A PER2 promoter fragment spanning nucleotides −500 to +50 upstream of the transcription start site was synthesized from human genomic DNA (Promega) via PCR with the primers 5′-ggG cgt agt gaa tgg aag gcg-3′ and 5′-cag cag ccc aag gaa ctt-3′. The PCR products were ligated into a T&A vector (RBC Bioscience, Taipei County, Taiwan), followed by enzyme digestion with KpnI and BglII and ligation into the corresponding sites of the pGL4.2(luc2P/minP/Hygro) vector (Promega), yielding the pPER2:luc2P bioluminescence reporter. HaCaT cells were transfected with pPER2:luc2P using the Nucleofector electroporation kit (Amaxa, Inc., Köln, Germany), followed by selection with 10 μg/mL hygromycin (Sigma-Aldrich). After four weeks, a stable bioluminescence reporter cell line (HaCaT/PER2:luc2P) was obtained.
T a vector
Manufactured by RBC Bioscience
The T&A vector is a plasmid-based genetic engineering tool used for the insertion and expression of target genes in various host organisms. The core function of the T&A vector is to provide a versatile platform for cloning and expressing recombinant proteins. It includes components necessary for DNA replication, selection, and transcription/translation initiation.
Automatically generated - may contain errors
Lab products found in correlation
Human genomic dna, by Promega (5 mentions)
Pgl4 basic vector, by Promega (3 mentions)
Tcs sp5, by Leica (2 mentions)
Hygromycin, by Merck Group (1 mentions)
Ezchange site directed mutagenesis kit, by Enzynomics (1 mentions)
Mouse genomic dna, by Promega (1 mentions)
Pgl3 basic, by Promega (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Omniscript rt kit, by Qiagen (1 mentions)
Dna sequencing, by Macrogen (1 mentions)
10 protocols using t a vector
1
Generating PER2 Bioluminescence Reporter
2
Construction and Mutation of CXCR3 Promoter Luciferase Reporters
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The methods used for the construction of the CXCL10 gene promoter-driving luciferase reporter, wild-type pCXCL10-Luc(−250/+8)) and disruption of the NF-κB-binding site by site-directed mutagenesis, pCXCR3-Luc(−178/+22)mtNF-κB, are described elsewhere [26 (link)].
For the generation of CXCR3 gene promoter-driving luciferase reporter constructs, the CXCR3 promoter fragment spanning nucleotides −178 to +22 upstream of the transcription start site was synthesized from human genomic DNA (Promega, Madison, WI, USA) via PCR using the primers 5′-ggtaccCATCCTCTGCCAGCTTTTCT-3′ (forward primer) and 5′-agatctCTTTGGTGCTTGTGGTTGGA-3′ (reverse primer). Small letters indicate inserted KpnI and BglII restriction sites. The PCR products ligated into a T&A vector (RBC Bioscience, Taipei county, Taiwan) were digested by KpnI and BglII and cloned into the KpnI and BglII sites of the pGL4-basic vector (Promega), yielding pCXCR3-Luc(−178/+22). Site-specific mutation of two NF-κB binding sites, mtNF-κB(I) and mtNF-κB(II), was performed using an EZchange Site-directed Mutagenesis Kit (Enzynomics, Daejeon, Republic of Korea), using the −178/+22 construct as a template plasmid. Primer sequences used to generate point mutations were asTable 2 .
RT-PCR was carried out by annealing at 55 °C for 25 cycles. The point mutation was verified by DNA sequencing (Macrogen, Seoul, Korea).
For the generation of CXCR3 gene promoter-driving luciferase reporter constructs, the CXCR3 promoter fragment spanning nucleotides −178 to +22 upstream of the transcription start site was synthesized from human genomic DNA (Promega, Madison, WI, USA) via PCR using the primers 5′-ggtaccCATCCTCTGCCAGCTTTTCT-3′ (forward primer) and 5′-agatctCTTTGGTGCTTGTGGTTGGA-3′ (reverse primer). Small letters indicate inserted KpnI and BglII restriction sites. The PCR products ligated into a T&A vector (RBC Bioscience, Taipei county, Taiwan) were digested by KpnI and BglII and cloned into the KpnI and BglII sites of the pGL4-basic vector (Promega), yielding pCXCR3-Luc(−178/+22). Site-specific mutation of two NF-κB binding sites, mtNF-κB(I) and mtNF-κB(II), was performed using an EZchange Site-directed Mutagenesis Kit (Enzynomics, Daejeon, Republic of Korea), using the −178/+22 construct as a template plasmid. Primer sequences used to generate point mutations were as
RT-PCR was carried out by annealing at 55 °C for 25 cycles. The point mutation was verified by DNA sequencing (Macrogen, Seoul, Korea).
