Novel animal model of combined generalized and focal epilepsy

Thioredoxin, encoded by Txn1, is a critical antioxidant that protects against oxidative damage by regulating the dithiol/disulfide balance of interacting proteins. We recently discovered the Adem rat, an epileptic rat harboring the Txn1-F54L mutation, characterized by wild running and vacuolar degeneration in the midbrain. This study aimed to characterize the classification of epilepsy in Adem rats. We performed simultaneous video-electroencephalographic recordings, magnetic resonance imaging, neurotransmitter measurements using gas chromatography–mass spectrometry (GC-MS), and immunohistochemistry. Adem rats exhibited absence, tonic, and focal seizures. The type of epilepsy was classified as combined generalized and focal epilepsy. Neurotransmitters in the midbrain and cortex were measured at 3 weeks of age, when neuronal cell death occurs in the midbrain. The results of GC-MS ruled out the dominance of the excitatory system in the midbrain and cortex of Adem rats. Activation of astrocytes and microglia was more pronounced at 5 weeks of age, at which time epileptic seizures occurred frequently. The underlying pathology in Adem rats remains unknown. However, glial cell activation and inflammation may play a significant role in the occurrence of epilepsy

Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

Background: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. Methodology/Principal Findings: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. Conclusions and Significance: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies

The Israeli strain IS-98-ST1 of West Nile virus as viral model for West Nile encephalitis in the Old World

West Nile virus (WNV) recently became a major public health concern in North America, the Middle East, and Europe. In contrast with the investigations of the North-American isolates, the neurovirulence properties of Middle-Eastern strains of WNV have not been extensively characterized. Israeli WNV strain IS-98-ST1 that has been isolated from a white stork in 1998, was found to be highly neuroinvasive in adult C57BL/6 mice. Strain IS-98-ST1 infects primary neuronal cells from mouse cortex, causing neuronal death. These results demonstrate that Israeli strain IS-98-ST1 provides a suitable viral model for WNV-induced disease associated with recent WNV outbreaks in the Old World

Therapy for hyperthermia-induced seizures in Scn1a mutant rats

Purpose: Mutations in the SCN1A gene, which encodes the alpha 1 subunit of voltage-gated sodium channels, cause generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). N1417H-Scn1a mutant rats are considered to be an animal model of human FS+ or GEFS+. To assess the pharmacologic validity of this model, we compared the efficacies of eight different antiepileptic drugs (AEDs) for the treatment of hyperthermia-induced seizures using N1417H-Scn1a mutant rats. Methods: AEDs used in this study included valproate, carbamazepine (CBZ), phenobarbital, gabapentin, acetazolamide, diazepam (DZP), topiramate, and potassium bromide (KBr). The effects of these AEDs were evaluated using the hot water model, which is a model of experimental FS. Five-week-old rats were pretreated with each AED and immersed in water at 45 degrees C to induce hyperthermia-induced seizures. The seizure manifestations and video-electroencephalographic recordings were evaluated. Furthermore, the effects of each AED on motor coordination and balance were assessed using the balance-beam test. Key Findings: KBr significantly reduced seizure durations, and its anticonvulsant effects were comparable to those of DZP. On the other hand, CBZ decreased the seizure threshold. In addition, DZP and not KBr showed significant impairment in motor coordination and balance. Significance: DZP and KBr showed potent inhibitory effects against hyperthermia-induced seizures in the Scn1a mutant rats, whereas CBZ exhibited adverse effects. These responses to hyperthermia-induced seizures were similar to those in patients with GEFS+ and SMEI. N1417H-Scn1a mutant rats may, therefore, be useful for testing the efficacy of new AEDs against FS in GEFS+ and SMEI patients

Assembly and Function of a Bioengineered Human Liver for Transplantation Generated Solely from Induced Pluripotent Stem Cells

The availability of an autologous transplantable auxiliary liver would dramatically affect the treatment of liver disease. Assembly and function in vivo of a bioengineered human liver derived from induced pluripotent stem cells (iPSCs) has not been previously described. By improving methods for liver decellularization, recellularization, and differentiation of different liver cellular lineages of human iPSCs in an organ-like environment, we generated functional engineered human mini livers and performed transplantation in a rat model. Whereas previous studies recellularized liver scaffolds largely with rodent hepatocytes, we repopulated not only the parenchyma with human iPSC-hepatocytes but also the vascular system with human iPS-endothelial cells, and the bile duct network with human iPSC-biliary epithelial cells. The regenerated human iPSC-derived mini liver containing multiple cell types was tested in vivo and remained functional for 4 days after auxiliary liver transplantation in immunocompromised, engineered (IL2rg−/−) rats.Fil: Takeishi, Kazuki. University of Pittsburgh; Estados UnidosFil: Collin de I'Hortet, Alexandra. University of Pittsburgh; Estados UnidosFil: Wang, Yang. University of Pittsburgh; Estados UnidosFil: Handa, Kan. University of Pittsburgh; Estados UnidosFil: Guzman Lepe, Jorge. University of Pittsburgh; Estados UnidosFil: Matsubara, Kentaro. University of Pittsburgh; Estados UnidosFil: Morita, Kazutoyo. University of Pittsburgh; Estados UnidosFil: Jang, Sae. University of Pittsburgh; Estados UnidosFil: Haep, Nils. University of Pittsburgh; Estados UnidosFil: Florentino, Rodrigo M.. University of Pittsburgh; Estados UnidosFil: Yuan, Fangchao. University of Pittsburgh; Estados UnidosFil: Fukumitsu, Ken. University of Pittsburgh; Estados UnidosFil: Tobita, Kimimasa. University of Pittsburgh; Estados UnidosFil: Sun, Wendell. University of Pittsburgh; Estados UnidosFil: Franks, Jonathan. University of Pittsburgh; Estados UnidosFil: Delgado, Evan R.. University of Pittsburgh; Estados UnidosFil: Shapiro, Erik M.. University of Pittsburgh; Estados UnidosFil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Duncan, Andrew W.. University of Pittsburgh; Estados UnidosFil: Yagi, Hiroshi. University of Pittsburgh; Estados UnidosFil: Mashimo, Tomoji. University of Pittsburgh; Estados UnidosFil: Fox, Ira J.. University of Pittsburgh; Estados UnidosFil: Soto Gutierrez, Alejandro. University of Pittsburgh; Estados Unido

CLICK:One-step generation of conditional knockout mice

Abstract Background CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. Results Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. Conclusions The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G⇔A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology