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BackgroundVertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mousePrdm9gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear.ResultsWe introducedPrdm9deletions into theRattus norvegicusgenome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-typePrdm9allele shared 88% hotspots but strains with differentPrdm9alleles only 3%. AfterPrdm9deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding toPrdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility,Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mousePrdm9mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants.ConclusionsWe hypothesize that the relative increased fertility of rat versus mousePrdm9mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation—spermatid development and reproductive age—that may help to explain male-specific hybrid sterility.

Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipid mediators with promising antidiabetic and anti-inflammatory properties that are formed in white adipose tissue (WAT) via de novo lipogenesis, but their biosynthetic enzymes are unknown. Using a combination of lipidomics in WAT, quantitative trait locus mapping, and correlation analyses in rat BXH/HXB recombinant inbred strains, as well as response to oxidative stress in murine models, we elucidated the potential pathway of biosynthesis of several FAHFAs. Comprehensive analysis of WAT samples identified ∼160 regioisomers, documenting the complexity of this lipid class. The linkage analysis highlighted several members of the nuclear factor, erythroid 2 like 2 (Nrf2)-mediated antioxidant defense system (Prdx6, Mgst1, Mgst3), lipid-handling proteins (Cd36, Scd6, Acnat1, Acnat2, Baat), and the family of flavin containing monooxygenases (Fmo) as the positional candidate genes. Transgenic expression of Nrf2 and deletion of Prdx6 genes resulted in reduction of palmitic acid ester of 9-hydroxystearic acid (9-PAHSA) and 11-PAHSA levels, while oxidative stress induced by an inhibitor of glutathione synthesis increased PAHSA levels nonspecifically. Our results indicate that the synthesis of FAHFAs via carbohydrate-responsive element-binding protein–driven de novo lipogenesis depends on the adaptive antioxidant system and suggest that FAHFAs may link activity of this system with insulin sensitivity in peripheral tissues.

Brown adipose tissue (BAT) has been suggested to play an important role in lipid and glucose metabolism in rodents and possibly also in humans. In the current study, we used genetic and correlation analyses in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), to identify genetic determinants of BAT function. Linkage analyses revealed a quantitative trait locus (QTL) associated with interscapular BAT mass on chromosome 4 and two closely linked QTLs associated with glucose oxidation and glucose incorporation into BAT lipids on chromosome 2. Using weighted gene coexpression network analysis (WGCNA) we identified 1,147 gene coexpression modules in the BAT from BXH/HXB rats and mapped their module eigengene QTLs. Through an unsupervised analysis, we identified modules related to BAT relative mass and function. The Coral4.1 coexpression module is associated with BAT relative mass (includes Cd36 highly connected gene), and the Darkseagreen coexpression module is associated with glucose incorporation into BAT lipids (includes Hiat1, Fmo5, and Sort1 highly connected transcripts). Because multiple statistical criteria were used to identify candidate modules, significance thresholds for individual tests were not adjusted for multiple comparisons across modules. In summary, a systems genetic analysis using genomic and quantitative transcriptomic and physiological information has produced confirmation of several known genetic factors and significant insight into novel genetic components functioning in BAT and possibly contributing to traits characteristic of the metabolic syndrome.

The spontaneously hypertensive rat (SHR), one of the most widely used model of essential hypertension, is predisposed to left ventricular hypertrophy, myocardial fibrosis, and metabolic disturbances. Recently, quantitative trait loci influencing blood pressure, left ventricular mass, and heart interstitial fibrosis were genetically isolated within a minimal congenic subline that contains only 7 genes, including mutant Plzf (promyelocytic leukemia zinc finger) candidate gene. To identify Plzf as a quantitative trait gene, we targeted Plzf in the SHR using the transcription activator-like effector nuclease technique and obtained SHR line harboring targeted Plzf gene with a premature stop codon. Because the Plzf targeted allele is semilethal, morphologically normal heterozygous rats were used for metabolic and hemodynamic analyses. SHR- Plzf +/− heterozygotes versus SHR wild-type controls exhibited reduced body weight and relative weight of epididymal fat, lower serum and liver triglycerides and cholesterol, and better glucose tolerance. In addition, SHR- Plzf +/− rats exhibited significantly increased sensitivity of adipose and muscle tissue to insulin action when compared with wild-type controls. Blood pressure was comparable in SHR versus SHR- Plzf +/− ; however, there was significant amelioration of cardiomyocyte hypertrophy and cardiac fibrosis in SHR- Plzf +/− rats. Gene expression profiles in the liver and expression of selected genes in the heart revealed differentially expressed genes that play a role in metabolic pathways, PPAR (peroxisome proliferator-activated receptor) signaling, and cell cycle regulation. These results provide evidence for an important role of Plzf in regulation of metabolic and cardiac traits in the rat and suggest a cross talk between cell cycle regulators, metabolism, cardiac hypertrophy, and fibrosis.

