The CRE BAG 2 cell line was initiated from the NIH 3T3 embryonic Swiss mouse fibroblast cell line by transfection of the previously established line with Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions. The genomes contained a deletion of the psi sequence needed for the efficient encapsidation of retroviral genomes into virus particles, as well as further alterations at the 3' end of the provirus.

CRE BAG2 cells produce a beta-galactosidase-transducing vector (BAG) and are similar in many ways to the psi 2 BAG alpha line. The fibroblast line can be utilized to package vectors derived from murine leukemia viruses and is positive for reverse transcriptase. CRE BAG 2 cells grow adherently to both glass and plastic surfaces in culture.

A stable enzyme, beta-galactosidase is responsible for initiating the cellular breakdown of lactose, a sugar present in milk. This enzymatic activity occurs through a hydrolysis reaction, in which a water molecule associates with the lactose in such a way that the oxygen bridge that holds the two carbon-based rings that comprise the enzyme together is broken, resulting in the simple sugars glucose and galactose. The beta-galactosidase enzyme is quite easy to detect through biochemical means and is, therefore, considered opportune for monitoring the expression level specified by a gene reporter region. Thus, the lacZ gene that encodes the enzyme is widely utilized as a reporter gene in the field of molecular biology. Reporter genes, such as lacZ, are extremely useful for determining the pattern and timing of gene expression, which, in turn, may reveal important clues regarding gene function.

The cell culture featured in the digital image presented above was fluorescently labeled with MitoTracker Red CMXRos and SYTOX Green, targeting the mitochondrial network and cell nuclei, respectively. In addition, Alexa Fluor 633 conjugated to phalloidin was utilized to label filamentous actin. Images were recorded in grayscale with a 12-bit digital camera coupled to either a Nikon E-600 or Eclipse 80i microscope equipped with bandpass emission fluorescence filter optical blocks. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles with the exception of Alexa Fluor 633, which was pseudocolored blue.

Triple Fluorophore Labeling of CRE BAG 2 Fibroblasts with MitoTracker Red CMXRos, Alexa Fluor 488, and DAPI - The adherent culture of CRE BAG 2 fibroblast cells featured in this section was stained with MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin, and DAPI, which bind with mitochondria, F-actin, and nuclear DNA, respectively.