Knockout
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Pho91 is a vacuolar phosphate transporter that exports phosphate from the vacuolar lumen to the cytosol in yeast cells. In this study, we have demonstrated the pleiotropic effects of the PHO91 gene knockout in the methylotrophic yeast Ogataea parapolymorpha (Hansenula polymorpha, Ogataea angusta). The content of both acid-soluble and acid-insoluble inorganic polyphosphate (polyP) in the ∆pho91 cells was slightly higher compared to the strain with wild-type PHO91, when the cells were cultivated on glucose. The pho91-Δ mutations both in O. parapolymorpha and in Saccharomyces cerevisiae diminished resistance to cadmium and increased resistance to manganese and peroxide stresses. The cells of the mutant strain of O. parapolymorpha were unable to consume methanol due to the lack of methanol oxidase activity. We speculate that these effects are associated with the inability of mutant cells to mobilize phosphate from the vacuolar pool and/or defects in the signaling pathways involving phosphate, polyP, and inositol polyphosphates.
- MeSH
- genový knockout MeSH
- polyfosfáty * MeSH
- Publikační typ
- časopisecké články MeSH
We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.
- MeSH
- cilie genetika MeSH
- ciliopatie * genetika MeSH
- faktografické databáze MeSH
- genový knockout MeSH
- myši knockoutované MeSH
- myši MeSH
- proteiny buněčného cyklu MeSH
- proteiny nervové tkáně MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.
- MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy * MeSH
- down regulace MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- genový knockout metody MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikro RNA genetika MeSH
- myši MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Analyze phenotypic data from knockout mice with late-adult retinal pathologic phenotypes to identify genes associated with development of adult-onset retinal diseases. METHODS: The International Mouse Phenotyping Consortium (IMPC) database was queried for genes associated with abnormal retinal phenotypes in the late-adult knockout mouse pipeline (49-80 weeks postnatal age). We identified human orthologs and performed protein-protein analysis and biological pathways analysis with known inherited retinal disease (IRD) and age-related macular degeneration (AMD) genes using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), PLatform for Analysis of single cell Eye in a Disk (PLAE), Protein Analysis Through Evolutionary Relationships (PANTHER), and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: Screening of 587 late-adult mouse genes yielded 12 with abnormal retinal phenotypes, which corresponded to 20 human orthologs. Three of the 12 mouse genes and two of the 20 human orthologs were previously implicated in retinal pathology or physiology in a literature review. Although all of the genes demonstrated retinal pathology when deleted from the mouse genome, most do not have established roles in human retinal disease. Furthermore, human protein-protein analysis and biological pathway analysis yielded only a few relationships between the candidate gene list and that of known IRD and AMD genes, suggesting they may represent novel retinal functions. CONCLUSIONS: We identified 12 mouse genes with significant late-adult abnormal retinal pathology, eight of which have not been previously implicated in either mouse or human retinal physiology or pathology. These serve as novel retinal disease gene candidates for late-onset retinal disease.
- MeSH
- fenotyp MeSH
- lidé MeSH
- makulární degenerace * genetika MeSH
- modely nemocí na zvířatech MeSH
- myši knockoutované MeSH
- myši MeSH
- oční proteiny * genetika MeSH
- retina * patologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: This study investigates genes contributing to late-adult corneal dystrophies (LACDs) in aged mice, with potential implications for late-onset corneal dystrophies (CDs) in humans. METHODS: The International Mouse Phenotyping Consortium (IMPC) database, containing data from 8901 knockout mouse lines, was filtered to include late-adult mice (49+ weeks) with significant (P < 0.0001) CD phenotypes. Candidate genes were mapped to human orthologs using the Mouse Genome Informatics group, with expression analyzed via PLAE and a literature review for prior CD associations. Comparative analyses of LACD genes from IMPC and established human CD genes from IC3D included protein interactions (STRING), biological processes (PANTHER), and molecular pathways (KEGG). RESULTS: Analysis identified 14 genes linked to late-adult abnormal corneal phenotypes. Of these, 2 genes were previously associated with CDs in humans, while 12 were novel. Seven of the 14 genes (50%) were expressed in the human cornea based on single-cell transcriptomics. Protein-protein interactions via STRING showed several significant interactions with known human CD genes. PANTHER analysis identified six biological processes shared with established human CD genes. Two genes (Rgs2 and Galnt9) were involved in pathways related to human corneal diseases, including cGMP-PKG signaling, mucin-type O-glycan biosynthesis, and oxytocin signaling. Other candidates were implicated in pathways such as pluripotency of stem cells, MAPK signaling, WNT signaling, actin cytoskeleton regulation, and cellular senescence. CONCLUSIONS: This study identified 14 genes linked to LACD in knockout mice, 12 of which are novel in corneal biology. These genes may serve as potential therapeutic targets for treating corneal diseases in aging human populations.
