A novel approach to genetic engineering of T-cell subsets by hematopoietic stem cell infection with a bicistronic lentivirus

Abstract

Here we introduce a novel lentiviral construct utilizing a bicistronic lentiviral vector harboring two promoters: a global promoter (mPGK) for the expression of the selection marker (mCherry) and a second T-cell specific promoter (either dLck or CD3δ) expressing eGFP to track T-cell subsets. A dual promoter expression cassette with a cell specific second promoter permits definitive lineage specific transgenesis to investigate mechanistic and biological relevance of hematopoietic subsets in inflammatory disease models. Material and methods Lentiviral construct. Designed bicistronic lentiviral vectors harbor two constructs driven by two independent promoters. A schematic overview of the two used lentiviral vectors is presented in Fig. 1A. mPGK was used in both constructs as a global promoter located towards the 3′ end and expressing mCherry, which allows identification of successfully transduced cells. Since the global promoter is active in every cell-progenitor, T-cells and non-T-cells-successfully transduced HSCs and all their descendent daughter cells will be mCherry positive, which will allow their enrichment with fluorescence activated cell sorting (FACS) based techniques. The second T-cell specific promoter is located towards the 5′ end and was either the CMV enhanced distal Lckpromoter (dLck; 9892 bp) 8 or the CD3δ-promoter (9218 bp) 9 each controlling eGFP expression. Therefore, only committed cells with activity of the T-cell specific promoter will turn green, while non-T-cells and progenitor cells will be eGFP negative.