Gene Knockout Rats Research Articles

Effect of lncRNA AK023948 on rats with postmenopausal osteoporosis via PI3K/AKT signaling pathway.

To investigate the effect of long non-coding ribonucleic acid (lncRNA) AK023948 (AK0) on rats with postmenopausal osteoporosis via the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Firstly, postmenopausal osteoporosis rat models were established to obtain osteoblasts. The phosphorylation level of AKT was analyzed by controlling the expression of AK0 gene in osteoblasts. Finally, XTT was used to analyze the proliferation of osteoblasts and the messenger ribonucleic acid (mRNA) expression level of caspase in AK0 gene knockout (KO) rat model. In the bone tissue of postmenopausal osteoporosis rats, the levels of phospho-PI3K (p-PI3K), p-Akt, and p-phosphoinositide-dependent kinase-1(PDK1) were significantly decreased (p<0.05). In rat model osteoblasts, the overexpression of AK0 gene upregulated the phosphorylation level of AKT, while the interference with small interfering RNA (siRNA) in AK0 gene decreased that of AKT. Knocking out AK0 gene led to the down-regulation of phosphorylation level of AKT in cells. Moreover, if the AK0 gene was re-expressed in the KO rat model cells, the phosphorylation level of AKT was restored to a certain extent, but still lower than that after the overexpression of AK0 gene. Although the proliferation rate of osteoblasts in estrogen deficiency-related osteoporosis rats was low, the growth rate of osteoblasts with AK0 KO was remarkably lower than that in blank control group (p<0.05). It was also found that there was a certain correlation between AK0 gene and osteoblast apoptosis. LncRNA AK0 can regulate the phosphorylation level of AKT in osteoblasts of rats with estrogen deficiency-related osteoporosis through the PI3K/AKT signaling pathway, thus regulating the proliferation of osteoblasts. It is speculated that lncRNA AK0 may be an important factor in regulating the PI3K/AKT signaling pathway.

C6orf120 gene deficiency may be vulnerable to carbon tetrachloride induced acute hepatic injury in rats.

The function of the C6orf120 gene, which encodes an N-glycosylated protein, remains unknown. The study was performed to characterize the utility of the C6orf120 gene in carbon tetrachloride-induced acute liver injury and to elucidate the potential underlying mechanisms by establishing a C6orf120 gene-knockout (C6orf120-/-) rat model. C6orf120-/- and wild-type (WT) rats were intraperitoneally administered with CCl4 (1 : 1 v/v in olive oil, 2 µl/g). Rats were sacrificed 24 h after CCl4 administration. Liver tissues were collected for H&E, IHC, qRT-PCR, and Western blot analysis. C6orf120 gene deficiency may be vulnerable to CCl4-induced acute liver injury in rats as indicated by the high levels of alanine aminotransferase (WT: 388.7 ±55.96 vs. C6orf120-/-: 915.9 ±118.8, p < 0.001) and greater degree of pathological damage. Quantitative reverse transcription polymerase chain reaction showed that the mRNA levels of inflammation-associated cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, in liver tissues were increased in C6orf120-/- rats compared with those in WT rats. Moreover, western blot showed that the protein expression of cytokines nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing 3 (NLRP3), caspase-1, IL-1β, nuclear factor-κB, c-Jun N-terminal kinases, and Bax were increased in C6orf120-/- rats compared with those in WT rats. C6orf120-/- rats were susceptible to CCl4-induced liver injury, which may be related to NLRP3 inflammasome and JNK signaling pathway activation.

The Mechanism of Downregulated Interstitial Fluid Drainage Following Neuronal Excitation.

The drainage of brain interstitial fluid (ISF) has been observed to slow down following neuronal excitation, although the mechanism underlying this phenomenon is yet to be elucidated. In searching for the changes in the brain extracellular space (ECS) induced by electrical pain stimuli in the rat thalamus, significantly decreased effective diffusion coefficient (DECS) and volume fraction (α) of the brain ECS were shown, accompanied by the slowdown of ISF drainage. The morphological basis for structural changes in the brain ECS was local spatial deformation of astrocyte foot processes following neuronal excitation. We further studied aquaporin-4 gene (APQ4) knockout rats in which the changes of the brain ECS structure were reversed and found that the slowed DECS and ISF drainage persisted, confirming that the down-regulation of ISF drainage following neuronal excitation was mainly attributable to the release of neurotransmitters rather than to structural changes of the brain ECS. Meanwhile, the dynamic changes in the DECS were synchronized with the release and elimination processes of neurotransmitters following neuronal excitation. In conclusion, the downregulation of ISF drainage following neuronal excitation was found to be caused by the restricted diffusion in the brain ECS, and DECS mapping may be used to track the neuronal activity in the deep brain.

