Abstract
The rat is an important animal model in biomedical research, but gene targeting technology is not established for this species. Therefore, we aimed to produce transgenic knockdown rats using shRNA technology and pronuclear microinjection. To this purpose, we employed a tetracycline-inducible shRNA expression system targeting the insulin receptor (IR). Doxycycline (DOX) treatment of the resulting transgenic rats led to a dose-dependent and reversible increase in blood glucose caused by ubiquitous inhibition of IR expression and signalling. We could neither detect an interferon response nor disturbances in microRNA processing after DOX treatment excluding toxic effects of shRNA expression. Low dose DOX treatment induced a chronic state of diabetes mellitus. In conclusion, we have developed a technology which allows the specific, inducible, and reversible suppression of any gene of interest in the rat. Our first transgenic rat line generated with this method represents an inducible model for diabetes mellitus.
Highlights
The rat is the preferred animal model in several areas of research including cardiovascular and neural biology
We first checked for the functionality of the tetracycline-inducible small hairpin RNA (shRNA) expression system by treating animals with a relatively high concentration of DOX (2 mg/mL)
After 4 days of treatment, shRNA expression was detected by ribonuclease protection assay (RPA) in white adipose tissue (WAT) (Figure 1B), brown adipose tissue (BAT), muscle, liver, kidney, heart, and brain of both lines
Summary
The rat is the preferred animal model in several areas of research including cardiovascular and neural biology. New animal models have been generated with blunted expression of a gene-of-interest by the use of expression cassettes for small hairpin RNA (shRNA) [2,3,4]. These methods were again based on germline-competent embryonic stem cells, which were not available in rats until recently [5,6]. These problems seemed to be overcome by two independent studies in which pronuclear-delivered shRNA constructs were successfully used to knockdown genes in mice [7,8]. The disadvantage of viral transgenesis is the multiplicity and the mosaicism of transgene integration into the genome rendering breeding of genetically pure lines tedious and time-consuming
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