Polymerase Chain Reaction:Article Title: Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase
Article Snippet: .. Vector construction The HIV-1-derived Flp substrate vector, pLV/FRT-hygro, was generated by replacing the eGFP gene (BamHI/XhoI digestion) of the third generation SIN-vector pCCL.WPS.PGK-eGFP.WHV (a kind gift from Dr. Patrick Aebischer, Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland) with the puromycin resistance gene, PCR-amplified from pT/PGK-Puro [ ] (creating pCCL.WPS.PGK-Puro.WHV), followed by insertion of the ATG-deficient FRT-hygro fusion gene (PCR-amplified from pcDNA5/FRT, Invitrogen) into the HpaI site located upstream of the cPPT The SB-based docking vector, pSBT/RSV-FGIP, was generated by amplifying the eGFP sequence (from peGFP.N1, Clontech) using a forward primer containing the 48-bp FRT sequence and inserting the resulting FRT.GFP fusion gene into MluI/XmaI-digested pSBT/RSV-hAAT [ ] (generating pSBT/RSV-FRT.GFP) prior to insertion of an IRES-puro cassette (PCR-amplified from pecoenv-IRES-puro, kindly provided by Dr. Finn Skou Pedersen, University of Aarhus, Denmark), into the XmaI site. pLV/PGK-Flp was generated by replacing the eGFP gene of pCCL.WPS.PGK-eGFP.WHV with the Flpx9 gene [ ] PCR-amplified from pCMV-Flp (obtained from A. Francis Stewart, University of California San Francisco, USA), the latter which contains the enhanced x9 Flp variant driven by a cytomegalovirus (CMV) promoter. pCMV-SB contains the SB10 transposase gene driven by a CMV promoter and has been described previously [ ]. .. Lentiviral vector production HEK-293 and 293T cells were cultured at 37°C in 5% (v/v) CO2 and maintained in Dulbecco's modified Eagle's medium (Cambrex, Verviers, Belgium) with D-glucose (4.500 mg/liter) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and L-glutamine (265 mg/liter).
Sequencing:Article Title: Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase
Article Snippet: .. Vector construction The HIV-1-derived Flp substrate vector, pLV/FRT-hygro, was generated by replacing the eGFP gene (BamHI/XhoI digestion) of the third generation SIN-vector pCCL.WPS.PGK-eGFP.WHV (a kind gift from Dr. Patrick Aebischer, Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland) with the puromycin resistance gene, PCR-amplified from pT/PGK-Puro [ ] (creating pCCL.WPS.PGK-Puro.WHV), followed by insertion of the ATG-deficient FRT-hygro fusion gene (PCR-amplified from pcDNA5/FRT, Invitrogen) into the HpaI site located upstream of the cPPT The SB-based docking vector, pSBT/RSV-FGIP, was generated by amplifying the eGFP sequence (from peGFP.N1, Clontech) using a forward primer containing the 48-bp FRT sequence and inserting the resulting FRT.GFP fusion gene into MluI/XmaI-digested pSBT/RSV-hAAT [ ] (generating pSBT/RSV-FRT.GFP) prior to insertion of an IRES-puro cassette (PCR-amplified from pecoenv-IRES-puro, kindly provided by Dr. Finn Skou Pedersen, University of Aarhus, Denmark), into the XmaI site. pLV/PGK-Flp was generated by replacing the eGFP gene of pCCL.WPS.PGK-eGFP.WHV with the Flpx9 gene [ ] PCR-amplified from pCMV-Flp (obtained from A. Francis Stewart, University of California San Francisco, USA), the latter which contains the enhanced x9 Flp variant driven by a cytomegalovirus (CMV) promoter. pCMV-SB contains the SB10 transposase gene driven by a CMV promoter and has been described previously [ ]. .. Lentiviral vector production HEK-293 and 293T cells were cultured at 37°C in 5% (v/v) CO2 and maintained in Dulbecco's modified Eagle's medium (Cambrex, Verviers, Belgium) with D-glucose (4.500 mg/liter) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and L-glutamine (265 mg/liter).
Generated:Article Title: Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase
Article Snippet: .. Vector construction The HIV-1-derived Flp substrate vector, pLV/FRT-hygro, was generated by replacing the eGFP gene (BamHI/XhoI digestion) of the third generation SIN-vector pCCL.WPS.PGK-eGFP.WHV (a kind gift from Dr. Patrick Aebischer, Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland) with the puromycin resistance gene, PCR-amplified from pT/PGK-Puro [ ] (creating pCCL.WPS.PGK-Puro.WHV), followed by insertion of the ATG-deficient FRT-hygro fusion gene (PCR-amplified from pcDNA5/FRT, Invitrogen) into the HpaI site located upstream of the cPPT The SB-based docking vector, pSBT/RSV-FGIP, was generated by amplifying the eGFP sequence (from peGFP.N1, Clontech) using a forward primer containing the 48-bp FRT sequence and inserting the resulting FRT.GFP fusion gene into MluI/XmaI-digested pSBT/RSV-hAAT [ ] (generating pSBT/RSV-FRT.GFP) prior to insertion of an IRES-puro cassette (PCR-amplified from pecoenv-IRES-puro, kindly provided by Dr. Finn Skou Pedersen, University of Aarhus, Denmark), into the XmaI site. pLV/PGK-Flp was generated by replacing the eGFP gene of pCCL.WPS.PGK-eGFP.WHV with the Flpx9 gene [ ] PCR-amplified from pCMV-Flp (obtained from A. Francis Stewart, University of California San Francisco, USA), the latter which contains the enhanced x9 Flp variant driven by a cytomegalovirus (CMV) promoter. pCMV-SB contains the SB10 transposase gene driven by a CMV promoter and has been described previously [ ]. .. Lentiviral vector production HEK-293 and 293T cells were cultured at 37°C in 5% (v/v) CO2 and maintained in Dulbecco's modified Eagle's medium (Cambrex, Verviers, Belgium) with D-glucose (4.500 mg/liter) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and L-glutamine (265 mg/liter).
