The hGFAP-driven conditional TSPO knockout is protective in a mouse model of multiple sclerosis.

The mitochondrial translocator protein (TSPO) has been implicated in CNS diseases. Here, we sought to determine the specific role of TSPO in experimental autoimmune encephalomyelitis (EAE), the most studied animal model of multiple sclerosis (MS). To fundamentally elucidate the functions of TSPO, we first developed a viable TSPO knockout mouse. A conditional TSPO knockout mouse was generated by utilizing the Cre-Lox system. We generated a TSPO floxed mouse, and then crossed this mouse with a Cre recombinase expressing mouse driven by the human glial fibrillary acidic protein (hGFAP) promoter. The resultant mouse was a neural linage line specific TSPO knockout. The loss of TSPO in the CNS did not result in overt developmental defects or phenotypes. The TSPO-/- mouse showed a decrease in GFAP expression, correlating with a decrease in astrogliosis in response to neural injury during EAE. This decrease in astrogliosis was also witnessed in the lessening of severity of EAE clinical scoring, indicating an in vivo functional role for TSPO in suppressing EAE. The TSPO-/- mouse could be a useful tool in better understanding the role of TSPO in CNS disease, and our results implicate TSPO as a potential therapeutic target in MS

Genome-wide screening of mouse knockouts reveals novel genes required for normal integumentary and oculocutaneous structure and function.

Oculocutaneous syndromes are often due to mutations in single genes. In some cases, mouse models for these diseases exist in spontaneously occurring mutations, or in mice resulting from forward mutatagenesis screens. Here we present novel genes that may be causative for oculocutaneous disease in humans, discovered as part of a genome-wide screen of knockout-mice in a targeted single-gene deletion project. The International Mouse Phenotyping Consortium (IMPC) database (data release 10.0) was interrogated for all mouse strains with integument abnormalities, which were then cross-referenced individually to identify knockouts with concomitant ocular abnormalities attributed to the same targeted gene deletion. The search yielded 307 knockout strains from unique genes with integument abnormalities, 226 of which have not been previously associated with oculocutaneous conditions. Of the 307 knockout strains with integument abnormalities, 52 were determined to have ocular changes attributed to the targeted deletion, 35 of which represent novel oculocutaneous genes. Some examples of various integument abnormalities are shown, as well as two examples of knockout strains with oculocutaneous phenotypes. Each of the novel genes provided here are potentially relevant to the pathophysiology of human integumentary, or oculocutaneous conditions, such as albinism, phakomatoses, or other multi-system syndromes. The novel genes reported here may implicate molecular pathways relevant to these human diseases and may contribute to the discovery of novel therapeutic targets

DNPIノックアウトマウスの作製

A gene-knockout mouse is a mouse in which a particular gene has been artificially destroyed and made dysfunctional. By analyzing symptoms which appear in the gene-knockout mouse's body, we are able to learn the essential function of the knocked-out gene in vivo. Utilizing a gene-knockout mouse, we can ana-lyze the differences between the mouse and a normal mouse of their growth, activity, or abnormalities of their tissues including brains, livers, kidneys and bones. These information from analyzing of a gene-knockout mouse is quite important. For instance, unknown gene functions or connections between genes and diseases can be clarified. Differentiation-associated Na+-dependent inorganic phosphate (Pi) cotransporter (DNPI) is protein that is reported to be associated with transdifferentiation of insulin-secreting cells and regulation of blood glucose levels. We tried to generate DNPI-knockout mouse. The analysis of this mouse clarifies the mech-anism of DNPI and leads to development of future diabetes medical treatment. This review article explained the background of this study and generation of knockout mouse

Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.

Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder

Untersuchung zur Expression und Funktion von HCN-Kanälen in Photorezeptoren und Bipolarzellen der Mäuseretina

The aim of this study was to examine the expression and the functional properties of HCN- channels (hyperpolarization-activated and cyclic nucleotide-gated) in photoreceptors and bipolar cells of the mouse retina. The expression pattern of the four HCN channel isoforms HCN1 - 4 was studied in retinal sections using immunocytochemistry with isoform-specific antibodies. The stainings were analysed using confocal laser scanning microscopy. In the mouse retina HCN1 is the predominantly expressed isoform. HCN1 could be detected in rods and cones as well as in type5 and type7 bipolar cells. HCN2, HCN3 and HCN4 are not abundantly expressed in the mouse retina. HCN4 was expressed in type 3 bipolar cells. HCN2 and HCN3 could not be found in photoreceptors and bipolar cells. These isoforms were mainly expressed in amacrine and ganglion cells. In retinal slices photoreceptors and bipolar cells were recorded electrophysiologically in the whole-cell mode of the patch-clamp technique to study the activation kinetics and the midpoint potential of activation of HCN channels. During the recording the cells were filled with dyes and were subsequently morphologically identified. The properties of the retinal HCN1 channels differ to some extend from those of heterologically expressed HCN1 channels. The properties of the HCN1 channels also vary between different cell types. This could mean, that HCN1 channels are modulated in the retina. To record retinal light-responses, a genetically modified mouse strain was used in which the gene coding for HCN1 has been deleted - “HCN1 knockout”. Electroretinogramms (ERG) were recorded in vitro from wildtype retinae and HCN1 knockout retinae. In HCN1 knockout retinae the B-wave of the ERG was slightly elongated. This could reflect extended lightresponses of the photoreceptors. In the scotopic flicker-ERG the frequency transfer is slightly reduced in knockout retinae. Application of the HCN channel blocker ZD7288 also slightly reduced the frequency transfer in wildtype retinae. The study provides first insights into the role of HCN1 channels in the generation of lightresponses

Agouti C57BL/6N embryonic stem cells for mouse genetic resources.

