The split-GFP, and splitHalo landing pad HEK293Ts, were generated from a published landing pad parent cell line 28 (link) seeded at 100k cells in a 12-well plate. To each well, 600 ng of BxbI Integrase Expression Vector (Addgene #51271) and 600 ng of each landing pad donor plasmid were co-transfected. Once cells are confluent, cells were split once and seeded in a T25 flask, and blasticidin (Gemini Bio-Products 400-165P) was added at 5 μg/mL for selection prior to FACS sorting integrated cell lines.
Protocol detail
Generation of split-GFP and splitHalo HEK293T cell lines
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Corresponding Organization : University of California, San Francisco
Other organizations : University of California, Berkeley, Tsinghua University
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Variable analysis
independent variables
- Amount of BxbI Integrase Expression Vector (Addgene #51271) transfected
- Amount of each landing pad donor plasmid transfected
- Integration of landing pad in the cells
- Parent cell line used to generate split-GFP and splitHalo landing pad HEK293Ts
- Cell seeding density (100k cells per well in a 12-well plate)
- Blasticidin selection concentration (5 μg/mL)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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