5-HT3 receptor signaling in serotonin transporter-knockout rats: a female sex-specific animal model of visceral hypersensitivity

The irritable bowel syndrome (IBS) is a functional gastrointestinal motor and visceral sensation disorder that is more common in women than men. Female serotonin transporter (SERT)-gene knockout (KO) rats exhibit hypersensitivity to colorectal balloon distention (CRD) that mimics colonic hypersensitivity occurring in female IBS patients. Alosetron (5-HT3 receptor antagonist) is used to treat diarrhea-predominant IBS in female patients. Other 5-HT3 receptor antagonists are ineffective at treating IBS symptoms. The visceromotor response (VMR) to CRD in SERT-KO and wild-type (WT) rats was measured following subcutaneous (sc), intracerobroventricular (icv), or intrathecal (it) treatment with 5-HT3 receptor antagonists and an agonist. Alosetron (sc) and granisetron (antagonists) caused a paradoxical increase in the VMR to CRD in SERT-KO female rats. Alosetron (sc) increased the VMR to CRD in WT male rats. Alosetron (it) increased the VMR to CRD in SERT-KO female rats only, and the 5-HT3 receptor agonist SR-52772 increased the VMR to CRD in SERT-KO male rats. Depletion of spinal 5-HT using 5,7-dihydroxytryptamine prevented the increase in VMR to CRD in SERT-KO female and male rats treated it with alosetron and SR-52772, respectively. Alosetron (icv) did not affect the VMR to CRD in WT or KO female rats, but it increased the VMR in male SERT-KO but not WT male rats. These data suggest that 5-HT3 receptor signaling at the dorsal spinal cord mediates visceral hypersensitivity in female SERT-KO rats. Such differences could facilitate development of sex-specific drug treatments for visceral pain. NEW & NOTEWORTHY We studied a model of female sex-specific visceral hypersensitivity using rats that had a loss of function of the serotonin transporter (SERT) caused by gene truncation. Female SERT-KO rats exhibited visceral hypersensitivity in response to colorectal balloon distention. We found that increased 5-HT signaling at dorsal spine 5-HT3 receptors was responsible for visceral hypersensitivity in female but not male SERT-KO rats.

Protein kinase A mediates scopolamine-induced mTOR activation and an antidepressant response

BackgroundClinical reports have shown that scopolamine produces a rapid (3–4 d) and potent anti-depressive response without severe adverse effects. Animal experiments have proven that scopolamine induces mTOR pathway activation in an AMPAR dependent manner. The present study aimed to determine the role of PKA in scopolamine-induced potentiation of AMPAR, as well as in mTOR pathway activation and rapid antidepressant effects. MethodsWe utilized electrophysiological recording, Western blotting, and behavior tests to examine the effects of scopolamine, the selective M2 cholinergic receptor antagonist methoctramine, and H89, a PKA specific inhibitor on AMPAR potentiation, mTOR pathway activation, and behavioral responses in a rat depression model of learned helplessness. ResultsScopolamine (1μM) rapidly increased AMPAR-fEPSP amplitudes and membrane GluA1 expression in CA1 region of hippocampal slices, both of which were abolished by H89. Moreover, scopolamine promoted AMPAR phosphorylation on GluA1 ser845, a PKA site involved in GluA1 membrane insertion. H89 disrupted both GluA1 ser845 phosphorylation and mTOR activation, as well as the antidepressant effects of scopolamine as determined via forced swim test. Additionally, methoctramine mimicked the effects of scopolamine on phosphorylation and counter-depressive action in a PKA-dependent manner. LimitationsOnly one test was used to evaluate depressive behavior, and gene knock-out rats were not yet utilized to refine our hypotheses. ConclusionOur findings revealed that PKA pathway is necessary for scopolamine-induced synaptic plasticity and mTOR pathway activation, and indicated that a potential M2-PKA mechanism underlies scopolamine's antidepressant effects. Such findings suggest that GluA1 ser845 phosphorylation may be a trigger event for scopolamine's actions, and that PKA may represent a novel target for the treatment of depressive symptoms.

Bile Salt Homeostasis in Normal and Bsep Gene Knockout Rats with Single and Repeated Doses of Troglitazone.

The interference of bile acid secretion through bile salt export pump (BSEP) inhibition is one of the mechanisms for troglitazone (TGZ)-induced hepatotoxicity. Here, we investigated the impact of single or repeated oral doses of TGZ (200 mg/kg/day, 7 days) on bile acid homoeostasis in wild-type (WT) and Bsep knockout (KO) rats. Following oral doses, plasma exposures of TGZ were not different between WT and KO rats, and were similar on day 1 and day 7. However, plasma exposures of the major metabolite, troglitazone sulfate (TS), in KO rats were 7.6- and 9.3-fold lower than in WT on day 1 and day 7, respectively, due to increased TS biliary excretion. With Bsep KO, the mRNA levels of multidrug resistance-associated protein 2 (Mrp2), Mrp3, Mrp4, Mdr1, breast cancer resistance protein (Bcrp), sodium taurocholate cotransporting polypeptide, small heterodimer partner, and Sult2A1 were significantly altered in KO rats. Following seven daily TGZ treatments, Cyp7A1 was significantly increased in both WT and KO rats. In the vehicle groups, plasma exposures of individual bile acids demonstrated variable changes in KO rats as compared with WT. WT rats dosed with TGZ showed an increase of many bile acid species in plasma on day 1, suggesting the inhibition of Bsep. Conversely, these changes returned to base levels on day 7. In KO rats, alterations of most bile acids were observed after seven doses of TGZ. Collectively, bile acid homeostasis in rats was regulated through bile acid synthesis and transport in response to Bsep deficiency and TGZ inhibition. Additionally, our study is the first to demonstrate that repeated TGZ doses can upregulate Cyp7A1 in rats.

Role of syndecan-4 in inflammatory responses of rats with ventilator-induced lung injury

Objective To evaluate the role of syndecan-4(SDC-4)in inflammatory responses of rats with ventilator-induced lung injury(VILI). Methods Twenty-four healthy male wild type Sprague-Dawley rats and 24 male SDC-4 gene knockout Sprague-Dawley rats, aged 2-3 months, weighing 200-220 g, were assigned into 2 groups(n=12 each)using a random number table: control group(group C)and VILI group.The animals were anesthetized with pentobarbital sodium and tracheostomized.The rats kept spontaneous breathing in group C. The rats were mechanically ventilated for 4 h in group VILI.Blood samples were taken from the femoral artery at the end of mechanical ventilation for detection of arterial oxygen partial pressure(PaO2). The animals were sacrificed after blood sampling.The left lung was lavaged, and the broncho-alveolar lavage fluid(BALF)was collected for determination of the tumor necrosis factor-α(TNF-α), interleukin-1beta(IL-1β)and IL-18 concentrations(by enzyme-linked immunosorbent assay)and total protein concentrations(by BCA assay). The right lungs were removed for determination of the wet to dry weight ratio(W/D ratio)and expression of TNF-α, IL-1β and IL-18 mRNA in lung tissues(by real-time polymerase chain reaction)and for examination of the pathological changes(with a light microscope). The lung injury scores were recorded. Results Compared with group C of wild type rats, PaO2 was significantly decreased, W/D ratio and lung injury scores were increased, the concentrations of total protein, TNF-α, IL-1β and IL-18 in BALF were increased, and the expression of TNF-α, IL-1β and IL-18 mRNA in lung tissues was up-regulated in group VILI of wild type rats(P 0.05). Compared with group C of gene knockout rats, PaO2 was significantly decreased, W/D ratio and lung injury scores were increased, the concentrations of total protein, TNF-α, IL-1β and IL-18 in BALF were increased, and the expression of TNF-α, IL-1β and IL-18 mRNA in lung tissues was up-regulated in group VILI of gene knockout rats(P<0.05). Compared with group VILI of wild type rats, PaO2 was significantly decreased, W/D ratio and lung injury scores were increased, the concentrations of total protein, TNF-α, IL-1β and IL-18 in BALF were increased, and the expression of TNF-α, IL-1β and IL-18 mRNA in lung tissues was up-regulated in group VILI of gene knockout rats(P<0.05). Conclusion SDC-4 can inhibit inflammatory responses of rats with VILI and is involved in the endogenous protective mechanism. Key words: Syndecan-4; Respiration, artificial; Respiratory distress syndrome, adult; Inflammation

Cystatin C Is a Crucial Endogenous Protective Determinant Against Stroke.

