eNews April 01, 2015

Introduction to fluorescent proteins and lacZ strains


GFP

Green Fluorescent Protein (GFP) is a single polypeptide gene product of 238 amino acids discovered in the jellyfish Aequorea victoria. The protein has a natural green fluorescence under specific illuminating conditions. The protein derives its bioluminescence from Ser-Tyr-Gly cyclization within its primary amino acid sequence. GFP is quite stable and withstands a number of chemical treatments and procedures.1 The first demonstration that this fluorescent protein could be expressed in a heterologous system was in C. elegans.2 GFP has since become a reporter gene of choice because it requires no biochemical transformation, contrast agent or the use of harmful ionizing radiation in order to be visualized.3 Since that initial report, GFP reporter gene expression has been reported in multiple organisms, including mice.4 In addition, under the direction of a specific promoter, transgenic GFP mice can express the fluorescent protein in a tissue and even a cell specific fashion. The non-invasive visualization is the key to being able to monitor physiological and biochemical processes in vivo and in real time.5

There are numerous ways to visualize the bioluminescence of GFP. One way to detect the fluorescence is with a hand-held UV light (365 nM). There are several models offered by Fisher in a price range of about $100 to $200. The hand-held UV light method does not work very well for the GFPX transgenic strain (Stock 003116). Dr. Andras Nagy, donating investigator of GFPX and GFPU transgenics (Stock 003115 and 003116), describes a viewer headset and a microscope adaptable filter that are now available commercially.

CFP and YFP

Recent advances have improved the characteristics and utility of GFP as a reporter gene. Enhanced GFP (EGFP) has been engineered to be expressed at higher levels in mammalian cells and to fluoresce more intensely. Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) are spectral variants of GFP that allow multiple cell types to be labeled simultaneously.

References

Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448-55.

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802-5.

Hoffman RM. 2002. Green fluorescent protein imaging of tumor cells in mice. Lab Animal 31(4): 34-41.

Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 1997. 'Green mice' as a source of ubiquitous green cells. FEBS Lett 407:313-9.

Yang M, Baranov E, Jiang P, Sun FX, Li XM, Li L, Hasegawa S, Bouvet M, Al-Tuwaijri M, Chishima T, Shimada H, Moossa AR, Penman S, Hoffman RM. 2000. Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci USA 97:1206-11.

lacZ

The use of a reporter gene can allow for examination of spatial patterns of gene expression of a particular promoter within a tissue, embryo, or adult mouse.1 The E. coli lacZ gene, when integrated into the mouse genome by transgenic techniques, can be used as a reporter gene under the control of a given promoter/enhancer in a transgene expression cassette. The lacZ gene encodes beta-galactosidase, which catalyzes the cleavage of lactose to form galactose and glucose. Beta-galactosidase activity can be identified by both in situ and in vitro techniques when incubated with the beta-galactosidase substrate X-gal. Beta-galactosidase cleaves X-gal, a chromogenic substrate, resulting in an insoluble blue dye, thus allowing for the identification of cells with lacZ activity.2 Transgenic animals can then be used to identify factors and conditions which modulate the expression profile of the promoter or enhancer.

References

Schmidt A, Tief K, Foletti A, Hunziker A, Penna D, Hummler E, Beermann F. 1998. lacZ transgenic mice to monitor gene expression in embryo and adult. Brain Res Brain Res Protoc 3(1):54-60.

Monastersky GM, Robl JM, eds. 1995. "Strategies in Transgenic Animal Science." American Society for Microbiology Press. Washington, DC.