Retroviral Transduction and CD2 Marker Selection

Bicistronic retroviruses coding for CD2 marker alone (pMIC), CD2 followed by full-length human Sp1 (Sp1) or CD2 followed by mutated Sp1 Zn^2,3 (mutations of the second and third Zn Fingers) (Zn). CD2 marker allow transduced cells identification as previously described [9 (link), 14 (link)]. Retroviral particles were generated in 293T HEK packaging cells by transfection with Sp1, Sp1 Zn^2,3 (Zn), or empty vector (pMIC) plasmid and concentrated with sucrose gradient separation procedure. Retroviral transduction was performed in 6-well plates, and selection of CD2 transduced cells was released at indicated time by magnetic selection using autoMACS technology (Miltenyi Biotec), according to the manufacturer’s protocol. Briefly, transduced cells were incubated 30 min with 4μg/ml PE-conjugated monoclonal Ab against murine CD2 (BD Biosciences) in Phosphate-buffered saline (PBS) containing 0,5% Bovine Serum Albumin (BSA) and 2mM EDTA. Cells were labelled with anti-PE microbeads (Miltenyi Biotec) and CD2 positive cells were magnetically selected by passing through an autoMACS (Miltenyi Biotec). The purity of positive fractions was evaluated by flow cytometry and was routinely above 95%.