Rats, Transgenic

The 22-kb mini human TDP gene was extracted from a BAC clone (RP11-829B14), and a M337V substitution was introduced into the mini TDP gene by homologous recombination in Escherichia coli[22] (link). The normal and mutant mini TDP transgenes were linearized by restriction digestion, purified from agarose gels, and then used to produce transgenic rats through microinjection. To generate Tet-regulatable TDP transgenic rats, we PCR-amplified the human TDP-43 ORF from a human brain cDNA pool (Invitrogen) and generated a mutant carrying the M337V substitution using site-directed mutagenesis (Stratagene). The mutated human TDP-43 cDNA gene was inserted downstream of a tTA-dependent promoter that was constructed by fusing seven tetracycline-responsive elements (TRE) with a minimal cytomegalovirus promoter (TRE-miniCMV). To enhance gene splicing and expression, we inserted the first intron of the human ubiquitin C gene between the TRE-miniCMV promoter and the TDP-43 ORF [24] .

To induce expression of shRNA, animals were treated with varying concentrations of doxycycline (DOX; Sigma) in the drinking solution. The DOX solution was freshly prepared each day and kept dark due to the light sensitivity of DOX. The drinking solution contained various percentages of sucrose depending on the DOX concentrations and was also given to the control animals.
To check the functionality of the system animals were treated with 2 mg/mL DOX in the drinking water containing 10% sucrose for 4 days.
In the reversibility tests, animals received different doses of DOX per day (20, 2 and 0.5 mg/kg body weight). To this end, rats were offered their daily dose of DOX in about 20 ml of 1% sucrose. After this volume was consumed they got normal water ad libitum. Once plasma glucose levels reached 250 to 300 mg/dL in the treated transgenic rats, DOX was withdrawn from their drinking solution.
To establish a chronic model of diabetes mellitus, a group of rats was treated daily with 5 µg/mL of DOX solution containing 1% sucrose. When blood glucose reached 300 mg/dL (after 8 days of treatment), the concentration was changed to 1 µg/mL DOX solution (in 1% sucrose) for in total 40 days.
During all experiments animals were regularly checked for drinking volume, body weight, blood glucose and insulin level. To collect urine for validation of urinary volume and albuminuria, experimental animals were kept in metabolic cages under standardized conditions for one day per week during a period of 3 weeks. After 24 hours, the volume of collected urine was determined. For quantification of albumin, the urine was centrifuged (600 g, 10 min, 4°C) and analysed by CellTrend using a specific ELISA.

All Sprague-Dawley rats were obtained from SLC Japan (Shizuoka, Japan). Transgenic rats carrying the human mutant SOD1 gene for H46R (SOD1^H46R) were housed and genotyped as previously.²¹ (link) The DNA of newborn rats was extracted from their tails, and PCR amplification (forward primer: 5′-TTGGGAGGAGGTAGTGATTA; reverse primer: 5′-AGCTAGCAGGATAACAGATGA; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles) was performed to identify the exogenous human SOD1 transgene DNA. Founder rats were mated with Sprague-Dawley rats. For western blotting or cDNA microarray experiments, rats were anesthetized intraperitoneally using the combination of medetomidine, midazolam, and butorphanol tartrate. Subsequently, they were intracardially perfused with PBS. For immunohistochemistry, 4% PFA (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was additionally used for tissue fixation.

TGR(A1-7)3292 rats show specific testicular expression of a cytomegalovirus promoter-driven transgene that results in a doubling of circulating Ang (1-7) compared to non-transgenic control rats [55 (link)]. The hypertensive (mRen-2)27 transgenic rat strain (TGR) shows strong expression of the murine Ren-2 transgene in extrarenal tissues, which induces and maintains hypertension through conventional angiotensin II (Ang II), and hypertension is readily controlled by inhibition of the renin–angiotensin system (RAS). This transgenic rat strain is now widely used to study a variety of conditions related to tissue RAS activation, including angiogenesis, cytokine activation, profibrotic and inflammatory pathologies, thus contributing to the understanding of the underlying processes causing severe hypertension [56 (link),57 (link)]. In the experimental model, 20 male TGR(A1-7)3292 laboratory rats, 10 male Hannover Sprague–Dawley (HSD) laboratory normotensive rats and 10 male (mRen-2)27 transgenic laboratory hypertensive rats (TGR) at the age of 8 weeks were used. HSD rats are standardly used as a normotensive control for TGR rats. The experimental animals came from the accredited laboratory breeding of the Center for Experimental Medicine, Institute of Clinical and Experimental Medicine in Prague, Czech Republic. All animal experiments were approved on 26 June 2017 by the Animal Care and Use Committee of the Institute for Clinical and Experimental Medicine, Prague; project number 50/2017; in accordance with guidelines and practices established by the Directive 2010/63/EU of the European Parliament on the Protection of Animals Used for Scientific Purposes. Laboratory animals TGR(A1-7)3292 were divided into two groups with sham surgery and ACF surgery, HSD and TGR with ACF surgery (Table 1). The animals were fed a standard laboratory diet, which, was available to them ad libitum, as well as drinking water. They were kept in air-conditioned rooms with a constant temperature of 22–24 °C, humidity of 40–60% and with a regular light regime of 12 h of darkness and 12 h of light. The ACF operation was performed under general anesthesia induced by isoflurane. After exposing the abdominal aorta and inferior vena cava, the aorta was occluded for 30 s in the area between the renal arteries and the iliac bifurcation. ACF was created by inserting a needle (diameter 1.2 mm) through the abdominal aorta into the inferior vena cava. The injection site was then sealed with cyanoacrylate tissue adhesive [58 (link)]. Five weeks after ACF induction, rats in the “compensated HF” phase were euthanatized by decapitation.