Knockout Rat

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Xin Wang - One of the best experts on this subject based on the ideXlab platform.

  • construction and characterization of crispr cas9 Knockout Rat model of carboxylesterase 2a gene
    Molecular Pharmacology, 2021
    Co-Authors: Jie Liu, Xuyang Shang, Yuanjin Zhang, Zongjun Liu, Shengbo Huang, Xin Wang
    Abstract:

    Carboxylesterase (CES) 2, an important metabolic enzyme, plays a critical role in drug biotransformation and lipid metabolism. Although CES2 is very important, few animal models have been geneRated to study its properties and functions. Rat Ces2 is similar to human CES2A-CES3A-CES4A gene cluster, with highly similar gene structure, function, and substRate. In this report, CRISPR-associated protein-9 (CRISPR/Cas9) technology was first used to knock out Rat Ces2a, which is a main subtype of Ces2 mostly distributed in the liver and intestine. This model showed the absence of CES2A protein expression in the liver. Further pharmacokinetic studies of diltiazem, a typical substRate of CES2A, confirmed the loss of function of CES2A both in vivo and in vitro. At the same time, the expression of CES2C and CES2J protein in the liver decreased significantly. The body and liver weight of Ces2a Knockout Rats also increased, but the food intake did not change. Moreover, the deficiency of Ces2a led to obesity, insulin resistance, and liver fat accumulation, which are consistent with the symptoms of nonalcoholic fatty liver disease (NAFLD). Therefore, this Rat model is not only a powerful tool to study drug metabolism mediated by CES2 but also a good disease model to study NAFLD. SIGNIFICANCE STATEMENT Human carboxylesterase (CES) 2 plays a key role in the first-pass hydrolysis metabolism of most oral prodrugs as well as lipid metabolism. In this study, CRISPR/Cas9 technology was used to knock out Ces2a gene in Rats for the first time. This model can be used not only in the study of drug metabolism and pharmacokinetics but also as a disease model of nonalcoholic fatty liver disease (NAFLD) and other metabolic disorders.

  • times new roman serif characterization of a novel cyp1a2 Knockout Rat model constructed by crispr cas9
    Drug Metabolism and Disposition, 2021
    Co-Authors: Dongyi Sun, Yuanjin Zhang, Yuanqing Guo, Jie Liu, Zongjun Liu, Bingyi Yao, Xin Wang
    Abstract:

    Cytochrome P450 1A2 (CYP1A2) as one of the most important CYP isoforms is involved in the biotransformation of many important endogenous and exogenous substances. CYP1A2 plays an important role in the development of many diseases because it is involved in the biotransformation of precancerous substances and poisons. Although the geneRation of Cyp1a2 Knockout (KO) mouse model has been reported, there are still no relevant Rat models for the study of CYP1A2-mediated pharmacokinetics and diseases. In this report, CYP1A2 KO Rat model was established successfully by using CRISPR/Cas9 without any detectable off-target effect. Compared with wild-type Rats, this model showed a loss of CYP1A2 protein expression in the liver. The results of pharmacokinetics in vivo and incubation in vitro of specific substRates of CYP1A2 confirmed the lack of function of CYP1A2 in KO Rats. In further studies of potential compensatory effects, we found that CYP1A1 was significantly up-regulated, and CYP2E1, CYP3A2 and LXRβ were down-regulated in KO Rats. In addition, CYP1A2 KO Rats exhibited a significant increase in serum cholesterol and free testosterone, accompanied by mild liver damage and lipid deposition, suggesting CYP1A2 deficiency affects lipid metabolism and liver function in Rats to some extent. In summary, we successfully constructed the CYP1A2 KO Rat model, which provides a useful tool for studying the metabolic function and physiological function of CYP1A2. Significance Statement Human CYP1A2 not only metabolizes clinical drugs and pollutants, but also mediates the biotransformation of endogenous substances, and plays an important role in the development of many diseases. However, there are no relevant CYP1A2 Rat models for the research of pharmacokinetics and diseases. This study successfully established CYP1A2 Knockout Rat model by using CRISPR/Cas9. This Rat model provides a powerful tool to study the function of CYP1A2 in drug metabolism and diseases.