Endogenous neuroprotection can be induced by ischemic and nonischemic preconditioning. However, not all subjects that undergo preconditioning exhibit similar favorable outcome. This study is to explore the molecules responsible for this phenomenon and find new therapeutic targets for stroke. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion. High-throughput proteomic technique, isobaric tag for relative and absolute quantification, was used to screen differentially expressed proteins in the rats that developed ischemic tolerance from hyperbaric oxygen (HBO) preconditioning. The proteomic results were verified by Western blot and ELISA. Then, short interfering RNA and gene knockout rats were used to further determine the pivotal role of candidate proteins in HBO preconditioning-induced endogenous neuroprotection. Finally, lysosomal permeability was tested to elaborate the mechanism underlying this intrinsic neuroprotective effect. Nine proteins differentially expressed in the serum of rats, which acquired benefits from HBO preconditioning, were screened and identified. Western blot and ELISA revealed that cystatin C (CysC) and mannose-binding lectin protein C were uniquely changed in rats with smaller infarction after HBO preconditioning and cerebral ischemia. Knockdown and knockout of CysC abolished HBO-induced neuroprotection. Moreover, HBO-induced endogenous CysC elevation preserved lysosomal membrane integrity after stroke in wild-type rats but not in CysC siRNA infusion or CysC-/- rats. Most importantly, exogenous CysC also induced neuroprotection against ischemic/reperfusion injury. CysC is a crucial determinant contributing to endogenous neuroprotection. It is also a novel candidate for stroke treatment through maintaining lysosomal membrane integrity.

Establishment of a rat model with diet-induced coronary atherosclerosis.

Coronary atherosclerotic disease is a serious disease in humans, but no suitable animal model is available currently for further studies. We used apolipoprotein E gene knockout (ApoE KO) rats to induce hypercholesterolemia through a special high cholesterol/bile salt diet (Paigen diet), then analyzed aortic and coronary atherosclerosis lesions and the myocardial injury in order to establish a novel small animal model of coronary atherosclerosis. Plasma cholesterol of ApoE KO rats increased 7.6-fold compared with wild-type rats after 8 weeks on the Paigen diet. After 10 to 12 weeks of subsisting on the Paigen diet, ApoE KO rats developed mild aortic atherosclerosis with severe coronary atherosclerosis. Hematoxilyn and eosin staining showed that 11 out of 12 ApoE KO male rats had right coronary artery atherosclerosis, 7 of them were>70% occluded. Oil Red O (Lipid Stain), Mac2 immuno-staining and Masson’s trichrome staining demonstrated substantial amounts of lipid, macrophages and collagen fibers in coronary atherosclerosis plaques. In addition, ApoE KO male rats had severe myocardial focal lesions with cholesterol ester as the main component in the lesions. In conclusion, ApoE KO rats developed severe hypercholesterolemia, coronary atherosclerosis and myocardial cholesterol ester deposition after subsisting on the Paigen diet and can be used as a novel animal model for studies on cholesterol metabolism and coronary atherosclerotic disease.

Generation of TALEN-mediated FH knockout rat model.

Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/−) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/− KO rats. There was no behaviour difference between the FH+/− KO and the control rats except that the FH+/− KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/− KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/− kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/− KO rat model, which will be useful for further functional FH studies.

Evidence for early and progressive ultrasonic vocalization and oromotor deficits in a PINK1 gene knockout rat model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease that leads to a wide range of motor and nonmotor deficits. Specifically, voice and swallow deficits manifest early, are devastating to quality of life, and are difficult to treat with standard medical therapies. The pathological hallmarks of PD include accumulation of the presynaptic protein α-synuclein (αSyn) as well as degeneration of substantia nigra dopaminergic neurons. However, there is no clear understanding of how or when this pathology contributes to voice and swallow dysfunction in PD. The present study evaluates the effect of loss of function of the phosphatase and tensin homolog-induced putative kinase 1 gene in rats (PINK1(-/-) ), a model of autosomal recessive PD in humans, on vocalization, oromotor and limb function, and neurodegenerative pathologies. Behavioral measures include ultrasonic vocalizations, tongue force, biting, and gross motor performance that are assayed at 2, 4, 6, and 8 months of age. Aggregated αSyn and tyrosine hydroxylase immunoreactivity (TH-ir) were measured at 8 months. We show that, compared with wild-type controls, PINK1(-/-) rats develop (1) early and progressive vocalization and oromotor deficits, (2) reduced TH-ir in the locus coeruleus that correlates with vocal loudness and tongue force, and (3) αSyn neuropathology in brain regions important for cranial sensorimotor control. This novel approach of characterizing a PINK1(-/-) genetic model of PD provides the foundational work required to define behavioral biomarkers for the development of disease-modifying therapeutics for PD patients.