Resistin has been originally identified as an adipokine that links obesity to insulin resistance in mice. In our previous studies in spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin ( Retn) transgene specifically in adipose tissue (SHR- Retn), we have observed an increased lipolysis and serum free fatty acids, ectopic fat accumulation in muscles, and insulin resistance. Recently, brown adipose tissue (BAT) has been suggested to play an important role in the pathogenesis of metabolic disturbances. In the current study, we have analyzed autocrine effects of transgenic resistin on BAT glucose and lipid metabolism and mitochondrial function in the SHR- Retn vs. nontransgenic SHR controls. We observed that interscapular BAT isolated from SHR- Retn transgenic rats compared with SHR controls showed a lower relative weight (0.71 ± 0.05 vs. 0.91 ± 0.08 g/100 g body wt, P < 0.05), significantly reduced both basal and insulin stimulated incorporation of palmitate into BAT lipids (658 ± 50 vs. 856 ± 45 and 864 ± 47 vs. 1,086 ± 35 nmol/g/2 h, P ≤ 0.01, respectively), and significantly decreased palmitate oxidation (37.6 ± 4.5 vs. 57 ± 4.1 nmol/g/2 h, P = 0.007) and glucose oxidation (277 ± 34 vs. 458 ± 38 nmol/g/2 h, P = 0.001). In addition, in vivo microPET imaging revealed significantly reduced 18F-FDG uptake in BAT induced by exposure to cold in SHR- Retn vs. control SHR (232 ± 19 vs. 334 ± 22 kBq/ml, P < 0.05). Gene expression profiles in BAT identified differentially expressed genes involved in skeletal muscle and connective tissue development, inflammation and MAPK and insulin signaling. These results provide evidence that autocrine effects of resistin attenuate differentiation and activity of BAT and thus may play a role in the pathogenesis of insulin resistance in the rat.

Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms of metabolic disturbances. Recent linkage analysis in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), identified two closely linked quantitative trait loci (QTL) associated with glucose oxidation and glucose incorporation into BAT lipids in the vicinity of Wars2 (tryptophanyl tRNA synthetase 2 (mitochondrial)) gene on chromosome 2. The SHR harbors L53F WARS2 protein variant that was associated with reduced angiogenesis and Wars2 thus represents a prominent positional candidate gene. In the current study, we validated this candidate as a quantitative trait gene (QTG) using transgenic rescue experiment. SHR-Wars2 transgenic rats with wild type Wars2 gene when compared to SHR, showed more efficient mitochondrial proteosynthesis and increased mitochondrial respiration, which was associated with increased glucose oxidation and incorporation into BAT lipids, and with reduced weight of visceral fat. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance in muscle and white adipose tissue. In summary, these results demonstrate important role of Wars2 gene in regulating BAT function and consequently lipid and glucose metabolism.

Chronic low-grade inflammation plays an important role in the pathogenesis of insulin resistance. In the current study, we tested the effects of salsalate, a non-steroidal anti-inflammatory drug, in an animal model of inflammation and metabolic syndrome using spontaneously hypertensive rats (SHR) that transgenically express human C-reactive protein (SHR-CRP rats). We treated 15-month-old male transgenic SHR-CRP rats and nontransgenic SHR with salsalate (200 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP and SHR rats were fed a standard diet without salsalate. In the SHR-CRP transgenic strain, salsalate treatment decreased circulating concentrations of the inflammatory markers TNF-α and MCP-1, reduced oxidative stress in the liver and kidney, increased sensitivity of skeletal muscles to insulin action and improved tolerance to glucose. In SHR controls with no CRP-induced inflammation, salsalate treatment reduced body weight, decreased concentrations of serum free fatty acids and total and HDL cholesterol and increased palmitate oxidation and incorporation in brown adipose tissue. Salsalate regulated inflammation by affecting the expression of genes from MAPK signalling and NOD-like receptor signalling pathways and lipid metabolism by affecting hepatic expression of genes that favour lipid oxidation from PPAR-α signalling pathways. These findings suggest that salsalate has metabolic effects beyond suppressing inflammation.

