In situ detection of transcriptionally active chromatin and genetic regulatory elements in individual viable mammalian cells.
Using a newly developed FACS method for quantifying the expression of the Escherischia coli lacZ reporter gene in viable mammalian cells, we have obtained cloned cell lines in which the expression of lacZ is under the control of native endogenous transcription elements. We infected the murine pre-B cell 70Z/3 with transcriptionally disabled retroviruses containing lacZ and employed the FACS-FDG technique to detect and sort rare lacZ+ cells in which we expect integration is near such endogenous transcription elements. After two rounds of enrichment we obtained a population of cells that was 80-90% positive for lacZ activity. Clones derived from the lacZ+ pool differ from each other with respect to their overall level of lacZ activity as well as in the pattern of lacZ expression among cells within an individual clone. Treatment of these lacZ+ 70Z/3 clones with lipopolysaccharide (LPS; which is known to stimulate differentiation of 70Z/3 from a pre-B cell to an IgM-expressing B cell) greatly decreased lacZ expression in one clone, 7e17. lacZ expression in this clone was 50-100 times lower within 24 hr of LPS addition and coincided with the acquisition of IgM kappa on the surface of 7e17. This suggests that a transcriptionally active domain of chromatin that harbors the lacZ construct is down-regulated during the transition induced by LPS stimulation.
Kerr
W G
WG
Department of Genetics, Stanford University, California, 94305.
Nolan
G P
GP
Herzenberg
L A
LA
D000818:Animals; D001402:B-Lymphocytes / Q000032:analysis*; D002469:Cell Separation; D002843:Chromatin / Q000502:physiology*; D002999:Clone Cells; D005434:Flow Cytometry; D007763:Lac Operon; D051379:Mice; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection
D002843:Chromatin; D014157:Transcription Factors