3
Mouse MKP3 Promoter Cloning
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A fragment of the mouse MKP3 gene spanning nucleotides −1597 to −10 (transcription start site numbered as +1) was amplified from mouse genomic DNA (Promega) by PCR using the primers 5'-agctcctttccctgggacc-3' (forward; −1597/−10) and 5'-agagaatgtatccattgagacgc-3' (reverse; −34/−10). The amplified PCR products were ligated into a T&A vector (RBC Bioscience), digested with HindIII, and then subcloned into the luciferase reporter plasmid pGL3-basic (Promega), yielding pMkp3-Luc (−1597/−10).
4
Localizing AtSNAT2 Protein with pER-mCherry
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We used the pER-mCherry vector [the kind gift of Dr. H.G. Kang (Texas State University, San Marcos, TX, USA)] to localize the AtSNAT2 protein. Full-length AtSNAT2 was PCR-amplified using a primer set containing AscI sites (GGCGCGCC). The resulting product was cloned into the T&A vector (RBC Bioscience) and the AtSNAT2 insert was digested with AscI and cloned into the AscI site of the binary vector, pER8-mCherry, containing the estrogen-inducible XVE promoter. The pER8-AtSNAT2-mCherry plasmid was transferred into Agrobacterium tumefaciens strain GV2260 using the freeze–thaw method. A confocal microscope (TCS-SP5; Leica, Wetzlar, Germany) was used to evaluate transient within-cell protein expression, as described previously [35 (link)].
5
Cloning of CCL5 promoter fragments
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A CCL5 promoter fragment spanning nucleotides –1074 to +45 upstream of the transcription start site was synthesized from human genomic DNA (Promega, Madison, WI, USA) by PCR using the primers 5′-GAG GGC AAC TGG GTT CTG AT-3′ (forward –1074F) and 5′-GAG GTC CAC GTG CTG TCT TG-3′ (reverse, +45R). The amplified PCR products were ligated to a T&A vector (RBC Bioscience, Taipei County, Taiwan) and digested with KpnI and BglII. The products were ligated at the KpnI and BglII sites of the pGL4 basic vector (Promega), yielding pCCL5-Luc(–1074/+45). Several deletion constructs of the human CCL5 promoter fragments were synthesized by PCR using the pCCL5-Luc(–1074/+45) construct as a template plasmid. The forward primer sequences were 5′-TGA GTG TGC TCA CCT CCT TT-3′ (−500/+45) and 5′-TGT GCA ATT TCA CTT ATG AT-3′ (–100/+45). One reverse primer, +45R, was used to generate all the deletion constructs. The amplified PCR products were ligated into the T&A vector and then digested using KpnI and BglII. The products were ligated into the KpnI and BglII sites of the pGL4 basic vector. The insert sequence of each construct was verified by DNA sequencing (Macrogen, Seoul, Republic of Korea).
6
Subcellular Localization of hNaa50
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The pER-mCherry binary vector for subcellular localization analysis of hNaa50 was kindly donated by Dr. H.G. Kang (Texas State University, San Marcos, TX, USA). Full-length synthetic hNaa50 DNA was amplified via PCR using a primer set containing AscI sites (AscI forward primer: 5′-GGC GCG CCA TGA AGG GCT CGC GCA TC-3′; AscI reverse primer: 5′-GGC GCG CCG GTT GTC CGT CTT CTG GAC-3′) with plasmid pBHA-hNaa50 as the template. The resulting hNaa50 PCR product was cloned into the TA vector (RBC Bioscience, New Taipei City, Taiwan), followed by AscI digestion. The AscI insert of hNaa50 was ligated into the AscI site of the binary vector pER8-mCherry containing the estrogen-inducible XVE promoter (Pxve), resulting in pER8-hNaa50-mCherry. The pER8-hNaa50-mCherry plasmid was transferred into Agrobacterium tumefaciens strain GV2260 using the freeze-thaw method. Agrobacterium-mediated transient expression of hNaa50-mCherry fusion protein and confocal microscopy (TCS-SP5; Leica, Wetzlar, Germany) were previously described [19 (link)].