It has been reported that the major function of the sterol regulatory element binding protein 2 (SREBP-2) is to activate preferentially cholesterol biosynthesis in liver and adipose tissue rather than fatty acid synthesis. In the current study, we analyzed the effects of overexpression of human dominant-positive SREBP-2 transgene under control of PEPCK promoter in the spontaneously hypertensive rat (SHR) on lipid and glucose metabolism. Transgenic overexpression of SREBP-2 was associated with significantly higher hepatic triglycerides (20.4+/-0.9 vs. 17.0+/-0.05 micromol/g, P

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mtF344 conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mtF344 conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractional shortening that was independent of blood pressure. In addition, conplastic rats exhibited reduced sensitivity of skeletal muscles to insulin action and impaired glucose tolerance. These results provide evidence that inherited alterations in mitochondrial genome, in the absence of variation in the nuclear genome and other confounding factors, predispose to insulin resistance, cardiac hypertrophy and systolic dysfunction.

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and accompanying metabolic disturbances. Recent advances in sequencing of genomes of BN-Lx and SHR progenitors of the BXH/HXB recombinant inbred (RI) strains as well as accumulation of multiple data sets of intermediary phenotypes in the RI strains, including mRNA and microRNA abundance, quantitative metabolomics, proteomics, methylomics or histone modifications, will make it possible to systematically search for genetic variants involved in regulation of gene expression and in the etiology of complex pathophysiological traits. New advances in manipulation of the rat genome, including efficient transgenesis and gene targeting, will enable in vivo functional analyses of selected candidate genes to identify QTL at the molecular level or to provide insight into mechanisms whereby targeted genes affect pathophysiological traits in the SHR.

BACKGROUND The spontaneously hypertensive rat (SHR) is the most widely used model of essential hypertension and is susceptible to left ventricular hypertrophy (LVH) and myocardial fibrosis. Recently, a quantitative trait locus (QTL) that influences heart interstitial fibrosis was mapped to chromosome 8. Our aim was to dissect the genetic basis of this QTL(s) predisposing SHR to hypertension, LVH, and interstitial fibrosis. METHODS Hemodynamic and histomorphometric analyses were performed in genetically defined SHR.PD-chr.8 minimal congenic strain (PD5 subline) rats. RESULTS The differential segment, genetically isolated within the PD5 subline, spans 788kb and contains 7 genes, including the promyelocytic leukemia zinc finger (Plzf) gene that has been implicated in hypertrophy and cardiac fibrosis. Mutant Plzf allele contains a 2,964-bp deletion in intron 2. The PD5 congenic strain, when compared with the SHR, showed significantly reduced systolic blood pressure by approximately 15mm Hg (P = 0.002), amelioration of LVH (0.23±0.02 vs. 0.39±0.02g/100g body weight; P < 0.00001), and reduced interstitial fibrosis (17,478±1,035 vs. 41,530±3,499 μm(2); P < 0.0001). The extent of amelioration of LVH and interstitial fibrosis was disproportionate to blood pressure decrease in congenic rats, suggesting an important role for genetic factors. Cardiac expression of Plzf was significantly reduced in prehypertensive (8 and 21 days) congenic animals compared with controls. CONCLUSIONS These results provide compelling evidence of a significant role for genetic factors in regulating blood pressure, LVH, and cardiac fibrosis and identify mutant Plzf as a prominent candidate gene.

