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, with incorporation of the TTAA footprint at a silent position (Leu104) and addition of a FLAG epitope (DYKDDDDK) at the protein Ct. To limit expression of the Cre Nt fragment prior to transposition, its coding sequence was positioned in frame (through the PB 5 0 TR) with a PEST degron (Li et al., 1998) followed by a translational stop. The membrane-restricted GFP was generated by adding a short Kras tethering sequence (Averaimo et al., 2016) at the Ct end of EGFP using annealed oligonucleotides. The TTAA-less LiOn* CAGNRFP vector was designed on the model of LiOn CAGNRFP with punctual substitutions intended not to affect vector replication and expression. The 3-color integration reporter was constructed by inserting in LiOn CAGNRFP two CMVdriven transcriptional units expressing mTurquoise2 and EYFP, 5 0 of CAG and 3 0 of the mRFP1 polyA, respectively. To assay Cre activity, we designed a floxed reporter ( Tol2 CAG::loxP-mCherry-loxP-EYFP, abbreviated as Tol2 CAG::RY) in which expression switches from mCherry to EYFP upon recombination, framed with Tol2 transposition endfeet to enable genomic integration. The LiOn-CAGNRFP-2A-NICD vector was assembled by introducing a P2A cleavage sequence between the RFP and NICD ORFs to enable their co-expression. As non-integrative control vectors, METHOD DETAILS DNA constructs A schematized map of the plasmids designed for this study can be found in Table S2, along with restriction sites available to exchange GOIs and promoters. All iOn and control piggyBac vectors were assembled in a pUC57-mini plasmid backbone (Genscript Inc) using a combination of DNA synthesis, p.2, 2002.
, For triple-color labeling experiments, we used 100 ng/well of each LiOn CAGNFP plasmid and 60 ng of PBase vector. To validate the LiOn CMVNCre transgene, 50 ng of the corresponding plasmid was co-transfected with 10 ng of PBase vector in a HEK293 cell line stably expressing the Tol2 CAG::RY reporter. This line was established by successive use of Tol2 transposition, drug selection with G418 (300 mg/ml, Sigma) and picking of RFP-positive clones. In some experiments, 50 ng of non-integrative plasmid expressing an FP marker distinct from the iOn vector (CMV::mTurquoise2, CMV::IRFP or CAG::GFP) were applied as transfection control. For FACS analysis, transfections were performed in 6-cm dishes with scaled up concentrations. HEK293 cell viability after iOn plasmids transfection was assessed by dye exclusion with Trypan blue solution (0.4%, Sigma). FP expression was either assayed by flow cytometry, epifluorescence or confocal microscopy, or an Arrayscan high-content system (Thermo Fisher Scientific) (see below). For fixed observations, cells grown on 13 mm coverslips coated with collagen (50 mg/ml, Sigma) were immersed in 4% paraformaldehyde (PFA), HEK293, HeLa and NIH 3T3 cell culture experiments iOn and piggyBac plasmids were transfected in human HEK293, HeLa or mouse NIH 3T3 cells using cationic lipids. Except when otherwise noted, 1 3 10 5 cells/well were plated in a 24-well dish and transfected at day 1 with 100 ng iOn vector with or without 20 ng of PBase-expressing plasmid (CAG::hyPBase) using 0.7 ml of Lipofectamine 2000 reagent (Invitrogen)
, 561 nm (RFP), 640 nm (IRFP). 10000 cells were analyzed for each condition; non-fluorescent controls were prepared from mock-transfected cells stained with DAPI. For clonal experiments, HEK293 cells were sorted as single cells two days after transfection. Selection windows were chosen to include most of the FP-positive population and exclude negative cells. For 3-color cell sorting, we first selected live dissociated cells and subsequently selected RFP+, IRFP+ cells within the GFP+ population. Cells were then plated as single cells in 96-well plates and grown for 7-10 days in 200 mL of 10% FBS/DMEM medium mixed 1:1 with filtrated HEK293-conditioned medium. FP expression was assayed by epifluorescence microscopy or Arrayscan High-Content imaging (see below). Some positive clones were expanded in larger dishes for sequencing. To this aim, genomic DNA was isolated from a confluent 3.5-or 10-cm dish with the Nucleospin Tissue Kit (Macherey-Nagel), HEK293 cells grown on 6-cm dishes were dissociated three days after transfection, stained with DAPI and analyzed on a MoFlo Astrios cell sorter (Beckman Coulter) using the following laser lines: 405 nm (DAPI), 488 nm (GFP)
, Episome extraction and characterization
, For the bacterial transformation assay, 5ng of episomal DNA was transformed into XL2-Blue Ultracompetent Cells (Stratagene); the TTAA footprint in activated LiOn CAGNRFP transgenes. Quantitative PCR analysis was performed on purified episomes using an Applied Biosystems realtime PCR machine (7500 Fast System) with PowerUp SYBR green master mix following the manufacturer's instructions. Results from PCR1 PCR2), Quantification of gene expression was based on the DeltaCt method in at least three independent biological experiments. Episomal DNA isolated from cells transfected with CAG::RFP was used as a reference, Extrachromosomal DNA was purified from HEK cells three days after transfection as in, 2009.
