Flp-In-3T3

BioVector NTCC質粒載體菌種細胞蛋白抗體基因保藏中心www.biovector.net

Description of Flp-In? cell linesAll of the Flp-In? cell lines (except Flp-In?-CHO; see the following section) contain a single integrated FRT site and stably express the lacZ-Zeocin? fusion gene from the pFRT/lacZeo plasmid under the control of the SV40 early promoter (see the following diagram). The location of the FRT site in each Flp-In? cell line has not been mapped, but is presumed to have integrated into a transcriptionally active genomic locus as determined by generation of a Flp-In? expression cell line containing the pcDNA?5/FRT/CAT or pEF5/FRT/GW-CAT control plasmid. The Flp-In? cell lines should be maintained in medium containing Zeocin? Selection Antibiotic (see Media for cell lines, page 7).For more information about pFRT/lacZeo, refer to the Flp-In? System manual. For information about pcDNA?5/FRT/CAT or pEF5/FRT/GW-CAT, refer to the pcDNA?5/FRT or pEF5/FRT/V5-DEST? manuals, respectively.  The Flp-In?-CHO cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin? fusion gene from the pFRT/lacZeo2 plasmid. Note that pFRT/lacZeo2 contains a mutated SV40 early promoter (PSV40Δ) which is severely abrogated in its activity. The SV40Δ early promoter in pFRT/lacZeo2 exhibits approximately 60-fold less activity than the wild-type SV40 early promoter in pFRT/lacZeo. Because of the minimal activity of the SV40Δ promoter, we expect that stable transfectants expressing the lacZ-Zeocin? gene from pFRT/lacZeo2 should contain FRT sites which have integrated into the most transcriptionally active genomic loci. The location of the FRT site in the Flp-In?-CHO cell line has not been mapped, but has been demonstrated to have integrated into a highly transcriptionally active genomic locus as determined by generation of a Flp-In? expression cell line containing the pcDNA?5/FRT/luc (luciferase-expressing) control plasmid. The Flp-In?-CHO cell line should be maintained in medium containing Zeocin? Selection Antibiotic (see Media for cell lines, page 7). For more information about pFRT/lacZeo2 and the SV40Δ early promoter, refer to the pFRT/lacZeo2 manual.    Important guidelines?FBS does not need to be heat inactivated for use with these cell lines.?Cell lines should be maintained in medium containing Zeocin? Selection Antibiotic at the concentrations listed in the previous section.?If adherent cells (e.g., Flp-In?-293, Flp-In?-CV-1, Flp-In?-CHO, Flp-In?-3T3, Flp-In?-BHK) are split at a 1:5 to 1:10 dilution, they will generally reach 80–90% confluence in 3–4 days.?Suspension Flp-In?-Jurkat cells will demonstrate optimal growth characteristics if maintained at a cell density between 1 × 105 cells/mL and 1 × 106 cells/mL.?When maintaining Flp-In?-Jurkat cells in suspension culture, do not allow the medium to turn yellow; this indicates that cells have reached too high a density or that the medium is depleted of nutrients. If this occurs, either add fresh complete media to the cells or passage them. BioVector NTCC質粒載體菌種細胞蛋白抗體基因保藏中心www.biovector.net    Flp-In?-293D-MEM (high glucose) 10% FBS* 2 mM L-glutamine 1% Pen-Strep (optional)100 μg/mL Zeocin? Selection Antibiotic90% complete medium10% DMSOFlp-In?-CV-1D-MEM (high glucose) 10% FBS* 2 mM L-glutamine 1% Pen-Strep (optional)100 μg/mL Zeocin? Selection Antibiotic90% complete medium10% DMSOFlp-In?-CHOHam’s F12 10% FBS* 2 mM L-glutamine 1% Pen-Strep (optional)100 μg/mL Zeocin? Selection Antibiotic90% complete medium10% DMSOFlp-In?-BHKD-MEM (high glucose) 10% FBS* 2 mM L-glutamine 1% Pen-Strep (optional)100 μg/mL Zeocin? Selection Antibiotic90% complete medium10% DMSOFlp-In?-3T3D-MEM (high glucose) 10% donor calf serum 2 mM L-glutamine 1% Pen-Strep (optional)100 μg/mL Zeocin? Selection Antibiotic90% complete medium10% DMSOFlp-In?-JurkatRPMI 1640 10% FBS* 2 mM L-glutamine 1% Pen-Strep (optional)100 μg/mL Zeocin? Selection Antibiotic90% complete medium10% DMSO      [Supplier來源]  http://www.biovector.net