  • geneRation and characterization of cytochrome p450 2j3 10 crispr cas9 Knockout Rat model
    Drug Metabolism and Disposition, 2020
    Co-Authors: Ang Chen, Mingyao Liu, Xuyang Shang, Yuanjin Zhang, Yuanqing Guo, Xin Wang
    Abstract:

    Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo In particular, a CYP2J gene Knockout (KO) Rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete Rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO Rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) Rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO Rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substRate of CYP2J, indicated that CYP2J was functionally inactive in KO Rats. The heart function indexes of WT and KO Rats were also measured and compared. The myocardial enzymes, including creatine kinase-muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK Ratio, of KO Rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO Rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease. SIGNIFICANCE STATEMENT: Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function in vivo. This study successfully developed a new CYP2J Knockout Rat model by using CRISPR/Cas9 technology. This Rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.

  • development and characterization of mdr1 mdr1a b crispr cas9 Knockout Rat model
    Drug Metabolism and Disposition, 2019
    Co-Authors: Chenmeizi Liang, Mingyao Liu, Xuyang Shang, Yuanjin Zhang, Junfang Zhao, Xin Wang
    Abstract:

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in Rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 (Mdr1a/b) double-Knockout (KO) Rat model was geneRated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO Rats. Further pharmacokinetic studies of digoxin, a typical substRate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of Mdr1a/b (−/−) Rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type Rats. In general, a new Mdr1a/b (−/−) Rat model has been successfully geneRated and characterized. This Rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.

  • crispr Knockout Rat cytochrome p450 3a1 2 model for advancing drug metabolism and pharmacokinetics research
    Scientific Reports, 2017
    Co-Authors: Yanjiao Shao, Xuan Qin, Mingyao Liu, Ang Chen, Daozhi Liu, Xin Wang
    Abstract:

    Cytochrome P450 (CYP) 3A accounts for nearly 30% of the total CYP enzymes in the human liver and participates in the metabolism of over 50% of clinical drugs. Moreover, CYP3A plays an important role in chemical metabolism, toxicity, and carcinogenicity. New animal models are needed to investigate CYP3A functions, especially for drug metabolism. In this report, Cyp3a1/2 double Knockout (KO) Rats were geneRated by CRISPR-Cas9 technology, and then were characterized for viability and physiological status. The Cyp3a1/2 double KO Rats were viable and fertile, and had no obvious physiological abnormities. Compared with the wild-type (WT) Rat, Cyp3a1/2 expression was completely absent in the liver of the KO Rat. In vitro and in vivo metabolic studies of the CYP3A1/2 substRates indicated that CYP3A1/2 was functionally inactive in double KO Rats. The Cyp3a1/2 double KO Rat model was successfully geneRated and characterized. The Cyp3a1/2 KO Rats are a novel rodent animal model that will be a powerful tool for the study of the physiological and pharmacological roles of CYP3A, especially in drug and chemical metabolism in vivo.

Michaela Serpi - One of the best experts on this subject based on the ideXlab platform.

  • the nucleotide prodrug cerc 913 improves mtdna content in primary hepatocytes from dguok deficient Rats
    Journal of Inherited Metabolic Disease, 2021
    Co-Authors: Mark A Vanden Avond, Hui Meng, Margaret Beatka, Daniel Helbling, Mariah J Prom, Jessica L Sutton, Rebecca A Slick, David Dimmock, Fabrizio Pertusati, Michaela Serpi
    Abstract:

    Loss‐of‐function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. in vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK‐deficient, patient‐derived fibroblasts and myoblasts. CERC‐913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC‐913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK‐deficient Rats. DGUOK Knockout Rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to 8 days in culture. CERC‐913 increased mtDNA content in DGUOK‐deficient hepatocytes up to 2.4‐fold after 4 days of treatment in a dose‐dependent fashion, which was significantly more effective than dGMP at similar concentRations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC‐913 treatment can improve mtDNA content in this model.

Stephanie W Watts - One of the best experts on this subject based on the ideXlab platform.

