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Confocal Microscopy Image Gallery
Swiss Mouse Embryo Moloney Murine Leukemia Virus Transfected Fibroblast Cells (CRE BAG 2 Line)
CRE BAG 2 is a fibroblast cell line that was developed from the NIH 3T3 embryonic Swiss mouse cell line, which was transfected with Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions. The genomes were altered at the 3’ end of the provirus and contained a deletion of the psi sequence needed for the efficient encapsidation of retroviral genomes into virions.
Cellular products of the CRE BAG 2 line include a beta-galactosidase-transducing vector (BAG) that is capable of infecting both mouse and rat cells. The cells can package vectors derived from murine leukemia viruses and are comparable to psi 2 BAG alpha cells. CRE BAG 2 fibroblasts exhibit adherent growth to both glass and polymer surfaces in culture.
The primary role of the enzyme beta-galactosidase is to instigate the cellular breakdown of the sugar lactose. This function involves a hydrolysis reaction, in which a molecule of water associates with the sugar in such a way that the oxygen linkage that binds the two carbon-based rings of the enzyme together is broken. As a result, the lactose is transformed into the simple sugars glucose and galactose. The biochemical detection of beta-galactosidase is relatively simple, and the enzyme is, consequently, considered well suited for monitoring the expression level specified by a gene reporter region. Accordingly, the lacZ gene that encodes beta-galactosidase is commonly used as a reporter gene by molecular biologists.
Networks of microtubules and cell nuclei are revealed in this confocal image of CRE BAG 2 fibroblasts stained with Alexa Fluor 555 (tubulin) and TO-PRO-3 (DNA). The cells were fixed with 3.7 percent paraformaldehyde, permeabilized with Triton X-100, blocked with 10 percent normal goat serum and treated with mouse anti-alpha-tubulin primary antibodies. Goat anti-mouse secondary antibodies conjugated to Alexa Fluor 555 were mixed with the nucleic acid stain. Images were recorded with a 60x oil immersion objective using a zoom factor of 1.5 and sequential scanning with the 543-nanometer line from a green helium-neon laser and the 633-nanometer line of a red helium-neon laser. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.
Additional Confocal Images of Transfected Fibroblast (CRE BAG 2) Cells
Targeting Tubulin in CRE BAG 2 Cells with Immunofluorescence - In order to visualize the microtubules present in a culture of transfected Swiss mouse fibroblasts, the cells were immunofluorescently labeled with anti-tubulin mouse monoclonal primary antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 555 (pseudocolored blue). In addition, the cells were labeled for DNA in the nucleus with the red-absorbing dye TO-PRO-3.
Contributing Authors
Nathan S. Claxton, Shannon H. Neaves, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.