7
Subcellular Localization of AtM3H Protein
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The pER8-mCherry for transgene localization analysis and pBIN61-GFP-HA (P35s:GFP-HA) plasmid for the cytoplasmic fluorescence marker were kindly provided by Dr. HG Kang (Texas state university, San Marcos, TX, USA). As for the subcellular localization study of AtM3H, a full-length AtM3H cDNA was amplified with PCR using a primer set containing an AscI site (forward, 5′-GGC GCG CCA TGG AAG CAA AAG GGG CAGC-3′, reverse, 5′-GGC GCG CCG GAT TCT CAA AGC ATC TAG-3′). The amplified PCR product was first introduced into the T&A vector (RBC Bioscience, New Taipei City, Taiwan), from which the AscI insert of AtM3H was prepared and ligated into the AscI site of the binary vector pER8-mCherry to generate pER8-AtM3H-mCherry. The resulting plasmids were transformed into the Agrobacterium Tumefaciens strain GV2260 using the freeze-thaw method. Leaves from four-week-old tobacco (Nicotiana benthamiana) plants, native Australian species, were infiltrated with Agrobacterium strain (about 0.2 OD), followed by infiltration with 10 μM β-estradiol, and then incubated for 24 h in a growth room. Images were obtained using a Leica TCS-SP5 confocal microscope (Leica, Wetzlar, Germany) using a ×63 oil-immersion objective. Images were processed using the Leica LAS-AF software version 1.8.2 (Leica, Wetzlar, Germany).
8
Constructing Chimeric F/HA Influenza Protein
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HA (Genbank AY676035) and M1 (Genbank AY676047) genes of A/chicken/Korea/ES/2003 and the F gene (Genbank AV630423) of KR-005/00 were extracted from virus infected allantoic fluid and amplified by two-step reverse transcription-polymerase chain reaction (RT-PCR; RNeasy® Mini Kit, Omniscript™ RT Kit, Qiagen, Germany). For cDNA synthesis, Uni12 primer was used for HA and M1 genes and a 5′-ATGGGCTCCAAACTTTC-3′ primer for F gene, as previously described [25 (link), 26 (link)]. To enhance the interaction between the ectodomain of the NDV F and the AIV M1 proteins in particle assembly, we constructed a chimeric F/HA protein. The ectodomain of the F gene was generated by PCR and the codon optimized HA TM/CT domain was synthesized by Bioneer, Korea. The two products were mixed equally and F/HA chimeric gene was constructed by overlap PCR using primers EcoRI-F-1 and HindIII-HA-TM/CT-2 (Fig 1A and Table 1 ). Sequences of primers used to amplify each segment are shown in Table 1 . Amplified genes were cloned into the TA vector (RBC Bioscience, Taiwan) and sequenced. The three resulting plasmid vectors were designated as vHA, vM1, and vF/HA.
9
Cloning and Characterizing TSLP Promoter
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A TSLP promoter fragment spanning nucleotides −1384 to +18 upstream of the transcription start site was synthesized from human genomic DNA (Promega) via PCR using the primers 5′-CGT CCA ACC TCC TTT CTC CG -3′ (forward −1384F) and 5′-TTG GAG TCT CCC TGA TGC TCC AG-3′ (reverse, +18R). The amplified PCR products were ligated to a T&A vector (RBC Bioscience, Taipei County, Taiwan) and digested using KpnI and HindIII. The products were ligated at the KpnI and HindIII sites of the pGL4-basic vector (Promega), yielding pTSLP-Luc(−1384/+18). Several deletion constructs of the human TSLP promoter fragments were synthesized using PCR, for which the pTSLP-Luc(−1384/+18) construct was used as the template. The forward primer sequences were as follows:
−1338F: 5′-GGA CCA GAG CGA TGC AGG-3′
−1214F: 5′-CAT GAG CCA AGC CAG GGA G-3′
−1017F: 5′-AAA TCT GAG CCC GCC ATC TC-3′
−369F: 5′-GGG ACA TAT GCA AGG ACT CC-3′
10
Cloning and Characterization of Human PROX1 Promoter
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The 5'-regulatory region of human PROX1 (ENST00000498508.6) spanning nucleotides (nt) -1077 to +33 was synthesized from human genomic DNA (Promega) using PCR with the following primers: 5'-AGATCTTTAAGAGCCACATTATCT-3' (forward) and 5'-CTCCGCTCCACAACAAGATT-3' (reverse). Hot-start PCR conditions used were as follows: hold for 10 min at 95 °C, followed by 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C for 35 cycles, according to the manufacturer's instructions (Enzynomics, Daejeon, Republic of Korea). PCR products were subcloned into a T&A vector (RBC Bioscience, New Taipei City, Taiwan), digested with restriction enzyme BglII (Enzynomics) and ligated into the pGL4.17 luciferase reporter vector (Promega), yielding pProx1-Luc(-1077/+33). The sequences of the promoter-reporter constructs were veri-
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