Judith R Homberg - One of the best experts on this subject based on the ideXlab platform.

  • bdnf overexpression in the ventral hippocampus promotes antidepressant and anxiolytic like activity in serotonin transporter Knockout Rats
    International Journal of Molecular Sciences, 2021
    Co-Authors: Danielle Mendes Diniz, Francesca Calabrese, Paola Brivio, Marco Riva, Joanes Grandjean, Judith R Homberg
    Abstract:

    BDNF plays a pivotal role in neuroplasticity events, vulnerability and resilience to stress-related disorders, being decreased in depressive patients and increased after antidepressant treatment. BDNF was found to be reduced in patients carrying the human polymorphism in the serotonin transporter promoter region (5-HTTLPR). The serotonin Knockout Rat (SERT-/-) is one of the animal models used to investigate the underlying molecular mechanisms of depression in humans. They present decreased BDNF levels, and anxiety- and depression-like behavior. To investigate whether upregulating BDNF would amelioRate the phenotype of SERT-/- Rats, we overexpressed BDNF locally into the ventral hippocampus and submitted the animals to behavioral testing. The results showed that BDNF overexpression in the vHIP of SERT-/- Rats promoted higher sucrose preference and sucrose intake; on the first day of the sucrose consumption test it decreased immobility time in the forced swim test and increased the time spent in the center of a novel environment. Furthermore, BDNF overexpression altered social behavior in SERT-/- Rats, which presented increased passive contact with test partner and decreased solitary behavior. Finally, it promoted decrease in plasma corticosterone levels 60 min after restraint stress. In conclusion, modulation of BDNF IV levels in the vHIP of SERT-/- Rats led to a positive behavioral outcome placing BDNF upregulation in the vHIP as a potential target to new therapeutic approaches to improve depressive symptoms.

  • bdnf overexpression in the ventral hippocampus promotes antidepressant and anxiolytic like activity in serotonin transporter Knockout Rats
    bioRxiv, 2020
    Co-Authors: Danielle Mendes Diniz, Francesca Calabrese, Paola Brivio, Marco Riva, Joanes Grandjean, Judith R Homberg
    Abstract:

    Brain-derived neurotrophic factor is one of the most studied proteins playing a pivotal role in neuroplasticity events and vulnerability and resilience to stress-related disorders. Most importantly, BDNF is decreased in depressive patients, and increased after antidepressant treatment. Additionally, BDNF was found to be reduced in a genetic subset of depression susceptible patients carrying the human polymorphism in the serotonin transporter promoter region (5-HTTLPR). The serotonin Knockout Rat (SERT-/-) is one of the animal models used to investigate the underlying molecular mechanisms behind the genetic susceptibility to depression in humans. SERT-/- Rats present decreased BDNF levels, especially BDNF exon IV, in the prefrontal cortex (PFC) and ventral hippocampus (vHIP), and display anxiety- and depression-like behavior. To investigate whether upregulating BDNF in the vHIP would melioRate the phenotype of SERT-/- Rats, we overexpressed BDNF locally into the Rat brain by means of stereotaxic surgery and submitted the animals to behavioral challenges, including the sucrose consumption, the open field, and forced swim tests. Additionally, we measured hypothalamus-pituitary-adrenal (HPA)-axis reactivity. The results showed that lentivirus-induced BDNF IV overexpression in the vHIP of SERT-/- Rats promoted higher sucrose preference and sucrose intake, on the first day of the sucrose consumption test, indicative for decreased anhedonia-like behavior. Moreover, it decreased immobility time in the forced swim test, suggesting adaptive passive coping. Additionally, BDNF upregulation increased the time spent in the center of a novel environment, implying decreased novel-induced anxiety-like behavior. Finally, it promoted a stronger decrease in plasma corticosterone levels 60 minutes after restraint stress. In conclusion, modulation of BDNF IV levels in the vHIP of SERT-/- Rats led to a positive behavioral outcome placing BDNF upregulation in the vHIP as a potential candidate for the development new therapeutic approaches targeting the improvement of depressive symptoms.

  • reduced inhibition within layer iv of sert Knockout Rat barrel cortex is associated with faster sensory integRation
    Cerebral Cortex, 2017
    Co-Authors: S M Miceli, Nael Nadif Kasri, Joep Joosten, C Huang, Larajane Kepser, Remi Proville, Martijn Selten, F Van Eijs, Alireza Azarfar, Judith R Homberg
    Abstract:

    Neural activity is essential for the matuRation of sensory systems. In the rodent primary somatosensory cortex (S1), high extracellular serotonin (5-HT) levels during development impair neural transmission between the thalamus and cortical input layer IV (LIV). Rodent models of impaired 5-HT transporter (SERT) function show disruption in their topological organization of S1 and in the expression of activity-regulated genes essential for inhibitory cortical network formation. It remains unclear how such alteRations affect the sensory information processing within cortical LIV. Using serotonin transporter Knockout (Sert-/-) Rats, we demonstRate that high extracellular serotonin levels are associated with impaired feedforward inhibition (FFI), fewer perisomatic inhibitory synapses, a depolarized GABA reversal potential and reduced expression of KCC2 transporters in juvenile animals. At the neural population level, reduced FFI increases the excitatory drive originating from LIV, facilitating evoked representations in the supragranular layers II/III. The behavioral consequence of these changes in network excitability is faster integRation of the sensory information during whisker-based tactile navigation, as Sert-/- Rats require fewer whisker contacts with tactile targets and perform object localization with faster reaction times. These results highlight the association of serotonergic homeostasis with formation and excitability of sensory cortical networks, and consequently with sensory perception.

  • the serotonin transporter Knockout Rat a review
    Experimental Models in Serotonin Transporter Research, 2010
    Co-Authors: Jocelien D A Olivier, Edwin Cuppen, A R Cools, Bart A Ellenbroek, Judith R Homberg
    Abstract:

    This chapter dicusses the most recent data on the serotonin transporter knock-out Rat, a unique Rat model that has been geneRated by target-selected N-ethyl-N-nitrosourea (ENU) driven mutagenesis. The knock-out Rat is the result of a premature stopcodon in the serotonin transporter gene, and the absence of the serotonin transporter has been confirmed at mRNA, protein, and functional levels. The serotonin transporter (SERT) plays a crucial role in serotonin reuptake and its absence has a huge effect on serotonin neurotransmission – exemplified by increased extracellular serotonin levels, reduced serotonin tissue/platelet/blood levels, and reduced evoked serotonin release – yet the animals appear normal and do not differ from wildtype littermates in respect to breeding and health. Behavioral phenotypes are only apparent when the animals are exposed to certain stimuli. For instance, the serotonin transporter knock-out Rat displays increased stress sensitivity in a variety of anxiety- and depression-like tests, such as the elevated plus maze test and the forced swim test. Also remarkable, while general activity is not changed, the knock-out Rats show a “neurotic-like” exploRatory pattern. In line with the serotonin hypothesis of impulsivity, which argues that there is an inverse relationship between the two, serotonin transporter knock-out Rats show reduced motor impulsivity in the five-choice serial reaction time task, and a reduction in social interaction during play and aggressive encounters. Interestingly, abdominal fat seems to be increased in the knock-out Rat, despite normal body weight. Pharmacological compounds also elicit genotype-dependent responses in the knock-out Rats.

  • stress induced hyperthermia and basal body tempeRature are mediated by different 5 ht 1a receptor populations a study in sert Knockout Rats
    European Journal of Pharmacology, 2008
    Co-Authors: Jocelien D A Olivier, Edwin Cuppen, Judith R Homberg, A R Cools, Berend Olivier, Bart A Ellenbroek
    Abstract:

