Inducible transgenic rat model for diabetes mellitus based on shRNA-mediated gene knockdown

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.

Figure 1. Generation of transgenic rats.

The transgene construct, pTet-shIR (A), contains two expression cassettes: One expresses shRNA against the insulin receptor (shIR) under the control of the human H1 promoter carrying a tetracycline operator (tetO) sequence. The second cassette consists of a tetracycline repressor (tetR) cDNA followed by a polyadenylation site (pA) and is driven by the CAGGS promoter. An RNase protection assay (RPA) probe was designed to bind to the loop and antisense strand of the hairpin. Primers TetRfor and TetRrev (arrowheads) were used for genotyping of rats. (B) Expression of the shRNA was detected by RPA in 20 µg of total RNA isolated from white adipose tissue (WAT) of wild-type (WT) and transgenic (Tet14 and Tet29) rats treated with doxycycline (DOX, 2 mg/mL) for 4 days. M: RNA Decade marker; Y: yeast RNA; Y-: yeast RNA without RNase digestion; nt: nucleotides. (C) Expression of insulin receptor (IR), tetracycline repressor (tetR), and ß-actin were detected by Western blot in 20 µg of WAT, brain and heart protein from the same rats.

Figure 2. Effect of shRNA expression on blood glucose levels and insulin signalling.

Blood glucose (A) and plasma insulin levels (B) were markedly increased in Tet14 and Tet29 transgenic rats after doxycyline treatment (DOX, 2 mg/mL for 4 days). Insulin sensitivity (C) and signalling (D) were blunted by the treatment. Blood glucose was measured before (open bars, C) and 15 min after i.p. injection of insulin (10 U/kg) (closed bars, C) after 4 days of DOX treatment. Values are given as % of baseline before insulin injection. In the same rats, total Akt and phospho Ser473-Akt (pAkt) (D) were determined by Western blot in 20 µg protein from the interscapular brown adipose tissue. * p<0.05; ** p<0.01 vs. baseline; # p<0.05; ## p<0.01 (Student's t-test). (E) The reversibility of insulin receptor knockdown was shown in three groups of Tet29 transgenic rats treated with different doses of DOX as indicated. DOX treatment was stopped when blood glucose levels reached values between 250 and 300 mg/dL and the further development of blood glucose was monitored.

Figure 3. Chronic diabetes mellitus model in rats.

Tet29 rats were treated with 5 µg/mL of doxycycline (DOX) for 8 days and with 1 µg/mL thereafter for in total 40 days. Blood glucose (A), body weight (B, BW), and drinking volume (C) were measured every second day; plasma insulin (D) was quantified by ELISA before and in the second week of DOX treatment, and urinary volume (E) and albumin (F) were determined weekly in the last three weeks. * p<0.05; ** p<0.01; *** p<0.001 vs. untreated Tet29 rats (Student's t-test).

Figure 4. Lack of toxicity of transgenic shRNA expression.

(A) Tet29 rats were treated with doxycycline (DOX) as described in Figure 3. At the end of the experiment, 20 µg of total RNA from liver was used in an RPA for detection of mir122. M: RNA Decade marker; Y: yeast RNA; Y-: yeast RNA without RNase digestion; nt: nucleotides. Protein kinase R (PKR) expression was used as marker for interferon response in white adipose tissue of acutely (B, as described in Figure 1) or in heart of chronically (C, as described in Figure 3) DOX treated wild-type (WT), Tet14, and Tet29 rats. PKR was detected by Western blot in 20 µg protein; an unspecific band (indicated by *) was used as loading control. HEKi: positive control, 20 µg of protein of HEK cells treated with 1 µM interferon-α2a for 24 hours.