May 18, 2009

New Proteins Help Peer Inside Living Animals

Image of mouse torso with glowing blue patch indicating the liver The liver of a live mouse glows with infrared fluorescent proteins (IFP), which can be seen through the animal's skin.Dr. Xiaokun Shu, University of California, San Diego.

Scientists have developed a new fluorescent molecule that emits infrared light bright enough to be detected deep within the tissues of a living mouse. With further development, this type of molecule could aid the study of cancer, infections and other biological processes in small animals.

For more than a decade, scientists have used jellyfish-derived green fluorescent protein (GFP) and other fluorescent proteins to tag molecules and watch how they move and interact. However, the relatively short, visible wavelengths of light used to excite these fluorescent molecules don't allow researchers to see deep into tissues.

To overcome this limitation, Dr. Roger Tsien and Dr. Xiaokun Shu of the University of California, San Diego, and their colleagues decided to develop fluorescent proteins that can be excited by much longer far-red light and emit infrared light. Unlike short-wavelength visible light, these longer wavelengths can deeply penetrate skin and underlying tissues without being so easily waylaid by blood, water and fat. The research was funded by NIH's National Institute of General Medical Sciences (NIGMS) and the Howard Hughes Medical Institute.

The scientists started with a light-absorbing photoreceptor from the bacterium Dienococcus radiodurans. When exposed to visible red light, this photoreceptor molecule—called a phytochrome—doesn't normally emit light, or fluoresce. Rather, it absorbs light and uses that energy to signal the activation of certain bacterial genes. However, a mutant version of the phytochrome was recently discovered to fluoresce.

As reported in the May 8, 2009, issue of Science, the researchers genetically modified the phytochrome to remove its ability to signal gene activation in response to light. This allowed the molecule to funnel even more light energy toward fluorescence rather than other processes.

When inserted into bacteria, the newly engineered phytochrome gene produced molecules that weakly fluoresced infrared light in response to far-red light. After several rounds of bacterial reproduction and directed evolution, the researchers ended up with an infrared fluorescent protein (IFP) that's about 4 times brighter than their original version.

To test the molecule, the scientists inserted an IFP gene into an adenovirus that infects mouse liver. Five days after the mice were injected with the virus, the liver emitted an infrared glow that could easily be detected through the living animal's skin.

As with GFPs, the new IFPs will likely have a big impact on basic research and animal studies. However, their clinical use will be limited because it would involve introducing fluorescent genes into humans. " Introducing such genes into people would pose scientific and ethical problems," says Tsien, who received the 2008 Nobel Prize in Chemistry for his role in developing GFP for biological research.

Tsien and his colleagues are now working to create improved IFPs from among the huge numbers of similar molecules harnessed from other organisms. More than 1,500 bacteriophytochrome-like gene sequences are already available from NIH's National Center for Biotechnology Information and other databases to aid their efforts.

—by Vicki Conte