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.
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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.
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Citations
Quantitative detection of lac-Z-transfected CC531 colon carcinoma cells in an orthotopic rat liver metastasis model.
TL;DR: An orthotopic liver metastases model based on CC531 rat colon adenocarcinoma cells which were transfected with a β-galactosidase gene as marker to facilitate their detection is established and it is envisaged that the model will have applications for various therapeutic strategies.
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Patent
Antibody constructs for flt3 and cd3
Tobias Raum,Jochen Pendzialek,Claudia Bluemel,Franziska Bott,Christoph Dahlhoff,Patrick Hoffmann,Elisabeth Nahrwold +6 more
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TL;DR: In this article, a bispecific antibody construct comprising a first binding domain which binds to human FLT3 on the surface of a target cell and a second binding domain binding to human CD3 on a T cell was described.
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HCV cDNA transfection to HepG2 cells
TL;DR: It is suggested that replication and translation of HCV were achieved for a prolonged time in HepG2 cells after transfection with HCV cDNA and may provide an in vitro system for HCV studies.
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A single-cell assay of beta-galactosidase in recombinant Escherichia coli using flow cytometry.
TL;DR: The feasibility of the flow cytometric assay for single E. coli cells is described and a direct relationship between β‐galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction.
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Simultaeous detection of β‐galactosidase activity and surface antigen expression in viable haematopoietic cells
TL;DR: Beta-galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells and is readily combined with fluorescently labelled antibodies against cell surface antigens.
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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.
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