DsRed-Express2) and YFP (Venus for mammalian expression and sYFP2 for yeast expression). Venus was attached to the C-terminal end of Rb by the linker sequence LESGSGKPGS. N-terminal UBL domains from S. cerevisiae Rad23 or human HR23B, N-end rule degrons or B. amyloliquefaciens barstar were connected to sYFP2 or DHFR by the linker sequence VDGGSGGGS. C-terminal tails were attached through a 2-amino acid linker (Pro-Arg). The amino acid sequences of the tails are provided in Supplementary Table 1 . The coding sequences were cloned into pcDNA3 or pIRES (Clontech) for culture cell transfection, into CEN plasmid YCplac33 for expression in yeast, or into pE3a (Novagen) for expression in vitro. The sequences of the primers used are given in Supplementary Table 3 .
10
Construction of Reporter Gene Constructs
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pIRES (Clontech, Mountain View, CA, USA), a parental eukaryotic expression vector used for hybrid construction, served as a control plasmid in this study. To generate the mono- or bi-cistronic reporter constructs pEGFPIRESφ, pφIRESEGFP, pRlucIRESφ, pφIRESRluc, pRlucIRESFluc, pFlucIRESRluc, pIL2IRESφ, pφIRESIL2, pIL2IRESIL4, and pIL4IRESIL2, the EGFP, Rluc, Fluc, IL-2, and IL-4 reporter gene coding regions were cloned into the pIRES vector using the appropriate restriction enzyme sites. In the nomination of constructs, the symbol, φ, indicates the multiple cloning sites without gene inserted. The pIRES used in this study is a “crippled” IRES in order to reduce IRES expression and increase expression of the upstream cap-dependent open reading frame [31 (link),32 (link)]. Plasmid DNA was purified from a transformed Escherichia coli (strain DH5α) using endotoxin-free Qiagen Plasmid Mega Kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The plasmid DNA was then stored at −80 °C as dry pellets before use. The plasmid DNA was reconstituted in sterile water at a concentration of 1 mg/mL prior to use in the transfection experiment [33 (link)]. One μg plasmid DNA was used for transfection.
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