BackgroundThe role of folate deficiency and associated hyperhomocysteinemia in the pathogenesis of metabolic syndrome is not fully established. In the current study, we analyzed the role of folate deficiency in pathogenesis of the metabolic syndrome in the spontaneously hypertensive rat (SHR).MethodsMetabolic and hemodynamic traits were assessed in SHR/Ola rats fed either folate-deficient or control diet for 4 weeks starting at the age of 3 months.ResultsCompared to SHRs fed a folate-replete diet, SHRs fed a folate-deficient diet showed significantly reduced serum folate (104 ± 5 vs. 11 ± 1 nmol/L, P < 0.0005) and urinary folate excretion (4.3 ± 0.6 vs. 1.2 ± 0.1 nmol/16 h, P < 0.0005) together with a near 3-fold increase in plasma total homocysteine concentration (4.5 ± 0.1 vs 13.1 ± 0.7 μmol/L, P < 0.0005), ectopic fat accumulation in liver, and impaired glucose tolerance. Folate deficiency also increased systolic blood pressure by approximately 15 mm Hg (P < 0.01). In addition, the low-folate diet was accompanied by significantly reduced activity of antioxidant enzymes and increased concentrations of lipoperoxidation products in liver, renal cortex, and heart.ConclusionsThese findings demonstrate that the SHR model is susceptible to the adverse metabolic and hemodynamic effects of low dietary intake of folate. The results are consistent with the hypothesis that folate deficiency can promote oxidative stress and multiple features of the metabolic syndrome that are associated with increased risk for diabetes and cardiovascular disease.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and left ventricular hypertrophy. Catecholamines play an important role in the pathogenesis of both essential hypertension in humans and in the SHR. Recently, we obtained evidence that the SHR harbors a variant in the gene for dopamine beta hydroxylase (Dbh) that is associated with reduced adrenal expression of Dbh mRNA and reduced DBH enzymatic activity which correlated negatively with blood pressure. In the current study, we used a transgenic experiment to test the hypothesis that reduced Dbh expression predisposes the SHR to hypertension and that augmentation of Dbh expression would reduce blood pressure. We derived 2 new transgenic SHR-Dbh lines expressing Dbh cDNA under control of the Brown Norway (BN) wild type promoter. We found modestly increased adrenal expression of Dbh in transgenic rats versus SHR non-transgenic controls that was associated with reduced adrenal levels of dopamine and increased plasma levels of norepinephrine and epinephrine. The observed changes in catecholamine metabolism were associated with increased blood pressure and left ventricular mass in both transgenic lines. We did not observe any consistent changes in brainstem levels of catecholamines or of mRNA levels of Dbh in the transgenic strains. Contrary to our initial expections, these findings are consistent with the possibility that genetically determined decreases in adrenal expression and activity of DBH do not represent primary determinants of increased blood pressure in the SHR model.

The spontaneously hypertensive rat (SHR), one of the most widely used model of essential hypertension, is predisposed to left ventricular hypertrophy, myocardial fibrosis, and metabolic disturbances. Recently, quantitative trait loci influencing blood pressure, left ventricular mass, and heart interstitial fibrosis were genetically isolated within a minimal congenic subline that contains only 7 genes, including mutant

Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50–64, 14–72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.—Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bősze, Z., Rülicke, T., Izsvák, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Common inbred strains of the laboratory rat can be divided into four different mitochondrial DNA haplotype groups represented by the SHR, BN, LEW, and F344 strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. LEW mitochondrial genomes by comparing the SHR to a new SHR conplastic strain, SHR-mtLEW; these strains are genetically identical except for their mitochondrial genomes. Complete mitochondrial DNA (mtDNA) sequence analysis comparing the SHR and LEW strains revealed gene variants encoding amino acid substitutions limited to a single mitochondrial enzyme complex, NADH dehydrogenase (complex I), affecting subunits 2, 4, and 5. Two of the variants in the mt-Nd4 subunit gene are located close to variants known to be associated with exercise intolerance and diabetes mellitus in humans. No variants were found in tRNA or rRNA genes. These variants in mt-Nd2, mt-Nd4, and mt-Nd5 in the SHR-mtLEW conplastic strain were linked to reductions in oxidative and nonoxidative glucose metabolism in skeletal muscle. In addition, SHR-mtLEW conplastic rats showed increased serum nonesterified fatty acid levels and resistance to insulin stimulated incorporation of glucose into adipose tissue lipids. These results provide evidence that inherited variation in mitochondrial genes encoding respiratory chain complex I subunits, in the absence of variation in the nuclear genome and other confounding factors, can influence glucose and lipid metabolism when expressed on the nuclear genetic background of the SHR strain.

