Figure 1

Stable Transgenesis and Sorting Systems. A: Tol2 transposition system: Cells are cotransfected with the plasmid carrying the Tol2 construct (Expression plasmid) and the helper plasmid, allowing a transient transposase expression (pCAGGS-T2TP). On the one hand, the Tol2 construct contains the expression cassette that should be integrated in the cellular genome, flanked by two Tol2 cis-sequences. On the other hand, the helper plasmid contains the transposase cDNA driven by a CAGGS promoter (a CMV-based constitutive promoter). The Tol2 construct is transposed by a cut-and-paste mechanism that requires the sequence-specific binding of the transposase to the Tol2 sequences on each extremity of the cassette. This transposition process involves the precise excision from the plasmid and integration of the cassette into the genomic DNA. B: Magnetic Cell Sorting (MACS): Cells are transfected with a plasmid construct harboring both the transgene and the ΔhCD4-coding sequence. The ΔhCD4 is a surface marker, derived from the human CD4 in which the cytoplasmic region was deleted. Positive cells, expressing both transgene and the ΔhCD4 marker, are magnetically labeled with an antibody, coupled to metallic microbeads, which recognizes the hCD4 extracellular region. After passing the heterogeneous cell population through a column placed on a magnetic field, labeled cells are retained by the column, whereas negative cells pass through. Positive cells are then easily eluted by just moving the column away from the magnet. C: The engineered pT2MIK empty plasmid with a multicloning site (MCS) and the pT2MIK-eGFP plasmid used throughout this study.