Corticotropin Releasing Hormone (CRH) Expression in an Animal Model of Visceral Hypersensitivity

Corticotropin releasing hormone (CRH) is an important mediator of the neuroendocrine stress response in many mammals, and has been implicated in stress-induced behavioral disorders such as anxiety and depression. Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder (FGID) that is often associated with stress-related behavioral disorders, which suggests dysfunctions in the neural circuitry between the brain and the gut. Both serotonin (5-HT) and CRH signaling may be altered in IBS patients. We studied serotonin transporter (SERT) gene knockout rats to determine if there is a difference in CRH protein expression compared to wildtype (WT) controls at the level of sensory afferent neurons. CRH protein expression in sensory neurons of SERT KO and wildtype female rats was visualized in lumbar and sacral dorsal root ganglia (DRG) using immunohistochemistry techniques. We found that female SERT KO rats display increased CRH protein expression in sensory neurons compared to female wildtypes, and increased CRH in small-diameter neurons may play a role in visceral hypersensitivity; an abdominal pain symptom characteristic of IBS.

Online High-throughput Mutagenesis Designer Using Scoring Matrix of Sequence-specific Endonucleases.

CRISPR Cas9 and other sequence-specific endonucleases are fundamental genome editors supporting gene knockout and gene therapy. A speedy and accurate computational allele designer is required for a high through-put gene mutagenesis pipeline using these new techniques. An automatic system, Cas9 online designer (COD), was created to screen Cas9 targets and off-targets, as well as to provide gene knockout and genotyping strategies. A gene knockout rat model was successfully created and genotyped under the direction of this online system confirming its ability to predict real targets and off-targets. Gene knockout strategies to mutate 72 rat cytochrome P450 genes were designed instantly by the system to demonstrate its high-throughput efficiency. Also, the system used an off-target scoring matrix which can be applied to any sequence-specific genome editing tools besides Cas9. The COD system (http://cas9.wicp.net) has established a speedy, accurate, flexible and high through-put computational gene knockout pipeline supporting the sequence-specific endonuclease induced mutagenesis.

AB75. Lower urinary tract dysfunction in a pink1 gene knockout rat model for Parkinson’s disease

Bladder dysfunction is a common non-motor disorder on Parkinson’s disease (PD) patients. A new transgenic Pink1 gene knockout rat model of PD has been developed recently. To explore lower urinary tract function in this newly established PD model and provide possible therapeutic base for PD urinary complications. Twelve Pink1 KO rats (pink1-/-, LEH-pink1tm 1sage, 40 weeks old) and twelve wide type rats (pink1+/+, LEH, 40 weeks old) were used in this project. After acclimation, the rats underwent 24 hours metabolic cage test, conscious cystometry, and leak point pressure test. Their bladders were harvested, weighted and then fixed for histological study.Total volume, micturition times, and voided volume per micturition were calculated for 24h metabolic cage test. Pressure parameters, micturition frequency, bladder capacity/compliance, volume parameters, leak point pressure (LPP) were analyzed according to the result of cystometry and LPP test. Body weight, bladder weight, and the general pathologic changes in bladder were also checked. Continuous variables were compared between groups using t-test. Categorical variables were compared using Chi-square test. P<0.05 was set as statistical significant.Pink1 KO rats acquired the functional disorder of detrusor over activity compared to wild type rats. Hyperplasia of smooth muscle and nerves were noted in the bladder wall of Pink1 KO rats. More specific nerve changes and specific causality among dopaminergic neuron loss, pathologic alterations of bladder, and functional changes should be investigated in the future. Pink1-/- rat model of PD exhibited bladder dysfunction at the age of 40 weeks old. Detrusor over activity was the main change. Smooth muscle and nerves in bladder wall displayed the alteration of hyperplasia. This is a better transgenic rat model of PD induced detrusor over activity for future study.

Brain penetration of WEB 2086 (Apafant) and dantrolene in Mdr1a (P-glycoprotein) and Bcrp knockout rats.