- MeSH
- dědičné dystrofie rohovky * genetika metabolismus MeSH
- fenotyp MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši knockoutované MeSH
- myši MeSH
- stárnutí * genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease.
- MeSH
- degenerace retiny etiologie metabolismus patologie MeSH
- hematoretinální bariéra MeSH
- hepcidiny fyziologie MeSH
- játra metabolismus patologie MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- přetížení železem etiologie metabolismus patologie MeSH
- retina metabolismus patologie MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Multiple gene knockouts play an important role in metabolic engineering. The flanked homology length, homologous to the region adjacent to the target gene, of the knockout fragments has a great effect on the efficiency of multiple gene knockouts, whereas the existing gene knockout methods can only supply a very short homology. This article presents a strategy of easily extending homologous sequence based on the available strain library through one-step PCR amplification (the one-step PCR method). In this approach, the library of single gene mutants was used as the templates for PCR to amplify knockout fragments. Thus, the flanked homology can be extended as long as possible by designing primers upstream and downstream far from the target gene. Based on the one-step PCR method, we studied the effect of the homology length and the number of mutations on the efficiency of multiple gene knockouts. Our results indicated that the one-step PCR method permitted rapid and efficient construction of multiple mutants continuously or simultaneously, and a length of 200-300 bp homologous sequence was equal for multiple gene knockouts.
Neurofibromatosis type 2 (NF-2) is a dominantly inherited genetic disorder that results from variants in the tumor suppressor gene, neurofibromin 2 (NF2). Here, we report the generation of a conditional zebrafish model of neurofibromatosis established by inducible genetic knockout of nf2a/b, the zebrafish homologs of human NF2. Analysis of nf2a and nf2b expression revealed ubiquitous expression of nf2b in the early embryo, with overlapping expression in the neural crest and its derivatives and in the cranial mesenchyme. In contrast, nf2a displayed lower expression levels. Induction of nf2a/b knockout at early stages increased the proliferation of larval Schwann cells and meningeal fibroblasts. Subsequently, in adult zebrafish, nf2a/b knockout triggered the development of a spectrum of tumors, including vestibular Schwannomas, spinal Schwannomas, meningiomas and retinal hamartomas, mirroring the tumor manifestations observed in patients with NF-2. Collectively, these findings highlight the generation of a novel zebrafish model that mimics the complexities of the human NF-2 disorder. Consequently, this model holds significant potential for facilitating therapeutic screening and elucidating key driver genes implicated in NF-2 onset.
- MeSH
- dánio pruhované * genetika embryologie MeSH
- geneticky modifikovaná zvířata MeSH
- genový knockout * MeSH
- larva metabolismus MeSH
- lidé MeSH
- modely nemocí na zvířatech * MeSH
- neurofibromatóza 2 genetika patologie metabolismus MeSH
- neurofibromatózy genetika patologie metabolismus MeSH
- neurofibromin 2 * genetika metabolismus nedostatek MeSH
- proliferace buněk MeSH
- proteiny dánia pruhovaného * genetika metabolismus nedostatek MeSH
- Schwannovy buňky metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N (6) -isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions.
- MeSH
- alkyltransferasy a aryltransferasy genetika metabolismus MeSH
- biologická evoluce * MeSH
- chloroplasty enzymologie genetika MeSH
- cytokininy biosyntéza MeSH
- fylogeneze * MeSH
- genový knockout MeSH
- mechy klasifikace enzymologie genetika MeSH
- molekulární sekvence - údaje MeSH
- RNA transferová genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- rostliny klasifikace enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.
- MeSH
- adenosintrifosfát metabolismus MeSH
- homozygot MeSH
- kardiomyopatie metabolismus MeSH
- membránové proteiny nedostatek genetika metabolismus MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondriální nemoci genetika metabolismus MeSH
- mitochondriální proteiny nedostatek genetika metabolismus MeSH
- mitochondriální protonové ATPasy biosyntéza genetika metabolismus MeSH
- mitochondrie metabolismus MeSH
- mutace MeSH
- myši knockoutované MeSH
- myši MeSH
- oxidativní fosforylace MeSH
- těhotenství MeSH
- vrozené poruchy metabolismu metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
