Color-engineered rats for experimental regenerative medicine

Rats with genetically encoded color probes such as green and red fluorescent protein have been developed for specific biomedical research applications.
21 June 2007
Eiji Kobayashi and Takashi Murakami

Until the mid-1990s, it was believed that only a limited number of organs and tissues could be regenerated after birth. For example, repair in the adult brain was thought to be restricted to a post-mitotic event, i.e. not involving cell division. However, recent advances in regeneration medicine show promise in the restoration of form and function to damaged tissues. For instance, demonstrations of functional tissue regeneration and the isolation of tissue precursor cells from adult organs have offered insights into a new therapeutic approach towards the repair of damaged tissues. However, knowing the fate of transplanted cells and tissues using appropriate markers is essential to delineate the scientific basis of such research.

Although the use of fluorescent dyes is relatively straightforward, a significant drawback is that the fluorescence intensity decreases during in vivo cell proliferation. One solution is to use genetically-encoded biological probes that can provide high performance tools to visualize cellular fate in living animals.2 In particular, fluorescent proteins such as the green fluorescent protein (GFP) and DsRed, a recently cloned fluorescent protein, are increasingly used as internal biological light sources to investigate a wide variety of biological processes in living cells.1

Like the mouse, the rat represents an important animal model for biology and medical research, and has enabled the acquisition of a wealth of physiological and pharmacological data. Its larger body size relative to that of the mouse allows the use of various physiological and surgical manipulations that may prove biologically significant and cannot be performed on a mouse. Using advances in genetic engineering, we recently started developing a color-engineered rat system.1

Since the production of the first generation GFP-transgenic (Tg) Wistar rat in 2001,3 we have already developed ten genetically marked rats (see Table 1). These Tg rats have been used worldwide to investigate particular biological events in biomedical research fields as varied as neuroscience, cardiology, dermatology, transplantation and tissue engineering. Besides our group, many researchers have demonstrated potential applications, such as a high performance cellular source for cellular trafficking, trans-differentiation and tissue repair.1 Since these marker rats are capable of crossbreeding, it is also possible to generate a double-marker rat, and to visualize cellular fate as both fluorescence and luminescence images in a breeding combination.1

Table 1. Color-engineered rat colonies

However, it is not always easy to obtain transgenic animals that ubiquitously express a particular gene even under a general promoter, since expression of the injected DNA is dependent on the integration site within the genome. Furthermore, despite advances in genetic engineering, it remains difficult to handle rat eggs for DNA microinjection. This is why the construction of a reliable Tg rat system represents a time-consuming endeavor. A remarkable advance in the color-engineered rat system was recently provided by the GFP-Tg Lewis rat.4 This new line strongly expresses GFP in all tissues examined, and the cell source (such as neural progenitor cells) provides a high performance tool for the investigation of cellular fate.

Some of our initial fluorescent Tg lines were derived from the Wistar rat. The GFP-Wistar line is also useful in leukocyte-trafficking studies. For instance, a liver-specific reporter Tg Wistar rat (Alb-DsRed2) revealed the role played by bone marrow-derived cells during liver regeneration studies. Similarly, DsRed2/GFP double-reporter Tg Wistar rats were used in studies on the control of gene expression under Cre/LoxP site-specific recognition, providing effective materials for the elucidation of the cellular fusion process. However, the Wistar-derived Tg rats have a drawback. Since they are derived from an outbred strain of Wistar rats, transplanted cells are occasionally destroyed by immune responses,1 which is why we now recommend the use of inbred Lewis-derived Tg rat lines.

Coupled with recent advances in optical imaging, this newly developed transgenic rat system can provide innovative tools and a new platform to expand the scope of biomedical research. Spatio-temporal information gleaned from using this Tg system should also accelerate the development of therapeutic strategies for human diseases.

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