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

Background: Cell transduction with multiple genes offers opportunities to i nvestigate specific gene interactions on cell function. Detection of multip le transduced genes in hematopoietic cells requires strategies to combine m easurements of gene expression with phenotypic cell discriminants. We descr ibe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopu lations using only a minor physical adjustment to a standard FACSCalibur. Methods: The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced y ellow fluorescent protein (EYFP) detection in mixtures of transduced and no ntransduced PG13 packaging cells were evaluated by flow cytometry. Retrovir al vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoiet ic cells derived from umbilical cord blood. The transduction efficiency int o subpopulations of hematopoietic cells was measured using multivariate flo w cytometry. Results: A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduce d cells than a retroviral vector encoding a pac-EGFP fusion protein. The se nsitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively. EGFP or EYFP was ex pressed in up to 95% of CD34(+) DR+ or CD34(+) 38 subpopulations in cord bl ood 48 h posttransduction. Simultaneous transduction with EGFP and EYFP vir al supernatants (1:1 mixture) led to coexpression of both GFP variants in 1 5% of CD34(+) DR+ and 20% of CD34(+) 38(+) cells. Conclusions: These results demonstrate simultaneous detection of EGFP and E YFP in immunophenotypically discriminated human hematopoietic cells. This t echnique will be useful to quantify transduction of multiple retroviral con structs in discriminated subpopulations. Cytometry 40:126-134, 2000. (C) 20 00 Wiley-Liss, Inc.