248 FLOW AND IMAGE CYTOMETRY, Montpellier, Decembcr 1992

RECOMBINANT RETROVIRUSES, B-GALACTOSIDASE AND FLOW CYTOMETRY: FOUR YEARS OF HAPPY MARRIAGE

H61~ne JOUIN

Unit6 de Parasitologle Exp6rimentale 25, rue du Docteur Roux - 75724 PARIS CEDEX 15 (FRANCE)

Retroviruses are RNA viruses whose life cycle includes their replication by a DNA intermediate that integrates into the host genome (1). Transcription of the provirus leads to the production of virions without any apparent cytopathic effect on the cells. Because integration into the host chromosome is an obligatory step in their life cycle, retr.oviruses are widely used as vectors.

In retroviral vectors, exogeneous genes replace portions of the retroviral genome. These recombinant viruses like the wild type, can integrate into the host genome and be inherited by daughter cells. Such retroviruses have been used to introduce a number of genes into cultured cells and post-implantation murine embryos. Because recombinant retroviruses are generally incapable of producing new virions without the aid of a helper virus, their genomes are only passed to the progeny of the infected cell. If the exogenous gene encodes a protein thai can be detected within single cells, the individual clonal descendants of the infected cell can be histochemically or immunohistochemically labelled.

The luciferase, chloramphenicol-acetyl- transferase, .neomycine resistance and E. Coil 8-galactosidase (Lac Z gene) genes are the most commonly utilized reporter genes.

However, in 1988 (2), a new probe for the detection of 13-galactosidase activity, fluorescein di-13-galactopyranoside (FDG) renewed interest in the Lac Z gene, since it allows one to viably stain, analyze and sort by flow cytometry, cells infected with a recombinant retrovirus expressing the lac Z gene. FDG, a non fluorescent substrate of rJ-galactosidase, is cleaved by the enzyme to yield fluorescein. As the cells are not significantly permeable, to FDG, the substrate is introduced by a hypotonic shock of a few minutes and the enzymatic reaction develops thereafter.

Accumulation of the fluorescent products in cells is linear with time and is directly correlated with enzymatic activity. By simply stipulating to the cell sorter the desired upper and lower limits of fluorescence, cells expressing Lac Z at different levels can be isolated.

This basic method has found application in a variety of studies: - the analysis of gene expression during mouse embryogenesis (3). - the study of interleukin 2 gene expression at the single cell level (4). - the titration of Human Immunodeficiency Virus (HIV) and HIV-infected cells (5).

In addition, the FDG assay has been adapted to bacteria and yeast (6).

These examples indicate that the association between recombinant retroviral vectors carrying the Lac Z gene and flow cytometry has an exciting future.

References

1. BALTIMORE D., 1970, 226, 1209-1211. 2. NOLAN G.P., FIERING S., NICOLAS J.F. and HERZENBERG L.A., 1988, PNAS, 85, 2603-2607. 3. NICOLAS J.F., BONNEROT C., KRESS C., JOUIN H., BRIAND P., GRIMBER P. and VERNET M., 1989. In vectors as tools for the study of normal and abnormal growth and differenciation, Springer-Verlag, Heidelberg, Berlin, 34, 33-45. 4. EMILIE D., PEUCHMAUR M., BARAD M., JOUIN H., MAILLOT M.C., COUEZ D/, NICOLAS J.-F. and MALISSEN B., 1989. Eur. J. Immunol., 19, 1619-1624. 5. ROCANCOURT D., BONNEROT C., JOUIN H., EMERMAN M. and NICOLAS J.-F., 1990. J. Virol., 64, 2660-2668. 6. NIR R., YISRAELI Y., LAMED R. and SAHAR E., 1990. Appl. Environ. Microbiol., 56, 3861-3866.