A nonsense mutation in Gnat1, encoding the alpha subunit of rod transducin, in spontaneous mouse models of retinal dysfunction.

ICR-derived retinal dysfunction (IRD) 1 and IRD2 mice are new spontaneous mouse models of rod-cone and rod dysfunctions, respectively. In this study, we investigated the cause of rod dysfunction in IRD1 and IRD2 mice. Gene expression of rod phototransduction proteins was analyzed by quantitative real-time RT-PCR. mRNA levels of Gnat1, which encodes the alpha subunit of rod transducin (Tralpha), were severely reduced. Tralpha protein was immunohistochemically undetectable in both IRD1 and IRD2 mice. Sequencing of Tralpha cDNA revealed a 48-base pair (bp) insertion between exons 4 and 5 in both mutant strains. The insertion changed codon 150 (TAC) to a stop codon (TAG) (Tyr150Ter). The truncated Tralpha protein was undetectable in the retinas of both mutants by western blot analysis using a primary antibody against the N-terminal region. A 57-bp deletion was identified in intron 4 of the Gnat1 gene, which encodes the Tralpha protein, and included the last two bases of the splice donor site of intron 4. Thus our results showed that IRD1 and IRD2 mice harbor a nonsense mutation in the Gnat1 gene, resulting in the absence or suppressed expression of the Tralpha protein, which is the likely cause of rod dysfunction in both mutants.

Gene expression profiling of functional murine embryonic stem cell-derived cardiomyocytes and comparison with adult heart: profiling of murine ESC-derived cardiomyocytes.

Although embryonic stem cell (ESC)-derived cardiomyocytes may be a powerful tool in drug discovery, their potential has not yet been fully explored. Nor has a detailed comparison with adult heart tissue been performed. We have developed a method for efficient production of cardiomyocyte-rich embryoid bodies (EBs) from murine ESCs. Analysis of global gene expression profiles showed that EBs on day 7 and/or 21 of differentiation (d7CMs and d21CMs, respectively) were similar to adult heart tissue for genes categorized as regulators of muscle contraction or voltage-gated ion channel activity, although d21CMs were more mature than d7CMs for contractile components related to morphological structures. Calcium and sodium channel blockers altered Ca2+ transients, and isoproterenol, a beta-adrenergic compound, increased the rate of beating in d7CMs and d21CMs. Our gene analytic system therefore enabled us to identify genes that are expressed in the physiological pathways associated with ion channels and structural components in d7CMs and d21CMs. We conclude that EBs might be of use for the basic screening of drugs that might affect contractile function through ion channels.

Visual electrophysiological features of two naturally occurring mouse models with retinal dysfunction.

PURPOSE: We developed two strains of mouse with retinal dysfunction, named the ICR-derived retinal dysfunction (IRD)1 and IRD2, from one male ICR mouse with a retinal dysfunction but a normal fundus. The purpose of this study was to describe the features of retinal dysfunction in both mutant mice. METHODS: Scotopic and photopic electroretinograms (ERGs) were recorded from IRD1 and IRD2 mice at 1 month of age to evaluate retinal function, and then the structures of the retinas in both mutant mice were observed by light microscopy at 1 and 3 months of age. In a mating study, the inheritance pattern and the genetic relation of IRD1 and IRD2 mice were defined. RESULTS: At 1 month of age, IRD1 mice showed affected scotopic and photopic ERGs, and IRD2 mice exhibited normal photopic but affected scotopic ERGs. The retinal structures of both mutant mice remained normal even at 3 months of age. The IRD1, and IRD2 phenotypes showed an autosomal recessive pattern of inheritance and in the IRD1 backcross offspring some mice that had only cone dysfunction were seen in addition to normal, IRD1, and IRD2 phenotypes. All F1 (IRD1 x IRD2) offspring exhibited IRD2 phenotype, rod dysfunction. CONCLUSIONS: IRD1 and IRD2 mice had affected rod systems caused by a homozygous mutation in the same rod function-related gene, and additionally IDR1 mice had affected cone systems caused by a homozygous mutation in the cone function-related gene, without apparent anatomical abnormalities in the retinas of either mutant mice even at 3 months of age. We believe that these mice could be new spontaneous animal models for the study of human inherited retinal disorders.

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness.

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had "negative-type" mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.

IRD1 and IRD2 mice, naturally occurring models of hereditary retinal dysfunction, show late-onset and progressive retinal degeneration.

PURPOSE: To elucidate whether Institute for Cancer Research (ICR) derived retinal dysfunction (IRD) 1 and IRD2 mice, spontaneous mouse models of rod-cone and rod dysfunction, respectively, develop age-related retinal degeneration. MATERIALS AND METHODS: Morphological and morphometric examinations were performed in the retinas of both mutants from 1 to 18 months of age. The rate of apoptotic cell death was determined by TUNEL techniques. Electroretinography was performed on the IRD2 mice at various ages. Age-matched ICR mice were used as controls. RESULTS: IRD1 and IRD2 mice showed a decrease in the thickness of the outer nuclear layer and a higher frequency of TUNEL-positive photoreceptor cells at 6 months of age compared with controls, and showed almost complete absence of the outer nuclear layer at 18 months of age. Light deprivation had no effect on the severity of the retinal degeneration. There were no differences in the number of cone nuclei among ICR, IRD1, and IRD2 mice at 12 months of age and the cones of the IRD2 mice were still functional at this age. CONCLUSIONS: IRD1 and IRD2 mice showed late-onset and progressive retinal degeneration.

Detection of cell-free, liver-specific mRNAs in peripheral blood from rats with hepatotoxicity: a potential toxicological biomarker for safety evaluation.

To verify the concept that cell-free organ/tissue-specific mRNAs leaking from drug-damaged organs/tissues into peripheral blood could be toxicological biomarkers for identification of the target organs of drug toxicity, we attempted to detect liver-specific mRNAs in peripheral blood from rats with chemical-induced hepatotoxicity. We selected alpha(1)-microglobulin/bikunin precursor (Ambp) and albumin mRNAs as tentative liver-specific biomarkers and successfully detected them by reverse transcription (RT)-PCR in peripheral blood 24 h after D-galactosamine HCl (D-gal) or acetaminophen administration. Moreover, albumin mRNA was detected 2 h after D-gal administration, although plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were still unchanged. On the other hand, in peripheral blood from rat with bupivacaine HCl-induced skeletal muscle damage, neither Ambp nor albumin mRNA was detectable while plasma creatine kinase, ALT, and AST levels prominently increased 2 or 12 h after dosing. Furthermore, Ambp mRNA was also detectable in filtered plasma from rats with liver damage, indicating that cell-free Ambp mRNA can be present in peripheral blood. In conclusion, cell-free, liver-specific Ambp, and albumin mRNAs were detectable in peripheral blood from rats with chemical-induced liver damage. It is believed that the detection of cell-free organ/tissue-specific mRNA in peripheral blood is a promising approach in the survey of toxicological biomarkers.