Bringing Science Back to the Laboratory Rat
“The derivation of rat sperm stem cell lines will allow Scientists to Produce New Genetic Models
for Human Disease in the Most Widely Studied Laboratory Mammal.”
More than 20 years have passed since the advent of genetically manipulating the mouse germline using cultures of pluripotent embryonic stem cells. Still, despite remarkable successes in the mouse, the application of stem cell cultures for transgenesis in other mammalian species has been comparatively non-existent. By focusing on the laboratory rat as a widely popular model species in science, our laboratory has tapped into the advantages of the spermatogonium as an alternative type of germline stem cell for transgenesis. We now have established protocols for isolating, propagating, genetically modifying and determining the germline transmission rates of spermatogonial cultures for the production of transgenic rats. Because the full potential of spermatogonia has yet to be realized in animal genetics, our laboratory is working enthusiastically to establish their application as novel germline vectors for transgenic/genetic technologies. Notably, gene manipulations directly in the spermatogonium could simplify production of germline founders while bypassing the intermediate need to produce and screen chimeric progeny that are first derived from micro-manipulated embryos. Once experimental conditions for producing genetically modified animals using clonal, genetically selected spermatogonial cultures are optimized, the approach holds potential to facilitate targeted germline modifications by gene replacement, gene-insertion and/or restriction endonuclease technologies in a diversity of mammals (Figure 8).
Genetic Screens in the Laboratory Rat
A most exciting breakthrough resulting from our studies on spermatogonial culture is that we have now invented a new technology for generating large panels of mutant rats that scientists can use to study the pathology of essentially any human disease.
To do this, we used a special DNA construct, termed the Sleeping Beauty transposon. When introduced into sperm stem cells by gene delivery methods, the Sleeping Beauty transposon incorporates preferentially into key expression elements of genes in the rat genome, and thereby disrupts their function (Figure 9). In essence, because Sleeping Beauty gets inserted randomly into all different classes of functional genes, we have now generated what could be described as “Sperm Stem Cell Libraries of Mutant Rats”. Such genomic libraries can literally shelve tens of thousands of mutant rats within just a few cryo-preservation vials of frozen sperm stem cells. Due to the potentially enormous impact that these mutant animals will have on biomedical research, we have focused a great deal of our effort over the last year to demonstrate that we can actually generate these libraries. Indeed, we have now shown that we can construct these libraries with stem cells cultured in our own specially formulated “Spermatogonial Medium” and then use them to produce clinically relevant models of mutant rats for studying human disease (see link to Knockout Rats)25.