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 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.

An in vitro messenger RNA binding assay as a tool for identifying hybridization-competent antisense oligonucleotides.

The apparent dissociation constants for 32 phosphodiester and 5 phosphorothioate antisense oligodeoxyribonucleotides (ODN) targeted to murine tumor necrosis factor-alpha (mTNF-alpha) mRNA were determined using a gel-shift binding assay. In this assay, radiolabeled ODN were hybridized in solution to a structured mRNA transcript generated in vitro. Free ODN was resolved from bound ODN on a two-phase discontinuous polyacrylamide gel. Excision of gel slices containing free ODN or bound ODN, followed by Cerenkov counting of the slices, was used to prepare apparent binding isotherms for each ODN. Apparent dissociation constants for the anti-mTNF-alpha ODN varied from > 100 microM to 0.4 nM. Slight differences in RNA target site position resulted in significant differences in apparent affinity, particularly for shorter (12-mer) ODN. This binding assay provides an empirical means for selecting ODN sequences possessing high affinity for a target RNA and lends itself to a high throughput assay in which all possible antisense sequences of a given length can be evaluated to obtain the better binders for use in cell culture or in vivo.

Antigene, ribozyme and aptamer nucleic acid drugs: progress and prospects.

Nucleic acids are increasingly being considered for therapeutic uses, either to interfere with the function of specific nucleic acids or to bind specific proteins. Three types of nucleic acid drugs are discussed in this review: aptamers, compounds which bind specific proteins; triplex forming (antigene) compounds; which bind double stranded DNA; and ribozymes (catalytic RNA), which bind and cleave RNA targets. The binding of aptamers to protein may involve specific sequence recognition, although this is not always the case. The interaction of triplex forming oligonucleotides or ribozymes with their targets always involves specific sequence recognition and hybridization. Early optimism concerning the possibility of designing drugs without a priori knowledge of the structure of the target (except a nucleotide sequence) has been tempered by the finding that target structure has a dramatic effect upon the hybridization potential of the nucleic acid drug. Other obstacles to the creation of effective nucleic acid drugs are their relative high molecular weight (> 3300) and their sensitivity to degradation. The molecular weight of these compounds has created a significant delivery problem which needs to be solved if nucleic acid drugs are to become effective therapies.

Single-stranded phosphodiester and phosphorothioate oligonucleotides bind actinomycin D and interfere with tumor necrosis factor-induced lysis in the L929 cytotoxicity assay.

Antisense oligonucleotides may prove useful tools to elucidate the biological functions of cytokines and to address regulatory control of cytokine expression. The functional activity of cytokines generally is determined by biological assays, and a standard biological assay for TNF activity measures the cytotoxic effect of TNF on actinomycin D-sensitized L929 cells (Matthews and Neale, 1987). We observed that phosphorothioate and phosphodiester oligonucleotides, at concentrations > 100 nM in supernatants tested in this bioassay, prevented TNF-induced lysis of L929 cells. This "protective" effect was due to an interaction of the single-stranded oligonucleotides with actinomycin D as demonstrated by UV spectra of an actinomycin D-oligonucleotide solution. Substitution of cycloheximide for actinomycin D in the L929 assay eliminated the protective effect of the oligonucleotide. Our results reinforce the importance of controlling for nonspecific effects of oligonucleotides, particularly when a functional assay for protein activity is used to screen for antisense-mediated reductions in target protein expression.

Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices.

Antisense oligonucleotides (ASOs) are designed to bind to a specific mRNA and selectively suppress its translation. To facilitate selection of optimal ASO targets, we have developed three thermodynamic indices to evaluate putative structural complexes important in ASO action. These indices are: a secondary structure score (Sscore), which estimates the strength of local mRNA secondary structures at the ASO target site; a duplex score (Dscore), which estimates the delta Gformation for the ASO:mRNA target sequence duplex; and a competition score (Cscore), which is the difference between the Dscore and the Sscore. We also present two histograms to graphically display these indices from different regions of the mRNA. The indices are compared to the inhibition reported in five studies of ASO-mediated suppression of gene expression. The Dscore is the most consistent predictor of ASO efficacy in four of the five studies (r2 from 0.44 to 0.99), while the results of the fifth study could not be predicted by any thermodynamic or physical index. Thus the Dscores and their histogram may prove useful in selection of ASO targets.