Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

Abstract

We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli fi-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescenceactivated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for 13-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of f-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescenceactivated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of (3-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.The Escherichia coli lacZ gene has previously been adapted for use in mammalian cells and has shown utility as a marker for the expression of chimeric genes (1, 2). In these previous studies, the chromogenic f3-galactoside analog o-nitrophenyl /3-D-galactopyranoside (ONPG) was used as an alternative to the chloramphenicol acetyltransferase assay (3) to quantitate gene expression from heterologous promoters fused 5' proximal to the lacZ structural gene. However, in both the chromogenic LacZ and radioactive chloramphenicol acetyltransferase assays, extracts of bulk populations of cells are used to quantitate an average promoter strength/enzyme activity and, thus, do not give any indication of the heterogeneous expression patterns that could exist within complex cell populations. These other assays also do not provide one with a means to select viable cells expressing known quantities of gene product.lacZ has also proven effective as a marker in the genetic tagging of cell lineage founders and subsequent tracing of progeny in tissue sections using a cytochemical stain for expression of LacZ (4, 5). Using recombinant, replicationdefective Moloney murine leukemia viruses (Mo-MuLV) in which the expression of lacZ is permitted from an internal simian virus 40 (SV40) promoter, Sanes et al. (4) Here, we use the f3-galactoside analog fluorescein di-,B-Dgalactopyranoside (FDG) (11) in a protocol, termed " 'ACS-FDG," that sensitively distinguishes LacZ' cells from LacZ-cells and allows time-dependent fluorescenceactivated cell analysis and sorting. FDG is cleaved by 03-galactosidase in LacZ' cells to yield fluorescein, which can be detected by FACS. We find that the kinetics of fluorescence accumulation within LacZ' cells, after FDG substrate loading, shows a linear dependence over time until maxima are approached because of...

Severity and Frequency of Proximal Tubule Injury Determines Renal Prognosis

“…We generated Ndrg1 CreERT2/+ :iDTR:USAG-1 +/LacZ mice and subjected these kidneys to FACS analysis, in which galactosidase cleaves fluorescein di-b-D-galactopyranoside (FDG) and releases a fluorescence product FITC. 31 Efficient sorting of distal tubules was confirmed by high expression of distal tubule markers (USAG-1 and uromodulin) and low expression of megalin, a proximal tubule marker, in LacZ-positive cells (P3) ( Figure 7C). Using this system, we showed the upregulation of Ngal and osteopontin in P3 after DT injection ( Figure 7D), confirming the presence of distal tubule injury.…”

“…Fluorescein di-b-D-galactopyranoside (SigmaAldrich) was used to distinguish LacZ-positive cells as described previously. 31 Coculture of NRK52E and NRK49F Cells NRK-52E and NRK49F cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured with DMEM/ 5%FCS. Transwell (Corning, Corning, NY) was used for coculture experiments.…”

A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells

“…Although we have not determined the absolute GFP reporter sensiadvantages over cell surface or enzymatic markers to evaluate and optimize gene transfer and expression in tivity, which is dependent upon background cellular human hematopoietic (and other target) cells. 32 In other selection schemes based on drug resistance in that an optimal drug selection window for the progenitor cells addition to eliminating specific antibody staining or enzymatic reactions (eg for lacZ) to detect the reporter by being assayed does not have to be determined a priori (indeed, this is not always readily achievable) and, as in flow cytometric analysis, 33 autonomous fluorescence of GFP enables facile monitoring of reporter expression in the case of the CAFC assay, detection of GFP expression does not require the existence of a drug-resistant stromal situ as illustrated herein. Although G418 resistance provides an estimation of gene transfer and expression in cell line.…”

VE‐cadherin‐CreERT2 transgenic mouse: A model for inducible recombination in the endothelium

“…Cells were then filtered (40 m) and transplanted into lethally irradiated (1,000 cGy) C57Bl mice (5 ϫ 10 6 cells by means of tail vein injection). A subset of the cells prepared for transplant also underwent FACS analysis as described (Nolan et al, 1988). In summary, bone marrow cells were hypotonically loaded with fluorescein di-␤-D-galactopyranoside (Molecu-lar Probes-Invitrogen) for LacZ expression and stained with 7AAD (BD Pharmingen) for viability.…”