CD36 fatty acid translocase plays a key role in supplying heart with its major energy substrate, long-chain fatty acids (FA). Previously, we found that the spontaneously hypertensive rat (SHR) harbors a deletion variant of Cd36 gene that results in reduced transport of long-chain FA into cardiomyocytes and predisposes the SHR to cardiac hypertrophy. In the current study, we analyzed the effects of mutant Cd36 on susceptibility to ischemic ventricular arrhythmias and myocardial infarction in adult SHR- Cd36 transgenic rats with wild-type Cd36 compared with age-matched SHR controls. Using an open-chest model of coronary artery occlusion, we found that SHR- Cd36 transgenic rats showed profound arrhythmogenesis resulting in significantly increased duration of tachyarrhythmias (207 ± 48 s vs. 55 ± 21 s, P < 0.05), total number of premature ventricular complexes (2,623 ± 517 vs. 849 ± 250, P < 0.05) and arrhythmia score (3.86 ± 0.18 vs. 3.13 ± 0.13, P < 0.001). On the other hand, transgenic SHR compared with SHR controls showed significantly reduced infarct size (52.6 ± 4.3% vs. 72.4 ± 2.9% of area at risk, P < 0.001). Similar differences were observed in isolated perfused hearts, and the increased susceptibility of transgenic SHR to arrhythmias was abolished by reserpine, suggesting the involvement of catecholamines. To further search for possible molecular mechanisms of altered ischemic tolerance, we compared gene expression profiles in left ventricles dissected from 6-wk-old transgenic SHR vs. age-matched controls using Illumina-based sequencing. Circadian rhythms and oxidative phosphorylation were identified as the top KEGG pathways, while circadian rhythms, VDR/RXR activation, IGF1 signaling, and HMGB1 signaling were the top IPA canonical pathways potentially important for Cd36-mediated effects on ischemic tolerance. It can be concluded that transgenic expression of Cd36 plays an important role in modulating the incidence and severity of ischemic and reperfusion ventricular arrhythmias and myocardial infarct size induced by coronary artery occlusion. The proarrhythmic effect of Cd36 transgene appears to be dependent on adrenergic stimulation.

Increased circulating levels of resistin have been proposed as a possible link between obesity and insulin resistance; however, many of the potential metabolic effects of resistin remain to be investigated, including systemic versus local resistin action. We investigated potential autocrine effects of resistin on lipid and glucose metabolism in 2- and 16-mo-old transgenic spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin under control of the aP2 promoter. To search for possible molecular mechanisms, we compared gene expression profiles in adipose tissue in 6-wk-old transgenic SHR versus control rats, before development of insulin resistance, by digital transcriptional profiling using high-throughput sequencing. Both young and old transgenic rats showed moderate expression of the resistin transgene in adipose tissue but had serum resistin levels similar to control SHR and undetectable levels of transgenic resistin in the circulation. Young transgenic rats exhibited mild glucose intolerance. In contrast, older transgenic rats displayed marked glucose intolerance in association with near total resistance of adipose tissue to insulin-stimulated glucose incorporation into lipids (6 ± 2 vs. 77 ± 19 nmol glucose·g−1·2 h−1, P < 0.00001). Ingenuity Pathway Analysis of differentially expressed genes revealed calcium signaling, Nuclear factor-erythroid 2-related factor-2 (NRF2)-mediated oxidative stress response, and actin cytoskeletal signaling canonical pathways as those most significantly affected. Analysis using DAVID software revealed oxidative phosphorylation, glutathione metabolism, pyruvate metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling as top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These results suggest that with increasing age autocrine effects of resistin in fat tissue may predispose to diabetes in part by impairing insulin action in adipose tissue.

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC(50) 4microM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.

The temporal relationship of hepatic steatosis and changes in liver oxidative stress and fatty acid (FA) composition to the development of non-alcoholic steatohepatitis (NASH) remain to be clearly defined. Recently, we developed an experimental model of hepatic steatosis and NASH, the transgenic spontaneously hypertensive rat (SHR) that overexpresses a dominant positive form of the human SREBP-1a isoform in the liver. These rats are genetically predisposed to hepatic steatosis at a young age that ultimately progresses to NASH in older animals. Young transgenic SHR versus SHR controls exhibited simple hepatic steatosis which was associated with significantly increased hepatic levels of oxidative stress markers, conjugated dienes, and TBARS, with decreased levels of antioxidative enzymes and glutathione and lower concentrations of plasma alpha- and gamma-tocopherol. Transgenic rats exhibited increased plasma levels of saturated FA, decreased levels of n-3 and n-6 polyunsaturated FA (PUFA), and increased n-6/n-3 PUFA ratios. These results are consistent with the hypothesis that excess fat accumulation in the liver in association with increased oxidative stress and disturbances in the metabolism of saturated and unsaturated fatty acids may precede and contribute to the primary pathogenesis of NASH.

