Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Apr;85(8):2603–2607. doi: 10.1073/pnas.85.8.2603

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

G P Nolan 1, S Fiering 1, J F Nicolas 1, L A Herzenberg 1
PMCID: PMC280046  PMID: 3128790

Abstract

We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated 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 beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-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 fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-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.

Full text

PDF
2603

Images in this article

2605Image
on p.2605
2605Image
on p.2605

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberti S., Parks D. R., Herzenberg L. A. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry. 1987 Mar;8(2):114–119. doi: 10.1002/cyto.990080203. [DOI] [PubMed] [Google Scholar]
  2. Bonnerot C., Rocancourt D., Briand P., Grimber G., Nicolas J. F. A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6795–6799. doi: 10.1073/pnas.84.19.6795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dick J. E., Magli M. C., Huszar D., Phillips R. A., Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell. 1985 Aug;42(1):71–79. doi: 10.1016/s0092-8674(85)80102-1. [DOI] [PubMed] [Google Scholar]
  4. Feinstein S. C., Ross S. R., Yamamoto K. R. Chromosomal position effects determine transcriptional potential of integrated mammary tumor virus DNA. J Mol Biol. 1982 Apr 15;156(3):549–565. doi: 10.1016/0022-2836(82)90266-2. [DOI] [PubMed] [Google Scholar]
  5. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hall C. V., Jacob P. E., Ringold G. M., Lee F. Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet. 1983;2(1):101–109. [PubMed] [Google Scholar]
  7. Hofmann J., Sernetz M. A kinetic study on the enzymatic hydrolysis of fluorescein diacetate and fluorescein-di-beta-D-galactopyranoside. Anal Biochem. 1983 May;131(1):180–186. doi: 10.1016/0003-2697(83)90151-3. [DOI] [PubMed] [Google Scholar]
  8. Jolly D. J., Willis R. C., Friedmann T. Variable stability of a selectable provirus after retroviral vector gene transfer into human cells. Mol Cell Biol. 1986 Apr;6(4):1141–1147. doi: 10.1128/mcb.6.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jongkind J. F., Verkerk A., Sernetz M. Detection of acid-beta-galactosidase activity in viable human fibroblasts by flow cytometry. Cytometry. 1986 Sep;7(5):463–466. doi: 10.1002/cyto.990070512. [DOI] [PubMed] [Google Scholar]
  10. Keller G., Paige C., Gilboa E., Wagner E. F. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature. 1985 Nov 14;318(6042):149–154. doi: 10.1038/318149a0. [DOI] [PubMed] [Google Scholar]
  11. MacGregor G. R., Mogg A. E., Burke J. F., Caskey C. T. Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene. Somat Cell Mol Genet. 1987 May;13(3):253–265. doi: 10.1007/BF01535207. [DOI] [PubMed] [Google Scholar]
  12. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  13. Norton P. A., Coffin J. M. Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication. Mol Cell Biol. 1985 Feb;5(2):281–290. doi: 10.1128/mcb.5.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Price J., Turner D., Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):156–160. doi: 10.1073/pnas.84.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. ROTMAN B. Measurement of activity of single molecules of beta-D-galactosidase. Proc Natl Acad Sci U S A. 1961 Dec 15;47:1981–1991. doi: 10.1073/pnas.47.12.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. ROTMAN B., ZDERIC J. A., EDELSTEIN M. Fluorogenic substrates for beta-D-galactosidases and phosphatases derived from flurescein (3,6-dihydroxyfluoran) and its monomethylether. Proc Natl Acad Sci U S A. 1963 Jul;50:1–6. doi: 10.1073/pnas.50.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanes J. R., Rubenstein J. L., Nicolas J. F. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. doi: 10.1002/j.1460-2075.1986.tb04620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Srienc F., Campbell J. L., Bailey J. E. Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations. Cytometry. 1986 Mar;7(2):132–141. doi: 10.1002/cyto.990070203. [DOI] [PubMed] [Google Scholar]
  19. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES