Pcdna5 frt to plasmid

Human SLX9, BYSL, and RIOK1 cell lines were generated as described previously (17 (link),18 (link)). In brief, the coding sequences were amplified from a cDNA library. In the case of SLX9, the SLX9 gene was inserted into a modified pcDNA5/FRT/TO plasmid (Invitrogen) containing an N-terminal Strep–Flag tag followed by a 3C cleavage site (pcDNA5-N-TAP-SLX9). In the case of BYSL, the BYSL gene was inserted into a separate modified pcDNA5/FRT/TO plasmid containing a 3C-Flag-Strep at the C terminus (pcDNA5-C-TAP-BYSL). In the case of RIOK1, first, a mutation (D324A) was introduced using site-directed mutagenesis by PCR. Then, an internal Strep–Flag tag following amino acid 496 was added before insertion into the original pcDNA5/FRT/TO vector. To generate the stable cell lines, HEK Flp-In 293 T-Rex cells (Thermo Fisher, R78007) were split one day before into a 10 cm dish and grown to 50% confluence. A mixture of 0.5 μg of the plasmid, 4.5 μg of the helper pOG44 plasmid and 20 μg of polyethylenimine (PEI) was incubated for 25 min then directly added into the culture. Selection was performed with 200 μg/ml hygromycin B (Thermo Fisher) supplied in normal DMEM medium.

The pcDNA5-FRT-TO plasmid was obtained from Invitrogen. cDNAs or sgRNAs, respectively for protein overexpression and gene deletions, were cloned in a dual promoter lentiviral vector, as described previously (van de Weijer et al., 2014 (link)).

V5-APEX2 was PCR-amplified from the pcDNA5-Mito-V5-APEX2 plasmid, which was kindly provided by Dr. Hyun-Woo Rhee (Seoul National University). V5-APEX2 alone or IPMK-V5-APEX2 were cloned into the pcDNA5/FRT/TO plasmid (Invitrogen). Flp-In T-REx–293 (Invitrogen) cells were seeded in six-well culture plates to 70% confluency, and then co-transfected with 0.25 µg of pcDNA5/FRT/TO and 2.25 µg of pOG44 Flp recombinase expression plasmid (Invitrogen) using Lipofectamine LTX with Plus Reagent (Invitrogen). After 48 hr, the cells were transferred to 90 mm culture dishes for negative selection with 50 µg/ml Hygromycin B (Gibco) until all non-transfected cells were dead. Surviving cells were seeded at low confluency to generate cellular clones on culture plates, after which each clone was individually screened for APEX2 construct expression with or without doxycycline (Sigma Aldrich) to search for optimal cell populations with minimal uncontrolled APEX2 expression and maximal APEX2 expression under stimulation. Selected clones were then expanded and stored in liquid nitrogen for downstream experiments.