Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ
TL;DR: 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 is indicated.
read more
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.
read more
Chat with Paper
AI Agents for this Paper
Find similar papers on Google Scholar, PubMed and Arxiv
Write a critical review of this paper
Analyze citations of this paper to find unaddressed research gaps
Citations
Purified lactases versus whole-cell lactases-the winner takes it all.
TL;DR: In this paper, the characteristics of β-galactosidase enzymes, their origin, and ways of use are discussed in light of their potential for hydrolyzing lactose.
7
Dermal Stem Cells Can Differentiate Down an Endothelial Lineage
Emma Bell,Gavin D. Richardson,Colin A.B. Jahoda,Karl Gledhill,Helen M. Phillips,Deborah J. Henderson,W. Andrew Owens,Nicholas Hole +7 more
TL;DR: Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle.
7
A novel reporter strain to follow Cre-mediated recombination in T and NK cells.
TL;DR: A novel reporter strain of mice is described that is designed to fulfill criteria of the Cre‐loxP system and it is shown that this strain permits the monitoring of Cre‐mediated recombination in both T cells and NK cells.
6
Patent
Method for detecting interaction of proteins
Xiaomei Yan,Lina Wu,Xu Wang +2 more
- 18 Jul 2012
TL;DR: In this paper, a method for detecting protein-protein interaction in a single-bacterium level was proposed. But the method is not suitable for the detection of proteins at the single-cell level.
6
Mechanisms of Lymphocyte Activation and Immune Regulation III
Sudhir Gupta,William E. Paul,Max D. Cooper,Ellen V. Rothenberg +3 more
- 01 Jan 1991
TL;DR: The specificity of the a{3 T cell receptor for either class I or class II thymic MHC molecules determines the CD4-g+ and CD4+gphenotype of mature T cells.
6
References
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
TL;DR: A series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells.
8.2K
Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus
TL;DR: PMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production, and can be used to produce helper-free stocks of natural or synthetic defective retroviruses.
1.9K
Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.
TL;DR: It is shown that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo and that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds.
1.2K
Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice
TL;DR: Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloids and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages.
704
Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors
TL;DR: Bone marrow cells infected with retroviral vectors carrying the bacterial neomycin resistance (neo) gene as a marker were used for long-term reconstitution of the haematopoietic system of irradiated mice, indicating that these lineages are derived from the same primitive multipotent cells.
629