Recently, the relationship of mitochondrial DNA (mtDNA) variants to metabolic risk factors for diabetes and other common diseases has begun to attract increasing attention. However, progress in this area has been limited because (1) the phenotypic effects of variation in the mitochondrial genome are difficult to isolate owing to confounding variation in the nuclear genome, imprinting phenomena, and environmental factors; and (2) few animal models have been available for directly investigating the effects of mtDNA variants on complex metabolic phenotypes in vivo. Substitution of different mitochondrial genomes on the same nuclear genetic background in conplastic strains provides a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Here we show that conplastic strains of rats with identical nuclear genomes but divergent mitochondrial genomes that encode amino acid differences in proteins of oxidative phosphorylation exhibit differences in major metabolic risk factors for type 2 diabetes. These results (1) provide the first direct evidence linking naturally occurring variation in the mitochondrial genome, independent of variation in the nuclear genome and other confounding factors, to inherited variation in known risk factors for type 2 diabetes; and (2) establish that spontaneous variation in the mitochondrial genome per se can promote systemic metabolic disturbances relevant to the pathogenesis of common diseases.

Metabolism of homocysteine and other sulfur amino acids is closely associated with metabolism of folates. In this study, we analyzed the possible role of folates and sulfur amino acids in the development of features of the metabolic syndrome in the BXH/HXB recombinant inbred strains derived from the spontaneously hypertensive rat (SHR) and Brown Norway progenitors. We mapped a quantitative trait locus for cysteine concentrations to a region of chromosome 1 that contains a cis -acting expression quantitative trait locus regulating mRNA levels of folate receptor 1 ( Folr1 ) in the kidney. Sequence analysis revealed a deletion variant in the Folr1 promoter region of the SHR. Transfection studies demonstrated that the SHR-promoter region of Folr1 is less effective in driving luciferase reporter gene expression than the Brown Norway promoter region of Folr1 . Results in the SHR.BN-chr.1 congenic strain confirmed that the SHR variant in Folr1 cosegregates with markedly reduced renal expression of Folr1 and renal folate reabsorption, decreased serum levels of folate, increased serum levels of cysteine and homocysteine, increased adiposity, ectopic fat accumulation in liver and muscle, reduced muscle insulin sensitivity, and increased blood pressure. Transgenic rescue experiments performed by expressing a Folr1 transgene in the SHR ameliorated most of the metabolic disturbances. These findings are consistent with the hypothesis that inherited variation in the expression of Folr1 in the kidney influences the development of the metabolic syndrome and constitutes a previously unrecognized genetic mechanism that may contribute to increased risk for diabetes mellitus and cardiovascular disease.

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.

Increased serum levels of resistin, a molecule secreted by fat cells, have been proposed as a possible mechanistic link between obesity and insulin resistance. To further investigate the effects of resistin on glucose metabolism, we derived a novel transgenic strain of spontaneously hypertensive rats expressing the mouse resistin gene under the control of the fat-specific aP2 promoter and also performed in vitro studies of the effects of recombinant resistin on glucose metabolism in isolated skeletal muscle. Expression of the resistin transgene was detected by Northern blot analysis in adipose tissue and by real-time PCR in skeletal muscle and was associated with increased serum fatty acids and muscle triglycerides, impaired skeletal muscle glucose metabolism, and glucose intolerance in the absence of any changes in serum resistin concentrations. In skeletal muscle isolated from non-transgenic spontaneously hypertensive rats, in vitro incubation with recombinant resistin significantly inhibited insulin-stimulated glycogenesis and reduced glucose oxidation. These findings raise the possibility that autocrine effects of resistin in adipocytes, leading to release of other prodiabetic effector molecules from fat and/or paracrine actions of resistin secreted by adipocytes embedded within skeletal muscle, may contribute to the pathogenesis of disordered skeletal muscle glucose metabolism and impaired glucose tolerance.

