从老鼠胚胎得到单倍体胚胎干细胞 大多数动物是双倍体，但也有全单倍体或雄性单倍体（如蜜蜂和蚂蚁）物种被报道。复杂生物的双倍体基因组限制了生物医学模式生物如老鼠等的遗传学操作。 为了解决这个问题，在鱼类中用实验手段诱导了单倍体。对斑马鱼以开展了单倍体发育的遗传学筛选。最近，青鳉鱼（Oryzias latipes）单倍体多能干细胞系也已建立。 相反的，哺乳动物的单倍体似乎与发育的兼容性较差。虽然单倍体细胞可以在孤雌生殖的小鼠胚胎卵圆柱期被观察到，胚胎中大多数生存的细胞将成为双倍体。这里我们描述了单倍体小鼠胚胎干细胞并展示了它们在正向遗传筛选中的应用。 Derivation of haploid embryonic stem cells from mouse embryos. Leeb M , Wutz A . Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK. Abstract Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouseembryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening. Nature. 2011 Sep 7. doi: 10.1038/nature10448.
美国批准用胚胎干细胞医治眼病试验 http://news.sciencenet.cn/htmlnews/2010/11/240557.shtm Stem cells could help blind patients to see within six weeks http://www.independent.co.uk/news/science/stem-cells-could-help-blind-patients-to-see-within-six-weeks-2140301.html http://www.gopubmed.org/web/gopubmed/1?WEB1mOWEB10O00d000j10020001000h00100090000 26 documents semantically analyzed Top Years Publications 2009 9 2010 6 2007 3 2006 3 2011 1 2008 1 2005 1 2002 1 2001 1 Top Countries Publications USA 12 United Kingdom 5 Japan 2 Germany 2 Australia 1 Israel 1 Brazil 1 Top Cities Publications London 4 Bethesda 3 Los Angeles 1 New York City 1 Kobe 1 Jerusalem 1 Santa Barbara 1 Newcastle upon Tyne 1 Portland, USA 1 San Francisco 1 Catanduva 1 Boston 1 Worcester 1 Salt Lake City 1 Omaha 1 Kyoto 1 Wrzburg 1 Cologne 1 1 2 Top Journals Publications Stem Cells 3 Hum Mol Genet 2 Invest Ophthalmol Vis Sci 1 Stem Cell Res 1 Pigment Cell Melanoma Res 1 Can J Ophthalmol 1 Transplantation 1 J Genet 1 Plos One 1 Cell Stem Cell 1 Bmc Med 1 Arq Bras Oftalmol 1 Crit Rev Biomed Eng 1 Exp Neurol 1 Prog Retin Eye Res 1 Sci Am 1 J Clin Invest 1 Method Enzymol 1 Cloning Stem Cells 1 Methods Mol Biol 1 1 2 1 2 3 ... 19 Top Terms Publications Macular Degeneration 26 Stem Cells 24 stem cell development 23 stem cell differentiation 23 Retinitis 22 Retinaldehyde 22 Embryonic Stem Cells 21 Pigmentation 19 pigmentation 19 Retinal Pigments 18 Humans 18 Pigment Epithelium of Eye 17 Animals 16 Photoreceptors 15 pigment cell differentiation 14 Epithelium 14 Cell Differentiation 12 Retinal Diseases 11 Transplants 10 Stem Cell Transplantation 10 1 2 3 ... 19 1 2 3 ... 7 Top Authors Publications Klimanskaya I 2 Miller S 1 Strunnikova N 1 Maminishkis A 1 Barb J 1 Wang F 1 Zhi C 1 Sergeev Y 1 Chen W 1 Edwards A 1 Stambolian D 1 Abecasis G 1 Swaroop A 1 Munson P 1 Tsang S 1 Wang N 1 Tosi J 1 Kasanuki J 1 Chou C 1 Kong J 1 1 2 3 ... 7 http://arrowsmith.psych.uic.edu/cgi-bin/arrowsmith_uic/edit_b.cgi?refresh=TID=14563 Start A-Literature C-Literature B-list Filter Literature A-query: embryonic stem cells and macular degen... 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Use Ctrl to select multiple B-terms. job id # 14563 started Wed Jan 5 02:19:04 2011 Max_citations: 50000 Stoplist: /var/www/html/arrowsmith_uic/data/stopwords_pubmed Ngram_max: 3 14563 Search ARROWSMITH A A_query_raw: embryonic stem cells and macular degenerationWed Jan 5 02:19:34 2011 A query = embryonic stem cells and macular degeneration started Wed Jan 5 02:19:34 2011 A query resulted in 26 titles 14563 Search ARROWSMITH C C_query_raw: light-sensitive retina cells Wed Jan 5 02:19:47 2011 C: light-sensitive retina cells 385 A: pubmed_query_A 26 AC: ( embryonic stem cells and macular degeneration ) AND ( light-sensitive retina cells ) 0 C query = light-sensitive retina cells started Wed Jan 5 02:19:49 2011 C query resulted in 385 titles A AND C query resulted in 0 titles 64 B-terms ready on Wed Jan 5 02:19:58 2011 B-list on Wed Jan 5 02:27:29 2011 1 visual function 2 model retinitis pigmentosa 3 retinitis pigmentosa 4 photoreceptor cell 5 retinal pigment epithelium 6 photoreceptor 7 pigment epithelium 8 retinal pigment 9 human retinal pigment 10 visual 11 stem cell 12 retinal disease 13 retinal cell 14 pigment 15 retina 16 retinal 17 epithelium 18 mouse model 19 degeneration 20 rescue 21 eye 22 cell survival 23 role retinal 24 mouse http://arrowsmith.psych.uic.edu/cgi-bin/arrowsmith_uic/show_sentences.cgi Start A-Literature C-Literature B-list Filter Literature AB literature B-term BC literature embryonic stem cells and mac... visual function light-sensitive retina cells 1: Transplantation of Reprogrammed Embryonic Stem Cells Improves Visual Function in a Mouse Model for Retinitis Pigmentosa.2010 Add to clipboard 2: Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.2006 Add to clipboard 1: Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells.2010 Add to clipboard 2: 2009 Add to clipboard 3: Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin.2008 Add to clipboard 4: Effects of potent inhibitors of the retinoid cycle on visual function and photoreceptor protection from light damage in mice.2006 Add to clipboard 5: The retinal pigment epithelium in visual function .2005 Add to clipboard