Transporter gene knockout rat models are attracting increasing interest for mechanistic studies of new drugs as transporter substrates or inhibitors in vivo. However, limited data are available on the functional validity of such models at the blood-brain barrier. Therefore, the present study evaluated Mdr1a [P-glycoprotein (P-gp)], Bcrp, and combined Mdr1a/Bcrp knockout rat strains for the influence of P-gp and breast cancer resistance protein (BCRP) transport proteins on brain penetration of the selective test substrates [(14)C]WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propanon) for P-gp and dantrolene for BCRP. Brain-to-plasma concentration ratios (BPR) were measured after intravenous coinfusions of 5.5 µmol/kg per hour [(14)C]WEB 2086 and 2 µmol/kg per hour dantrolene for 2 hours in groups of knockout or wild-type rats. Compared with wild-type controls, mean BPR of [(14)C]WEB 2086 increased 8-fold in Mdr1a knockouts, 9.5-fold in double Mdr1a/Bcrp knockouts, and 7.3-fold in zosuquidar-treated wild-type rats, but was unchanged in Bcrp knockout rats. Mean BPR of dantrolene increased 3.3-fold in Bcrp knockouts and 3.9-fold in double Mdr1a/Bcrp knockouts compared with wild type, but was unchanged in the Mdr1a knockouts. The human intestinal CaCo-2 cell bidirectional transport system in vitro confirmed the in vivo finding that [(14)C]WEB 2086 is a substrate of P-gp but not of BCRP. Therefore, Mdr1a, Bcrp, and combined Mdr1a/Bcrp knockout rats provide functional absence of these efflux transporters at the blood-brain barrier and are a suitable model for mechanistic studies on the brain penetration of drug candidates.

Evaluation of the moderately obese apoe gene knockout rat as a new model for impaired glucose tolerance and atherosclerosis

Evaluation of the moderately obese apoe gene knockout rat as a new model for impaired glucose tolerance and atherosclerosis

Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Promising future for the transgenic rat in transplantation research.

The rat is the most widely used animal species in surgical research and offers distinct advantages over the mouse in transplantation models due to its size and close genetic similarity to humans. Sequencing of the rat genome and successful application of transgenic technologies which had only been available for mice have since led to a resurgence of the use of rat models. Transplantation provides the possibility to deliver transgenes through a variety of routes which can potentially offer treatment modalities for post-transplant dysfunction and rejection. Moreover, the use of genetically encoded fluorescent light probes has enabled in vivo visualization of organs and tissue in living animals. In recent years, generation of gene knockout rats via the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies has offered alternatives to the sophisticated embryonic stem cell based gene-targeting. In this review, we aim to provide an overview of transplantation studies involving transgenic techniques using rat models and recent advances in methods to modify the rat genome. Through novel gene modification techniques, precise, complete and conditional knockout and knockin rat models have become available which can provide promising new treatment options and opportunities for studying human transplant-related pathophysiology.

Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (ApoE, B2m, Prf1, and Prkdc) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the off-target mutagenesis and mosaicism are rarely detected by comprehensive analysis. Thus, the CRISPR/Cas9 system makes it possible to efficiently and reliably generate gene knock-out rats.

Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effector nuclease (TALEN)-mediated gene inactivation

Genetically engineered mice are a valuable resource for studies of the behavioral effects of ethanol. However, for some behavioral tests of ethanol action, the rat is a superior model organism. Production of genetically engineered rats has been severely hampered due to technical limitations. Here we utilized a promising new technique for efficient site-specific gene modification to create a novel gene knockout rat line. This approach is based on transcriptional activator-like effector nucleases (TALENs). TALENs function in pairs and bind DNA in a sequence-specific manner. Upon binding to the target sequence, a functional nuclease is reconstituted that creates double-stranded breaks in the DNA that are efficiently repaired by non-homologous end joining. This error-prone process often results in deletions of varying lengths at the targeted locus. The toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and neurodegeneration, as well as multiple ethanol-induced behavioral effects. To generate Tlr4 knockout rats, a pair of TALEN constructs was created that specifically target Exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. The mutated allele consisted of a 13 base-pair deletion that was predicted to create a frameshift mutation after amino acid 25. This founder rat successfully transmitted the mutation to F1 offspring. Heterozygous F1 offspring were interbred to produce homozygous F2 animals. Homozygous mutants expressed the 13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. Thus, the mutant Tlr4 allele generated by TALEN-mediated gene inactivation represents a null allele. This knockout rat line will be valuable for studies of ethanol action as well as more general inflammatory conditions including septic shock. In conclusion, TALEN-mediated gene targeting in rat zygotes represents an inexpensive, efficient, and rapid method for creating genetically engineered rats.