We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background

Deletion of Gpr27 in vivo reduces insulin mRNA but does not result in diabetes.

Gpr27 is a highly conserved, orphan G protein coupled receptor (GPCR) previously implicated in pancreatic beta cell insulin transcription and glucose-stimulated insulin secretion in vitro. Here, we characterize a whole-body mouse knockout of Gpr27. Gpr27 knockout mice were born at expected Mendelian ratios and exhibited no gross abnormalities. Insulin and Pdx1 mRNA in Gpr27 knockout islets were reduced by 30%, but this did not translate to a reduction in islet insulin content or beta cell mass. Gpr27 knockout mice exhibited slightly worsened glucose tolerance with lower plasma insulin levels while maintaining similar insulin tolerance. Unexpectedly, Gpr27 deletion reduced expression of Eif4e3, a neighboring gene, likely by deleting transcription start sites on the anti-sense strand of the Gpr27 coding exon. Our data confirm that loss of Gpr27 reduces insulin mRNA in vivo but has only minor effects on glucose tolerance

Neuromuscular synaptic function in mice lacking major subsets of gangliosides

Gangliosides are a family of sialylated glycosphingolipids enriched in the outer leaflet of neuronal membranes, in particular at synapses. Therefore, they have been hypothesized to play a functional role in synaptic transmission. We have measured in detail the electrophysiological parameters of synaptic transmission at the neuromuscular junction (NMJ) ex vivo of a GD3-synthase knockout mouse, expressing only the O- and a-series gangliosides, as well as of a GM2/GD2-synthase*GD3-synthase double-knockout (dKO) mouse, lacking all gangliosides except GM3. No major synaptic deficits were found in either null-mutant. However, some extra degree of rundown of acetylcholine release at high intensity use was present at the dKO NMJ and a temperature-specific increase in acetylcholine release at 35 °C was observed in GD3-synthase knockout NMJs, compared with wild-type. These results indicate that synaptic transmission at the NMJ is not crucially dependent on the particular presence of most ganglioside family members and remains largely intact in the sole presence of GM3 ganglioside. Rather, presynaptic gangliosides appear to play a modulating role in temperature- and use-dependent fine-tuning of transmitter output

Reduced body weight is a common effect of gene knockout in mice

<p>Abstract</p> <p>Background</p> <p>During a search for obesity candidate genes in a small region of the mouse genome, we noticed that many genes when knocked out influence body weight. To determine whether this was a general feature of gene knockout or a chance occurrence, we surveyed the Jackson Laboratory Mouse Genome Database for knockout mouse strains and their phenotypes. Body weights were not available for all strains so we also obtained body weight information by contacting a random sample of investigators responsible for a knockout strain.</p> <p>Results</p> <p>We classified each knockout mouse strain as (1) lighter and smaller, (2) larger and heavier, or (3) the same weight, relative to control mice. We excluded knockout strains that died early in life, even though this type of lethality is often associated with a small embryo or reduced body size. Based on a dataset of 1,977 knockout strains, we found that that 31% of viable knockout mouse strains weighed less and an additional 3% weighed more than did controls.</p> <p>Conclusion</p> <p>Body weight is potentially a latent variable in about a third of experiments that use knockout mice and should be considered in interpreting experimental outcomes, e.g., in studies of hypertension, drug and hormone metabolism, organ development, cell proliferation and apoptosis, digestion, heart rate, or atherosclerosis. If we assume that the knockout genes we surveyed are representative then upward of 6,000 genes are predicted to influence the size of a mouse. Body weight is highly heritable, and numerous quantitative trait loci have been mapped in mice, but "multigenic" is an insufficient term for the thousands of loci that could contribute to this complex trait.</p

A viable mouse model of factor X deficiency provides evidence for maternal transfer of factor X.

BackgroundActivated factor X (FXa) is a vitamin K-dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal.ObjectiveWe sought to generate a viable mouse model of FX deficiency.MethodsWe used a socket-targeting construct to generate F10-knockout mice by eliminating F10 exon 8 (knockout allele termed F10(tm1Ccmt), abbreviated as '-'; wild-type '+'), and a plug-targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4-9% normal in humans carrying the defect, Pro343--&gt;Ser, termed FX Friuli (mutant allele termed F10(tm2Ccmt), abbreviated as F)].ResultsF10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of approximately 5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age-dependent iron deposition and cardiac fibrosis. Interestingly, F10 (-/F) mice with FX activity levels of 1-3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival.ConclusionsWe demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1-3% is sufficient to rescue the lethal phenotype. This viable low-FX mouse model will facilitate the development of FX-directed therapies as well as investigation of the FX role in embryonic development