That do not contain bacterial remnants of plasmid DNA.MethodsVector constructionThat do not contain bacterial remnants

That do not contain bacterial remnants of plasmid DNA.MethodsVector construction
That do not contain bacterial remnants of plasmid DNA.MethodsVector 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 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28298493 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 [12] (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/XmaIdigested pSBT/RSV-hAAT [12] (generating pSBT/RSVFRT.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.PGKeGFP.WHV with the Flpx9 gene [13] 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 [14]. Lentiviral vector production HEK-293 and 293T cells were cultured at 37 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 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 10 fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and Lglutamine (265 mg/liter). When selection was applied, puromycin (Sigma, St. Louis, MO) or hygromycin B (GW9662 web Invitrogen, Carlsbad, CA) was added to the growth medium to a final concentration of 1 g/ml or 200 g/ml, respectively.Page 2 of(page number not for citation purposes)BMC Biotechnology 2008, 8:http://www.biomedcentral.com/1472-6750/8/VSV-G-pseudotyped lentiviral vectors were produced by CaPO4-transfection of 293T cells (seeded at 3 ?106 cells/ dish in 10-cm dishes) with 3 g pRSV-Rev, 3.75 g pMD.2G (VSV-G), 13 g pMDLg/pRRE (or 13 g pMDLg/ pRREintD64V [8] for production of integration-defective vectors) and 13 g lentiviral vector plasmid. The supernatant was harvested two days post-transfection and polybrene was added to a final concentration of 8 g/ml prior to transfer to target cells.Generation and transfection of FRT-tagged cell lines FRT-tagged HEK-293-derived cell lines were generated by transfecting HEK-293 cells (seeded at 2 ?105 cells/well in 6 well plates) with 1.5 g pSBT/RSV-FGIP and 0.5 g pCMV-SB [14] (using 4 l Fugene-6; Roche, Basel, Switzerland) and selecting for puromycin resistance. Resistant clones were isolated and expanded. For evaluating the efficacy of Flp-mediated insertion of transgenes into the engineered FRT docking site, FRT-tagged cell lines (seeded at 7 ?105 cells/dish in 10-cm dishes, n = 3) were CaPO4transfected with 2 g pLV/FRT-hygro and 10 g pCMV-Flp or 10 g pUC19 (negative control). Two days after transfection the cell lines were split, diluted, and selected for 10 days with hygromycin B prior to counting of hygromycin B-resistant colonies. Quantification of viral DNA forms in transduced cells HEK/FGIP1 cells (seeded at 3 ?1.