Molecular cloning was performed as previously described (Moyer et al., 2015 (link)). All constructs were cloned into a pcDNA5/FRT/TO vector backbone (Life Technologies) and expressed from a CMV promoter. DNA constructs for Drosophila S2 transfection were cloned into a pAc vector backbone (Invitrogen).
Pcdna5 frt to vector backbone
Manufactured by Thermo Fisher Scientific
The PcDNA5/FRT/TO vector backbone is a plasmid vector designed for controlled gene expression in mammalian cell lines. It contains an FRT site for Flp recombinase-mediated integration and a tetracycline-regulated promoter system for inducible transgene expression.
Automatically generated - may contain errors
Lab products found in correlation
Blasticidin, by Thermo Fisher Scientific (2 mentions)
Pmaxgfp vector, by Lonza (2 mentions)
Pog44 flp recombinase expression vector, by Thermo Fisher Scientific (2 mentions)
Hek flp in t rex cells, by Thermo Fisher Scientific (2 mentions)
4d nucleofector in, by Lonza (2 mentions)
Amaxa 4d nucleofector protocol for hek293, by Lonza (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
6 protocols using pcdna5 frt to vector backbone
1
Molecular Cloning and Expression Vectors
2
Tetracycline-Inducible Gene Expression
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All DNA constructs were cloned into a pcDNA5/FRT/TO vector backbone (Life Technologies) and expressed from a CMV promoter under the control of two tetracycline operator sites. All constructs were full-length proteins unless otherwise noted.
3
Inducible Expression of CEP85 Fragments
4
Tetracycline-Inducible PLK4 Reporter
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All PLK4 reporter constructs were cloned into a pcDNA5/FRT/TO vector backbone (Life Technologies) and expressed from a CMV promoter under the control of two tetracycline operator sites. The detailed configuration of each is indicated in the figure and figure legend for each experiment. The ecDHFR-sfGFP-NLS cassette was subcloned from a plasmid purchased from Addgene (67929).
5
Stable Transgenic HEK Cell Line Generation
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RNA from HEK cells was isolated using the DirectZol RNA MiniPrep Kit (Zymo) according to the manufacturer's protocol. cDNA was generated using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific), and coding regions of RPS27A without the N‐terminal ubiquitin sequence was PCR amplified and cloned into a pcDNA5/FRT/TO vector backbone (Invitrogen) that had been previously modified to have a constitutive CMV promoter and C‐terminal SBP‐tag.
To generate stable transgenic cell lines, 1 × 106 HEK Flp‐In T‐Rex Cells (Invitrogen) were electroporated using a Lonza 4D Nucleofector in according to the Amaxa 4D‐Nucleofector™ Protocol for HEK293 (Lonza) for large cuvettes with 1.8 μg pOG44 Flp‐Recombinase Expression Vector (Invitrogen) and 0.2 μg pCMV‐RPS27A‐SBP. Two days after electroporation, cells were passaged and placed on media containing 5 μg·mL−1 blasticidin (Invitrogen) and 10 μg·mL−1 Hygromycin B (Thermo Fisher Scientific) until all cells from a control plate electroporated with pmaxGFP™ Vector (Lonza) were dead. Flp‐In cell lines were validated using anti‐SBP westerns and Sanger sequencing of the transgenic insert.
To generate stable transgenic cell lines, 1 × 106 HEK Flp‐In T‐Rex Cells (Invitrogen) were electroporated using a Lonza 4D Nucleofector in according to the Amaxa 4D‐Nucleofector™ Protocol for HEK293 (Lonza) for large cuvettes with 1.8 μg pOG44 Flp‐Recombinase Expression Vector (Invitrogen) and 0.2 μg pCMV‐RPS27A‐SBP. Two days after electroporation, cells were passaged and placed on media containing 5 μg·mL−1 blasticidin (Invitrogen) and 10 μg·mL−1 Hygromycin B (Thermo Fisher Scientific) until all cells from a control plate electroporated with pmaxGFP™ Vector (Lonza) were dead. Flp‐In cell lines were validated using anti‐SBP westerns and Sanger sequencing of the transgenic insert.
6
Generating Stable Cell Lines with RPS27A-SBP
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