HEK293 Flp-In cells with stable expression of ABCB1 wild-type were generated previously [10 (link)]. The cDNAs encoding ABCB1-Q347/990A and ABCB1-Q347/725/990A were excised from their parent pCI-neo plasmids using BamHI/NotI restriction endonuclease double digests and subcloned into the equivalent sites of pcDNA5/FRT (ThermoFisher Sci, Waltham, MA, USA). The resulting pcDNA5/FRT-ABCB1-Q347/990A and pcDNA5/FRT-ABCB1-Q347/725/990A (Qtriple) were used to co-transfect HEK293 Flp-In cells (ThermoFisher Sci, Waltham, MA, USA) along with the pOG44 (ThermoFisher Sci, Waltham, MA, USA) as a source of Flp recombinase as described by the manufacturer. Stable transfected cells (Flp-In ABCB1-Q347/990A, Flp-In ABCB1-Q347/725/990A (Qtriple) and pcDNA/FRT as a vector-only negative control) were selected with hygromycin (200 μg/mL) and, once uniform expression of the mutant ABCB1 were confirmed, maintained in hygromycin (100 μg/mL).
Pcdna5 frt
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA5/FRT is a mammalian expression vector designed for generating stable cell lines. It contains an FRT site for Flp-mediated recombination and a hygromycin resistance gene for selection of stable transfectants.
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Lab products found in correlation
Pcdna3, by Thermo Fisher Scientific (8 mentions)
Pog44, by Thermo Fisher Scientific (5 mentions)
In fusion hd cloning kit, by Takara Bio (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Flp in 293 cells, by Thermo Fisher Scientific (3 mentions)
Flp in system, by Thermo Fisher Scientific (3 mentions)
Platinum pfx dna polymerase, by Thermo Fisher Scientific (2 mentions)
Coelenterazine, by Thermo Fisher Scientific (2 mentions)
Hygromycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pcdna 5 frt 5xms2, by Thermo Fisher Scientific (2 mentions)
Psicheck 2, by Promega (1 mentions)
Oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Tetracycline free fbs, by Lonza (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Lonza (1 mentions)
Pcag flpe, by Addgene (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
47 protocols using pcdna5 frt
1
Stable Expression of ABCB1 Mutants in HEK293 Cells
2
ABCC1 and ABCC2 Expression Vectors
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ABCC1 gene was subcloned from a previous pcDNA3.1(-)-MRP1, using the restriction enzyme XbaI and NotI to extract the ABCC1 insert. The insert included both an upstream Kozak sequence (G/A)NNATGG (where N = T or C) to promote the initiation of the translation and a downstream poly-histidine tag (10-His) to allow Ni-agarose affinity purification of the protein product. Correct subcloning of ABCC1 insert was attested by Nhe I and Not I digestion profile of the vectors and sequencing of the vector. The MRP2 expression vector pcDNA5/FRT-MRP2 was constructed by cloning the ABCC2 gene into the eukaryotic expression vector pcDNA5/FRT (Life Technologies) as described previously59 with an additional Kozak sequence added at the beginning of the gene.
3
Molecular Cloning and Purification of Key Proteins
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Human SFXN1 cDNA was amplified from HEK293 mRNA by using GoScript™ Reverse Transcription Mix, Oligo(dT) (Promega). Human AGK was copied from HeLa cDNA. CNAP-SFXN1, SFXN1-HA, and AGKG126E were generated by overlap-extension polymerase chain reaction (PCR) using Platinum™Pfx DNA polymerase (Thermo Fisher Scientific). All were cloned into pcDNA5/FRT (Invitrogen) and sequenced before use for transfection. To purify recombinant AGK and recombinant ANT2, human AGK and ANT2 were cloned into pET28a vector (Novagen) containing a N-terminal hexahistidine tag. Protein induction, extraction and affinity purification were carried out as described previously (Lu et al., 2017 (link)).
4
Luciferase Reporting of Oct4 3'UTR
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The 3 'UTR sequence of Oct4 was synthesized by PCR from a human genomic template. This sequence was cloned into a fusion plasmid with the reporter genes for Firefly and Renilla luciferase controlled by CMV promoter. The fusion plasmid called “pc5/Psi” was cloned previously by using parts of pcDNA5/FRT (Invitrogen) and psiCHECK-2 (Promega, catalog-no. C8021). Renilla luciferase was coupled with the 3’UTR serving as reporter gene, and Firefly luciferase served as cell number control.
5
Zebra Finch tgTas2r1 cDNA Cloning
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The cDNA of zebra finch tgTas2r1 was synthesized by MWG Operon and subcloned into vector pcDNA5FRT (Invitrogen). Sequence analysis to verify the integrity of constructs was done by double stranded Sanger sequencing (MWG Eurofins).
6
Cytokine Gene Variants Optimization
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The cytokine genes human mip-1α (humip-1α), murine mip-1α (mmip-1α), human gm-csf (hugm-csf) and murine mgm-csf (mgm-csf) were modified in silico with respect to codon usage and CpG amount, synthesized via stepwise PCR from oligonucleotides (GeneArt/life technologies) and inserted into the eukaryotic expression vector pcDNA3.1 (+) (Invitrogen). Based on each wild type (wt) gene sequence, three gene variants with varying CpG content were generated. The resulting plasmids were named phuMIP-wt/-11/-0/-43, pmMIP-wt/-13/-0/-42, phuGM-CSF-wt/-12/-0/-63 and pmGM-CSF-wt/-21/-0/-61. The codon adaptation index (CAI) of the gene variants was calculated as described previously (21 (link)). For infection/transfection experiments, mmip-1α gene variants were cloned into the plasmid pPCR-Script Amp SK (+) (Stratagene) using HindIII and EcoRI, resulting in plasmids pT7-mMIP-wt, −0, −13 and −42. For the generation of stable cell lines, mmip-1α variants were inserted into pcDNA5/FRT (Invitrogen) via the restriction sites HindIII and BamHI, resulting in the plasmids pFRT-mMIP-wt, pFRT-mMIP-0, pFRT-mMIP-13 and pFRT-mMIP-42.
