Figure 4.

Structure of monocistronic and bicistronic constructs inserted in lentiviral vectors for combined expression of the luciferase 2 and truncated rat CD2 proteins. Lentiviral vectors were designed for expression of the luciferase 2 protein combined with a truncated form of the rat CD2 (tCD2) protein either as a fusion protein (monocistronic insert) or as two distinct proteins (bicistronic insert). Both types of constructs are inserted at an EcoR1 site in the pro-lentiviral plasmid under the control of the EF-1 alpha promoter and flanked by the WPRE sequence. Recombinant lentiviral particles were produced by tri-transfection in 293T cells as explained in the Materials and Methods section. A) The tCD2-luc2 insert contains the first 232 codons of the rat CD2 gene fused to 2 codons GAA (glutamic acid) and GTC (valine) added for cloning purpose (EcoR1 site) in frame with the full length luciferase 2 gene starting at codon 236. B) The bicistronic insert - luc2-IRES-tCD2 - contains the full length luciferase 2 gene linked by an IRES (Internal Ribosome Entry System sequence) to the truncated rat CD2 gene. This IRES (573 base pairs) was derived from the pQCXIX plasmid (Clontech).