Inflammation and oxidative stress have been implicated in the pathogenesis of metabolic disturbances. Esters of fumaric acid, mainly dimethyl fumarate, exhibit immunomodulatory, anti-inflammatory, and anti-oxidative effects. In the current study, we tested the hypothesis that fumaric acid ester (FAE) treatment of an animal model of inflammation and metabolic syndrome, the spontaneously hypertensive rat transgenically expressing human C-reactive protein (SHR-CRP), will ameliorate inflammation, oxidative stress, and metabolic disturbances. We studied the effects of FAE treatment by administering Fumaderm, 10 mg/kg body weight for 4 weeks, to male SHR-CRP. Untreated male SHR-CRP rats were used as controls. All rats were fed a high sucrose diet. Compared to untreated controls, rats treated with FAE showed significantly lower levels of endogenous CRP but not transgenic human CRP, and amelioration of inflammation (reduced levels of serum IL6 and TNFα) and oxidative stress (reduced levels of lipoperoxidation products in liver, heart, kidney, and plasma). FAE treatment was also associated with lower visceral fat weight and less ectopic fat accumulation in liver and muscle, greater levels of lipolysis, and greater incorporation of glucose into adipose tissue lipids. Analysis of gene expression profiles in the liver with Affymetrix arrays revealed that FAE treatment was associated with differential expression of genes in pathways that involve the regulation of inflammation and oxidative stress. These findings suggest potentially important anti-inflammatory, anti-oxidative, and metabolic effects of FAE in a model of inflammation and metabolic disturbances induced by human CRP.

Summary Aims Statins have antiinflammatory effects and are known to decrease risk of cardiovascular events and to reduce serum levels of C-reactive protein (CRP), a widely studied biomarker and potential mediator of inflammation and heart disease. However, it is unclear whether statins can block pro-inflammatory effects of human CRP independent of their ability to reduce serum levels of human CRP. Here, we investigated whether rosuvastatin could block pro-inflammatory effects of human CRP without reducing circulating levels of human CRP. Methods and Results We studied the antiinflammatory effects of rosuvastatin in spontaneously hypertensive rats (SHR) transgenically expressing human CRP (CRP-transgenic SHR) and in nontransgenic SHR lacking human CRP (nontransgenic SHR). The CRP-transgenic SHR is characterized by increased serum levels of human CRP and inflammation. In the CRP-transgenic strain, we found that rosuvastatin treatment decreased circulating levels of inflammatory response markers IL6 and TNFα without decreasing circulating levels of human CRP. In contrast, in the nontransgenic strain lacking human CRP, rosuvastatin treatment had little or no effect on IL6 and TNFα levels. Rosuvastatin also reduced cardiac inflammation and oxidative tissue damage, reduced epididymal fat mass, and improved adipose tissue lipolysis much more in the CRP-transgenic strain than in the nontransgenic strain. Conclusion Rosuvastatin can protect against pro-inflammatory effects of human CRP in a manner that is not dependent on achieving a reduction in circulating levels of human CRP.

Inflammation and oxidative stress have been implicated in the pathogenesis of metabolic disturbances. Esters of fumaric acid, mainly dimethyl fumarate, exhibit immunomodulatory, anti-inflammatory, and anti-oxidative effects. In the current study, we tested

Mouse and rabbit preimplantation embryos with intact zona pellucida were incubated for 3 hr with DNA-carrying constructs containing insulin as an internalizable ligand: (insulin-polylysine)-DNA and (insulin-polylysine)-DNA-(streptavidin-polylysine)-(biotinylated adenovirus). Video-intensified microscopy demonstrated that the constructs penetrated the zona pellucida and accumulated in the blastomere perinuclear space. The percentage of blastocysts formed was about 70% after incubation of zygotes and two-cell embryos with the constructs. Foreign DNA was detected after 51 hr in 80% of rabbit embryos and after 96 hr in 73% of mouse embryos. Inclusion of various adenoviruses into the construct improved foreign DNA preservation in early embryos. Blot hybridization revealed genome-integrated foreign DNA in 12- and 15-day mouse embryos and in a newborn. Thus, the ligand-mediated mechanism can be employed for introducing foreign genetic material into early mammalian embryos; insulin provides for delivery inside the cell and to the nucleus, while adenoviruses ensure release from endosomes.