7
Stable Expression of Bitter Taste Receptors
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For functional expression of the human bitter taste receptor hTAS2R39, HEK293 T-Rex Flp-In cells (Invitrogen, San Diego, CA, USA) were used, stably expressing the chimeric G-protein α-subunit Gα16-gust44 (cloned into pcDNA4 (Invitrogen)) [17] and the human bitter receptor genes for hTAS2R39 (cloned into pcDNA5/FRT (Invitrogen)). The bitter receptor gene contained a DNA sequence encoding the first 45 amino acids of rat somatostatin receptor type 3 at its 5′ end (the receptor expression was achieved according to [18] with exception of the HSV-tag), in order to improve membrane targeting of the receptor protein. The same procedure was applied for stable expression of hTAS2R14. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) and 10% (v/v) tetracycline-free FBS (both Lonza, Verviers, Belgium) supplemented with blasticidin (5 μg/mL), geneticin (400 μg/mL) and hygromycin (100 μg/mL) (all from Invitrogen). Cells were grown and maintained at 37 °C and 5% (v/v) CO2.
8
Flp-In System for Inducible CD20 Expression
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The vector pcDNA5/FRT (Invitrogen) was used for chromosomal integration via the Flp-In system. The codon optimized CD20 coding sequence (33 (link)) was integrated into pcDNA5/FRT using the restriction sites for NheI and SacI resulting in the construct pFRT/CD20. Afterward, the bgl2 intron containing the tc aptamer-controlled exon 2 was cloned from the luciferase construct L2 into the CD20 open reading frame (46 bp downstream of the start codon) using the flanking regions 5′-CGCCGAGC//ATTTATGG-3′. This insertion resulted in the vector pFRT/CD20-C2. Cloning was done via overlap extension PCR. All constructs were integrated into the cell line HF1–3 via the Flp-In system (18 (link)).
All primer and vector sequences can be provided upon request.
All primer and vector sequences can be provided upon request.
9
Cloning and Tagging of Human ANT Proteins
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Human ANT1 and ANT2 were amplified by PCR using cDNA from HeLa cells and subcloned first into pBSK (Stratagene) and then into pcDNA5/FRT (Invitrogen). Tagged-ANT1 and ANT2 amplicons were generated via primer extension containing an N-terminal CNAP tag (amino acid sequence Met-DYKDDDDK -GGAGG-EDQVDPRLIDGK ; FLAG epitope tag underlined and Protein C tag in bold) using untagged ANT plasmids as template and then subcloned into pcDNA5/FRT. Human ANT3 was first amplified by PCR using genomic DNA from yeast expressing yNhANT3 (Hamazaki et al., 2011 (link)) and then replacing the first 10 amino acids with the correct human ANT3 N-terminal nucleotides by PCR, followed by subcloning into pcDNA5/FRT. The fidelity of every construct was verified by DNA sequencing. TALEN binding pairs were designed to recognize sequences in ANT2 (SLC25A5) exon 1 that are separated by 16 nucleotides and encompass the start site (Life Technologies). The two constructs, which are fused to the truncated Fok1 nuclease, were subcloned into pcDNA3.1 and pEF6/V5-HisA (Life Technologies), generating pcDNA3ANT2-R and pEF6AANT2-L, respectively.
10
GCN2 Mutant Plasmid Construction
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The pCas-Guide-AAVS1(Cat #GE100023) and pAAVS1-EF1a-Puro-DNR (Cat #GE100046) plasmids were obtained from Origene. A segment of DNA encoding a FRT site and eGFP CDS were sub cloned into pAAVS1-EF1a-Puro-DNR using an In-Fusion HD cloning kit (Takara, #638920) to produce pAAVS1-EF1a-FRT-eGFP-Puro-DNR. Flp recombinase expression plasmid pCAG-Flpe (plasmid #13787) was purchased from Addgene (32 (link)). Plasmid pCDNA5-FRT was purchased from Invitrogen (Cat #V601020) and GCN2 cDNA plasmid pENTR223-1 GCN2 (NM_001013703) was purchased from Transomic Technologies (Huntsville, AL). The cDNA encoding the human GCN2 coding sequence was sub-cloned from pENTR223-1 GCN2 into pCDNA5-FRT and an amino-terminal 3x FLAG tag was added using an In-Fusion HD cloning kit in a three-way cloning reaction. The resulting plasmid pCDNA5-FRT-GCN2 is referred to in the text as FLAG-WT-GCN2. This plasmid was used as a template to construct the mutant version pCDNA5-FRT-GCN2-m2 by site directed mutagenesis using an In-Fusion HD cloning kit (33 (link)). This mutant plasmid is referred to in the text as FLAG-GCN2-m2 with TCC/AGG → CTG/CTG encoding residue substitutions F1143L/R1144L.
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