Variant Assay:Article Title: Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase
Article Snippet: .. Vector construction The HIV-1-derived Flp substrate vector, pLV/FRT-hygro, was generated by replacing the eGFP gene (BamHI/XhoI digestion) of the third generation SIN-vector pCCL.WPS.PGK-eGFP.WHV (a kind gift from Dr. Patrick Aebischer, Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland) with the puromycin resistance gene, PCR-amplified from pT/PGK-Puro [ ] (creating pCCL.WPS.PGK-Puro.WHV), followed by insertion of the ATG-deficient FRT-hygro fusion gene (PCR-amplified from pcDNA5/FRT, Invitrogen) into the HpaI site located upstream of the cPPT The SB-based docking vector, pSBT/RSV-FGIP, was generated by amplifying the eGFP sequence (from peGFP.N1, Clontech) using a forward primer containing the 48-bp FRT sequence and inserting the resulting FRT.GFP fusion gene into MluI/XmaI-digested pSBT/RSV-hAAT [ ] (generating pSBT/RSV-FRT.GFP) prior to insertion of an IRES-puro cassette (PCR-amplified from pecoenv-IRES-puro, kindly provided by Dr. Finn Skou Pedersen, University of Aarhus, Denmark), into the XmaI site. pLV/PGK-Flp was generated by replacing the eGFP gene of pCCL.WPS.PGK-eGFP.WHV with the Flpx9 gene [ ] PCR-amplified from pCMV-Flp (obtained from A. Francis Stewart, University of California San Francisco, USA), the latter which contains the enhanced x9 Flp variant driven by a cytomegalovirus (CMV) promoter. pCMV-SB contains the SB10 transposase gene driven by a CMV promoter and has been described previously [ ]. .. Lentiviral vector production HEK-293 and 293T cells were cultured at 37°C in 5% (v/v) CO2 and maintained in Dulbecco's modified Eagle's medium (Cambrex, Verviers, Belgium) with D-glucose (4.500 mg/liter) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and L-glutamine (265 mg/liter).
Plasmid Preparation:Article Title: Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase
Article Snippet: .. Vector construction The HIV-1-derived Flp substrate vector, pLV/FRT-hygro, was generated by replacing the eGFP gene (BamHI/XhoI digestion) of the third generation SIN-vector pCCL.WPS.PGK-eGFP.WHV (a kind gift from Dr. Patrick Aebischer, Swiss Federal Institute of Technology, EPFL, Lausanne, Switzerland) with the puromycin resistance gene, PCR-amplified from pT/PGK-Puro [ ] (creating pCCL.WPS.PGK-Puro.WHV), followed by insertion of the ATG-deficient FRT-hygro fusion gene (PCR-amplified from pcDNA5/FRT, Invitrogen) into the HpaI site located upstream of the cPPT The SB-based docking vector, pSBT/RSV-FGIP, was generated by amplifying the eGFP sequence (from peGFP.N1, Clontech) using a forward primer containing the 48-bp FRT sequence and inserting the resulting FRT.GFP fusion gene into MluI/XmaI-digested pSBT/RSV-hAAT [ ] (generating pSBT/RSV-FRT.GFP) prior to insertion of an IRES-puro cassette (PCR-amplified from pecoenv-IRES-puro, kindly provided by Dr. Finn Skou Pedersen, University of Aarhus, Denmark), into the XmaI site. pLV/PGK-Flp was generated by replacing the eGFP gene of pCCL.WPS.PGK-eGFP.WHV with the Flpx9 gene [ ] PCR-amplified from pCMV-Flp (obtained from A. Francis Stewart, University of California San Francisco, USA), the latter which contains the enhanced x9 Flp variant driven by a cytomegalovirus (CMV) promoter. pCMV-SB contains the SB10 transposase gene driven by a CMV promoter and has been described previously [ ]. .. Lentiviral vector production HEK-293 and 293T cells were cultured at 37°C in 5% (v/v) CO2 and maintained in Dulbecco's modified Eagle's medium (Cambrex, Verviers, Belgium) with D-glucose (4.500 mg/liter) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and L-glutamine (265 mg/liter).
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