Identifying GFP-transgenic animals by flashlight.

In recent years, GFP has proven to be a valuable tool in many biological systems. Wild-type GFP has a major absorption in the UV region at 398 nm and a minor absorption in the blue region at 475 nm (3). Recently, several groups have independently carried out amino acid modifications of the GFP protein and have optimized these optical characteristics (6). For a comprehensive review on the photophysical behavior of GFP and GFP mutants, see Reference 7. Two widely used mutant forms of GFP, eGFP (BD Biosciences Clontech, Palo Alto, CA, USA) and mmGFP6 (6), have greatly enhanced absorbance at 475 nm, thus allowing excitation with blue light alone. Many lines of transgenic mice expressing GFP and GFP fusion proteins have been described. Commonly, such mice are maintained by intercrossing of heterozygotes, a breeding strategy that necessitates the identification of transgenic and non-transgenic progeny. In animals that express GFP widely, this can be done by examining tissue under a suitable fluorescence microscope. Small pieces of tissue removed from animals during ear-notching (for identification purposes) are suitable. However, this approach has several drawbacks. Ear-notching is an invasive procedure that must be performed under appropriate animal husbandry practise, is open to errors of misidentifying from which mice biopsies have come, and is a relatively time-consuming way of screening large numbers of litters. In addition, ears are not sufficiently developed to allow ear-notching before around three weeks of age. Previously, a genotyping protocol has been described that uses a UV light source and filters to visualize GFP in exposed tissue (1). Using this as a starting point, we investigated the possibility of using blue light excitation of GFP as a means to genotype GFP organisms. As a model, we used the tauGFP expressing transgenic line TgTP6.3 developed in our laboratory (4). These animals express a fusion protein in which GFP is joined to the microtubule-binding protein tau. The transgene is expressed at high levels and in most tissues. We decided not to use UV light, since we wanted to identify the GFP fluorescence in living animals and UV light is a hazard to both the operator and the animals. In addition, there were a number of other criteria for our GFP visualization equipment. We wanted it to be (i) noninvasive, (ii) easily brought in to the animal care facility, (iii) amenable to disinfection, (iv) quick to use (without significant warm-up time), (v) to be readily available, and (vi) inexpensive. A survey of commercially available macroscopic GFP visualization equipment found that the cheapest was more than $1100 for a system that satisfies points i, ii, iii, and iv, and v. However, these systems are designed to high specifications that far exceed our requirements. Blue light GFP visualization works by illuminating the tissue with light with a peak intensity at 475 nm and a steep decline in intensity at other wavelengths. Thus, the greatest possible amount of light from extraneous wavelengths is excluded. This is usually achieved by using an appropriate filter. The tissue is then visualized through a second barrier filter that excludes the blue light and passes only the emitted green fluorescent light. This is typically achieved using a filter that cuts out light from wavelengths less than 500 nm. These filters should also help to reduce the background autofluorescence. A “homemade” system for GFP detection has been previously described (5). This system used a single blue light emitting diode (LED) to visualize GFP in E. coli transformed with a GFP containing plasmid. However, we considered that a single blue LED would be unlikely to generate enough light to excite GFP to detectable levels in the transgenic animals in vivo. In addition, this system used a photomultiplier tube (PMT) to convert photons to an electrical signal for computer analysis. PMTs are sensitive detectors for low-intensity applications such as fluorescence and typically can create millions of electrons for each photoelectron detected. This suggests that without the PMT amplification the GFP signal would not be strong enough from a single blue LED. We have identified a commercially available blue LED flashlight (InovaX5; Emissive Energy, Warwick, RI, USA) with an emission wavelength of 470 nm. However, there are many available, and any blue LED flashlight with a wavelength of 470 nm would be suitable. The flashlight used here has five blue LEDs. Blue LEDs are also available from many semiconductor suppliers and a homemade flashlight could be produced. Several different types of blue LED are available. The compounds used in the LED manufacture govern the wavelength of the light. The majority of gallium nitride and indium gallium nitride on Al2O3 LEDs have a peak wavelength of around 470 nm: the exact wavelength is given in the Benchmarks