HEK 293T cells were used to generate the lenti-landing pad line derived from LLP-Int-BFP-IRES-iCasp9-Blast as previously described [10 (link)]. Landing pad cells expressing Bxb1 integrase with a nuclear localization signal to allow for transport into the nucleus were recombined in either 24-well or 6-well plates. In the 24-well plate, 120,000 cells were transfected with 254 ng of attB recombination plasmid mixed with 0.96 μL of Fugene 6 reagent in D10-dox media. In the 6-well plate, 600,000 cells were transfected with 1,200 ng of attB recombination plasmid mixed with 5 μL of Fugene 6 reagent in D10-dox media.
Upon attB-plasmid transfection, negative selection of nonrecombined landing pad cells was performed with the addition of 10 nM AP1903 (ApexBio, B4168) to activate iCasp9. Positive selection of recombined cells was achieved with the addition of 1 μg/mL puromycin (InvivoGen, ANTPR1). Recombined cells were maintained in D10-dox with 1 μg/mL puromycin to prevent transgene silencing.
Upon attB-plasmid transfection, negative selection of nonrecombined landing pad cells was performed with the addition of 10 nM AP1903 (ApexBio, B4168) to activate iCasp9. Positive selection of recombined cells was achieved with the addition of 1 μg/mL puromycin (InvivoGen, ANTPR1). Recombined cells were maintained in D10-dox with 1 μg/mL puromycin to prevent transgene silencing.
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