    Disturbances in the serotonergic system are implicated in many central nervous system disorders. The serotonin transporter (SERT) regulates the serotonin homeostasis in the synapse. We recently developed a Rat which lacks the serotonin transporter (SERT(-/-)). It is likely that adaptive changes take place at the level of pre- and postsynaptic 5-HT receptors. Because autonomic responses are often used to measure 5-HT(1A) receptor function, we analysed these responses by examining the effects of a 5-HT(1A) receptor agonist and antagonist under in vivo conditions in the SERT(-/-) Rat. Moreover, we studied the effect of a mild stressor on the body tempeRature (stress-induced hyperthermia) because of the known involvement of 5-HT(1A) receptors in this phenomenon. Results show that core body tempeRature did not differ between genotypes under basal, non-stressed conditions. Compared to SERT(+/+) Rats, stress-induced hyperthermia was reduced in SERT(-/-) Rats. The 5-HT(1A) receptor agonist [R(+)-N-(2[4-(2,3-dihydro-2-2-hydroxy-methyl-1,4-benzodioxin-5-yl)-1-piperazininyl]ethyl)-4-fluorobenzoamide HCl (flesinoxan) reduced stress-induced hyperthermia in both genotypes. The flesinoxan-induced hypothermia in SERT(+/+) Rats was blocked by the 5-HT(1A) receptor antagonist [N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridinyl) cyclohexane carboxamide 3HCl (WAY100635). Moreover, WAY100635-induced hyperthermia in SERT(-/-), but not in SERT(+/+) Rats. In SERT(-/-) Rats, WAY100635 completely blocked the flesinoxan-induced reduction of stress-induced hyperthermia. Interestingly, flesinoxan-induced hypothermia was absent in SERT(-/-) Rats. It is concluded that the SERT Knockout Rat reveals that 5-HT(1A) receptors modulating stress-induced hyperthermia belong to a population of receptors that differs from that involved in hypothermia.

Hui Meng - One of the best experts on this subject based on the ideXlab platform.

  • the nucleotide prodrug cerc 913 improves mtdna content in primary hepatocytes from dguok deficient Rats
    Journal of Inherited Metabolic Disease, 2021
    Co-Authors: Mark A Vanden Avond, Hui Meng, Margaret Beatka, Daniel Helbling, Mariah J Prom, Jessica L Sutton, Rebecca A Slick, David Dimmock, Fabrizio Pertusati, Michaela Serpi
    Abstract:

    Loss‐of‐function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. in vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK‐deficient, patient‐derived fibroblasts and myoblasts. CERC‐913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC‐913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK‐deficient Rats. DGUOK Knockout Rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to 8 days in culture. CERC‐913 increased mtDNA content in DGUOK‐deficient hepatocytes up to 2.4‐fold after 4 days of treatment in a dose‐dependent fashion, which was significantly more effective than dGMP at similar concentRations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC‐913 treatment can improve mtDNA content in this model.

  • sdha Rats display minimal muscle pathology without significant behavioral or biochemical abnormalities
    Journal of Neuropathology and Experimental Neurology, 2018
    Co-Authors: Emily M Siebers, Hui Meng, Margaret Beatka, Daniel Helbling, Melinda J Choi, Jennifer Tinklenberg, Samuel Ayres, Akiko Takizawa, Brian Bennett, Alexander M Garces
    Abstract:

    Mitochondrial diseases (MDs) result from alteRation of the mitochondrial respiRatory chain (MRC) function. Despite the prevalence of MDs in the population, the paucity of animal models available limits the understanding of these disorders. Mutations in SDHA, a gene that codes for the alpha subunit of succinate dehydrogenase (SDH), can cause some forms of MD. SDHA is a crucial contributor to MRC function. In order to expand the range of MD animal models available, we attempted to geneRate a Sdha Knockout Rat. Since homozygous Sdha-/- Rats could neither be identified in newborn litters, nor as early as embryonic day 14, we evaluated wild-type (WT) and heterozygous Sdha+/- genotypes. No differences in behavioral, biochemical, or molecular evaluations were observed between WT and Sdha+/- Rats at 6 weeks or 6 months of age. However, 30% of Sdha+/- Rats displayed mild muscle fiber atrophy with rare fibers negative for cytochrome oxidase and SDH on histochemical staining. Collectively, our data provide additional evidence that modeling SDH mutations in rodents may be challenging due to animal viability, and heterozygous Rats are insufficiently symptomatic at a phenotypic and molecular level to be of significant use in the study of SDH deficiency.

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