A team of investigators from TaconicArtemis, the University of Wurzberg, and the University of Gottingen Medical School last month published a report describing how they developed an inducible and reversible gene-silencing system in transgenic rats.
In the paper, which appeared last month in the Proceedings of the National Academy of Sciences, the team used the system to silence in rats the insulin receptor gene, which triggered an increase in blood glucose while impairing glucose regulation and insulin signaling. Re-activation of the gene eliminated these diabetes symptoms.
"Up to now the availability of tools for loss-of-function mutations in rats was limited,” Jost Seibler, head of the RNAi research department at TaconicArtemis, said in a statement. "The possibility to modify gene function in transgenic rats … opens up novel scientific possibilities in academic and pharmaceutical research."
“Contract breeding and genetic characterization … will allow us to make these models available to academia and pharmaceutical companies,” TaconicArtemis CEO Peter Stadler added. Taconic, TaconicArtemis’ parent company, offers such services, he noted.
While traditional knockouts have been widely used to study gene function in mice, there are disadvantages to the technology, including its inapplicability to other species and inability to re-activate genes, according to the paper.
Aiming to address these issues, the researchers turned to RNAi, although they were not the first to do so: Other previously developed in vitro inducible shRNA-expression systems for the temporal inactivation of genes using RNA polymerase III promoters that contain operator sequences of the E. coli tetracycline-resistance operon, the authors note.
More recently, Seibler and colleagues at TaconicArtemis published a paper describing a mouse model in which shRNA-based gene silencing could be tightly controlled using the inductor doxycycline.
Specifically, that team generated a mouse model of reversible insulin resistance using an insulin receptor-specific shRNA. “Upon induction, mice develop severe hyperglycemia within seven days … [with] the onset and progression of the disease [correlating] with the concentration of doxycycline,” they wrote in that report. “The phenotype returns to baseline shortly after withdrawal of the inductor.”
Building on this approach, which TaconicArtemis uses in its so-called Artemice transgenic mice, the company and its academic collaborators developed for use in rats a lentiviral single-vector system composed of an shRNA cassette containing the H1 promoter with tetO sequences and a cloning site for insulin receptor-specific shRNA.
Constitutively expressing the tetracycline resistance operon repressor blocks shRNA transcription in the absence of doxycycline, the authors wrote in PNAS. Adding doxycycline releases the tetR, “thereby initiating shRNA expression.”
"Up to now the availability of tools for loss-of-function mutations in rats was limited. The possibility to modify gene function in transgenic rats … opens up novel scientific possibilities in academic and pharmaceutical research."
After testing the lentiviral system in mouse myoblast cells, the team used the technology to generate transgenic rats, which were given doxycycline in their drinking water. The result was a “strong increase in blood glucose … comparable to the degree of hyperglycemia observed in BB/OK rats, a model for spontaneous hereditary type I diabetes mellitus,” according to the paper.
Transgenic and wild-type control rats, meanwhile, displayed normal glucose levels.
Additionally, treating the transgenic rats with doxycycline led to the down-regulation of insulin receptor in muscle, liver, pancreas, kidney, and brown adipose tissue “to almost undetectable levels.” After 14 days of treatment, the researchers observed a “considerable degree” of insulin receptor silencing in the rat brains that received higher doses of doxycycline.
“To provide evidence for the specificity of the observed changes in the … knockdown rats, we generated transgenic rats expressing an irrelevant shRNA,” the researchers wrote. “Transgene expression was induced by adding [doxycycline] to the drinking water … for 14 days.”
Levels of insulin receptor protein in the liver and serum glucose concentrations in these rats was unaltered, and no effect on the growth, behavior, or well-being of the animals was observed.
“This indicates that the observed phenotype of the … knockdown rats was indeed specific and therefore related to the induced expression” of the insulin receptor-specific shRNA, they wrote.
In light of a report in 2006 showing that sustained, high-level shRNA expression can led to severe toxicity and death in mice due to the oversaturation of the cellular RNAi machinery (see RNAi News, 5/25/2006), the researchers analyzed the expression of the liver-specific microRNA miR-122 in the rats before and after treatment with doxycycline. “The level of miR-122 was unaltered after treatment and indistinguishable from that of [wild-type] rats,” they noted.
To demonstrate the reversible nature of the system, the investigators treated transgenic rats with doxycycline for seven days, then withheld the agent for 21 days. Following the removal of the doxycycline, “glucose levels consistently declined and … returned to basal levels after three weeks,” they wrote.
“Water consumption increased over the course of [doxycycline] treatment and then normalized again after removal,” they added. Further, insulin receptor silencing in the transgenic rats “resulted in severe growth retardation that was only slowly overcome after [doxycycline] removal.”
Despite the benefits of the system, the study’s authors concede that it does have two main limitations. The first is limited accessibility to the brain, in which a significantly higher dose of doxycycline was required to achieve strong insulin receptor silencing compared with other organs and tissues.
“This indicates that the system needs to be adapted to the individual aim of each study: high concentrations of [doxycycline] favor broad and strong gene inactivation, whereas low concentrations are required to revert the phenotype,” they wrote.
The second limitation, the researchers noted, is the fact that “the kinetics of reversibility differ between individual tissues.” For instance, in the liver, brown adipose tissue, and pancreas, levels of insulin receptor were mostly restored 21 days after doxycycline treatment was discontinued. In muscle, however, insulin receptor re-expression was incomplete, most likely as a result of the concentration of doxycycline being highest in that tissue type.
Still, the system represents “a simple tool … [for] inducible and reversible gene silencing in transgenic rats and, presumably, other species,” they concluded. “We therefore believe that this technology holds great promise for the generation of additional animal models to study human diseases in various species, exploiting each of their individual advantages as model organisms.”