Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations.

Abstract

Background: Cell transduction with multiple genes offers opportunities to investigate specific gene interactions on cell function. Detection of multiple transduced genes in hematopoietic cells requires strategies to combine measurements of gene expression with phenotypic cell discriminants. We describe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopulations using only a minor physical adjustment to a standard FACSCalibur.

Methods: The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP) detection in mixtures of transduced and nontransduced PG13 packaging cells were evaluated by flow cytometry. Retroviral vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoietic cells derived from umbilical cord blood. The transduction efficiency into subpopulations of hematopoietic cells was measured using multivariate flow cytometry.

Results: A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduced cells than a retroviral vector encoding a pac-EGFP fusion protein. The sensitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively. EGFP or EYFP was expressed in up to 95% of CD34(+) DR(-) or CD34(+) 38(-) subpopulations in cord blood 48 h posttransduction. Simultaneous transduction with EGFP and EYFP viral supernatants (1:1 mixture) led to coexpression of both GFP variants in 15% of CD34(+) DR(-) and 20% of CD34(+) 38(-) cells.

Conclusions: These results demonstrate simultaneous detection of EGFP and EYFP in immunophenotypically discriminated human hematopoietic cells. This technique will be useful to quantify transduction of multiple retroviral constructs in discriminated subpopulations.