Masaru Okabe, Ph.D. & Masahito Ikawa, M.S.
Research Institute for Microbial Disease & Faculty of Pharmaceutical Sciences,
Osaka University, Japan
Abstract
Since the first reports of expression of green fluorescent protein (GFP) in transgenic Caenhorhabditis elegans (1), Drosophila melanogaster (2), and zebrafish (3), this novel and simple reporter has attracted much interest for its potential as an in vivo marker in a wide variety of biological systems. Here we describe the establishment of stable lines of GFP transgenic mice and the ready detection of GFP fluorescence in living newborn mice and by various methods in many tissues.
Generation of transgenic mice
To test the utility of GFP as a marker in transgenic mice, we PCR-amplified the GFP coding sequence from pGFP10.1 (1, 4) and inserted it into the pCAGGS expression vector (5). The resulting plasmid contains the GFP coding sequence downstream of a Kozak consensus ribosome-binding site and under the control of the chicken beta-actin promoter and the immediate early enhancer of cytomegalovirus. A fragment of the plasmid was injected into the male pronuclei of 166 fertilized eggs of the B6C3F1 mouse strain, and the eggs were transferred into pseudopregnant ICR females.
The characteristic green fluorescence of GFP was readily detected upon the birth of three mice. As seen in the figure below, the toes of newborn transgenic mice were green when observed with a fluorescence microscope. The presence of the GFP transgene in the three founder mice was confirmed by PCR amplification of DNA isolated from tail tissue at age three weeks. All three mice were successfully bred to establish transgenic lines, each of which demonstrated stable, Mendelian transmission of the GFP transgene and 100% penetrance of GFP expression (6).
 | GFP expression in toes of living newborn transgenic mice. Fluorescence in toes of newborn founder mice was readily detectable under a fluorescence microscope. Examination of sectioned toes revealed that the fluorescence is due to GFP expression in the underlying muscle (data not shown). |
Widespread fluorescence detected by various methods
 | GFP expression in pancreas of living newborn transgenic mice. Pancreatic tissue was excised from transgenic descendants, mounted directly on glass slides under glass coverslips, and examined under a fluorescence microscope with UV excitation. No fluorescence was detected in nontransgenic litter mates. |
We also monitored GFP fluorescence in supernatants of homogenized tissues using a fluorometer (seen figure below). Fluorescence was detectable at ~200 ng/ml of total protein--a level comparable to more complicated assays for CAT. Finally, GFP fluorescence was detected in frozen thin-sections (fixed and untreated) of whole mice and various organs using a fluorescence microscope with a FITC-filter set (data not shown).
 | Fluorometric detection of GFP. Indicated organs were dissected from GFP transgenic (GFP-TG) or control mice and homogenized in PBS. The homogenates were centrifuged at 11,000 x g for 10 min, the supernatants were diluted, total protein was measured, and fluorescence was measured in a fluorometer using excitation at 490 nm and detection at 509 nm. |
GFP versus other transgenic markers
Even when invasive detection methods are required, GFP is generally easier to assay, and the sensitivity is comparable, at least to a first approximation, to other reporters. The utility of GFP transgenes is further enhanced by the recent demonstration that cells expressing GFP or red-shifted GFP can be readily separated from nonexpressing cells using fluorescence-activated cell-sorting (FACS; 7, 8). While our results demonstrate that fluorescence is easily detected in various tissues when the wild-type GFP coding sequence is expressed from a strong promoter/enhancer combination, the sensitivity may be significantly increased through the use of red-shifted (9) and/or codon-optimized variants.
References
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