, Human iPS cell transfection and differentiation For iOn labeling of differentiating iPS cells, colonies were dissociated with Accutase (Life Technologies) and replated in 96-well plates coated with poly-L-ornithine (20 mg/ml, Sigma P4957) and laminin (3 mg/ml, Sigma 23017-015), 2015.
, 1 mass ratio) using Lipofectamine 2000 reagent. 48 hrs after transfection, GFP-positives cells (1.5%) were sorted using an Astrios MoFlo EQ cell sorter and plated at low density (10 3 cells/ 10-cm dish) on feeder cells. After eight days, GFP-positives clones were picked under a fluorescent stereomicroscope, Mouse ES cell transfection and clone selection KH2 ES cells were transfected with LiOn CAGNGFP-Kras and CAG::hyPBase plasmids, vol.4
, A DNA mix containing 1-1.2 mg/ml of iOn vector, 0.5-1.2 mg/ml of non-integrative control plasmid and 0.2 mg/ml of CAG::hyPBase plasmid supplemented with fast green dye was injected with a glass capillary pipette into one lateral ventricle or one eye of E12.5 or E14.5 mice, or the optic cup or central spinal cord canal of E1.5 or E2 chick embryos, respectively. For multicolor labeling, the mix contained 1 mg/ml of each LiOn CAGNFP vector and 0.6 mg/ml of PBase vector. Embryos were left to develop until sacrifice. Tissues were fixed in 4% PFA. Postnatal mouse brains were sectioned at 200-mm thickness with a vibrating microtome (VT1000, Leica), while mouse retinas, chick E6 spinal cords and E6-E8 retinas were flat-mounted on glass slides, Mouse and chicken embryonic electroporation In utero and in ovo electroporation in mouse and chicken embryos were performed as previously described, 2009.
, Immunostaining For cell cultures: HEK293 cells plated on glass coverslips or iPS cells were fixed with 4% PFA, followed by washing in PBS and a 20-60 min blocking step at room temperature. Blocking solution for HEK293 and iPS cells respectively contained 10% normal goat serum (Sigma) or fetal bovine serum (Eurobio) and 0.5% or 0.2% Triton X-100 (Sigma)
, 1:100) in PBS-0.1% Triton-1% NDS. Following PBS washes, slides were incubated 1 hr with secondary antibody (Alexa 647 donkey anti-mouse or anti-rabbit, Invitrogen, 1:500) in the above buffer, washed and mounted with Vectashield medium. For whole chicken spinal cords: embryos were fixed overnight in 4% PFA. Spinal cords were dissected, washed in PBS and incubated with Alexa 647-conjugated phalloidin (Molecular Probes, 1:250) in PBS-0.1% Triton-1% NDS for 3 hr at room temperature. Following PBS washes, spinal cords mounted in Vectashield medium. Fluorescence imaging and image analysis Epifluorescence images were, C with primary antibody diluted in blocking solution (rabbit anti-FLAG, Sigma, 1:250 or mouse anti-Tuj1, Biolegend, 1:500). After washing in PBS and incubation with secondary antibody (Alexa 647 anti-goat IgG, 1:500, or Alexa 488 anti-goat IgG, 1:1000, vol.40, p.3