Nucleolar transcription was analysed in permeabilized pre-implantation mouse embryos at the four-cell, eight-cell, morula and early blastocyst stages using confocal microscopy to detect incorporated 5-bromouridine. The results demonstrated that the patterns of nucleolar transcription sites were common for all embryonic stages studied. They consisted most frequently of tightly associated groups of transcription foci similar to those encountered in somatic interphase cells. In addition, the nucleologenesis accompanying each cell cycle apparently gave rise to a different fluorescent pattern, that is to spatially separated fluorescent foci in the cells just after the resumption of rRNA synthesis. An immunoelectron microscopic analysis of the nucleolar transcription was also performed in the eight-cell embryos. A signal, usually consisting of clustered gold particles, was found specifically within nucleolar dense fibrillar components. This result was in agreement with established findings, which identify dense fibrillar component as the major site of nucleolar transcription in somatic cells.

Background The angiotensin receptor blocker telmisartan has unique chemical properties that enable it to partially activate the peroxisome proliferator activated receptor gamma (PPARG) as well as block angiotensin II type 1 receptors. Methods To directly test whether some of the metabolic effects of telmisartan require the presence of PPARG, we studied mice in which the gene (Pparg) for PPARG had been deleted in fat or in muscle. Results We found that knockout of Pparg in fat tissue greatly impaired the ability of telmisartan to increase adiponectin levels and to enhance sensitivity to insulin-stimulated glucose incorporation into adipose tissue lipids. In contrast, muscle-specific Pparg knockout had relatively little or no impact on the ability of telmisartan to increase adiponectin levels or affect glucose metabolism either in fat or muscle. These findings provide compelling evidence that the ability of telmisartan to increase adiponectin levels and stimulate glucose use in adipose tissue may depend on the presence of PPARG in fat. Conclusions We conclude that PPARG in adipose tissue is required for at least several of the metabolic actions of telmisartan.

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-3H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-3H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-3H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.

Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its “pleiotropic” effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action–studied by gene expression profiling in the liver–revealed deregulated genes from inflammatory and insulin signaling, AMP-activated protein kinase (AMPK) signaling and gluconeogenesis pathways. It can be concluded that in the presence of high levels of human CRP, metformin protects against inflammation and oxidative and dicarbonyl stress in the heart, but not in the kidney. Accordingly, these cardioprotective effects of metformin might be especially effective in diabetic patients with high levels of CRP.

Summary Completely concordant distributions of Cd36 and Rt8 deletion/null and wild-type alleles among inbred and congenic strains, together with Western blot analysis of RT8/CD36 proteins, indicated that the CD36 protein functions as an immunogenic domain of the RT8 alloantigen.

Major controversy exists as to whether increased C-reactive protein (CRP) contributes to individual components of the metabolic syndrome or is just a secondary response to inflammatory disease processes. We measured blood pressure and metabolic phenotypes in spontaneously hypertensive rats (SHRs) in which we transgenically expressed human CRP in the liver under control of the apolipoprotein E promoter. In transgenic SHRs, serum levels of human CRP approximated the endogenous levels of CRP normally found in the rat. Systolic and diastolic blood pressures measured by telemetry were 10 to 15 mm Hg greater in transgenic SHRs expressing human CRP than in SHR controls ( P P P P P P P P

Congenic strains are routinely used for positional mapping of quantitative trait loci; while conplastic strains, derived by substitution of different mitochondrial genomes on the same nuclear genetic background of inbred rodent strains, provide a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Derivation of congenic or conplastic strains using a traditional backcross breeding strategy (10 backcrosses) takes more than 3 years. There are two principal strategies to speed up this process: (1) marker-assisted derivation of "speed" congenic/conplastic strains and (2) derivation of "supersonic" congenic/conplastic strains using in each backcross generation embryos obtained from 4-week-old superovulated females; thus, each backcross generation takes only 7 weeks. Both strategies could also be combined. In the current chapter, a method for derivation of "supersonic" congenic/conplastic rat strains is described.

Congenic strains are routinely used for positional mapping of quantitative trait loci; while conplastic strains, derived by substitution of different mitochondrial genomes on the same nuclear genetic background of inbred rodent strains, provide a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Derivation of congenic or conplastic strains using a traditional backcross breeding strategy (10 backcrosses) takes more than 3 years. There are two principal strategies to speed up this process: (1) marker-assisted derivation of "speed" congenic/conplastic strains and (2) derivation of "supersonic" congenic/conplastic strains using in each backcross generation embryos obtained from 4-week-old superovulated females; thus, each backcross generation takes only 7 weeks. Both strategies could also be combined. In the current chapter, a method for derivation of "supersonic" congenic/conplastic rat strains is described.