http://www.gopubmed.org/web/gopubmed/1?WEB0yneywov8hor4I3I1I00f01000j10040001rl 8 documents semantically analyzed top author statistics Top Years Publications 2010 1 2009 1 2008 1 2007 1 2006 1 2005 1 2002 1 1998 1 Top Countries Publications USA 5 Taiwan 1 United Kingdom 1 Japan 1 Switzerland 1 Top Cities Publications Taipei 1 New York 1 London 1 Philadelphia 1 New Orleans 1 Omaha 1 Boston 1 Kyoto 1 Zrich 1 Top Journals Publications Transplantation 1 Mol Vis 1 Invest Ophthalmol Vis Sci 1 Mutat Res-fund Mol M 1 Methods Mol Biol 1 Invest Ophth Vis Sci 1 P Natl Acad Sci Usa 1 J Neurosci 1 1 2 3 Top Authors Publications Tsang S 1 Wang N 1 Tosi J 1 Kasanuki J 1 Chou C 1 Kong J 1 Parmalee N 1 Wert K 1 Allikmets R 1 Lai C 1 Chien C 1 Nagasaki T 1 Lin C 1 Gregory-Evans C 1 Gregory-Evans K 1 Chang F 1 Hodges M 1 Pierce E 1 Graziotto J 1 Inglehearn C 1 1 2 3 1 2 3 ... 12 Top Terms Publications Retinitis Pigmentosa 8 Mice 8 Retinitis 7 Animals 7 stem cell differentiation 6 Retina 6 Genes 6 Proteins 5 stem cell development 5 Photoreceptors 5 Cell Differentiation 4 Stem Cells 4 Mutation 4 Embryonic Stem Cells 3 Injections 3 Retinal Degeneration 3 Cell Line 3 Cell Transplantation 2 Epithelial Cells 2 Cell Survival 2 1 2 3 ... 12 documents Title: Transplantation of Reprogrammed Embryonic Stem Cells Improves Visual Function in a Mouse Model for Retinitis Pigmentosa . PMID: 20164818 Related Articles Authors: Wang, N , Tosi, J , Kasanuki, J M , Chou, C L , Kong, J , Parmalee, N , Wert, K J , Allikmets, R , Lai, C , Chien, C , Nagasaki, T , Lin, C , Tsang, S H Journal: Transplantation , 2010 Abstract: BACKGROUND.: To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa : Rpe65 /Rpe65 C57BL6 mice . METHODS.: Yellow fluorescent protein ( YFP )-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice . Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RESULTS.: RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. CONCLUSIONS.: ES cells can differentiate, morphologically, and functionally, into RPE-like cells . Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials. Affiliation: 1Department of Ophthalmology, Columbia University, New York City, NY. 2Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan . 3Retina Division, Chang Gung University College of Medicine, Taoyuan, Taiwan . 4Department of Anatomy and Cell Biology, National Taiwan University, Taipei , Taiwan . 5Department of Genetics and Development, Columbia University, New York, NY. 6Institute of Human Nutrition, Columbia University, New York, NY. 7Department of Pathology and Cell Biology, Columbia University, New York, NY. 8Regenerative Medicine Division, Bernard and Shirlee Brown Glaucoma Laboratory, Columbia University, New York City , NY . Wikipedia: Age-related maculopathy , Benign neoplasm , Cancer , Cell differentiation , Colony-forming unit , Colony forming unit , Critique , Dysembryoma , Embryonic Stem Cell , Epithelial cell , Evaluation , Evaluation research , Fluorescence , House Mouse , House mice , Injectable , Injection , Laboratory mice , Laboratory mouse , Macular Degeneration , Mature teratoma , Mice , Mother cell , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Neoplasm , Pigmentary retinopathy , Pigmentation , Progenitor cell , Proteins , Retinal Detachment , Retinal detachments , Retinal pigments , Retinitis , Retinitis Pigmentosa , Salinity , Stem Cell , Stem cell differentiation , Teratoma , Therapeutic , Treatment , Tumor Title: Ex vivo gene therapy using intravitreal injection of GDNF -secreting mouse embryonic stem cells in a rat model of retinal degeneration. PMID: 19461934 Related Articles Authors: Gregory-Evans, K , Chang, F , Hodges, M D , Gregory-Evans, C Y Journal: Mol Vis , Vol. 15 , 2009 Abstract: PURPOSE: Safe and prolonged drug delivery to the retina is a key obstacle to overcome in the development of new medicines aimed at treating progressive retinal disease. We took advantage of the ability of embryonic stem cells to survive long-term in foreign tissue and used these cells to deliver neuroprotectant molecules to the retina of the rhodopsin TgN S334ter-4 rat model of retinitis pigmentosa (RP). METHODS: Mouse embryonic stem (mES) cells , derived from the pluripotent embryonic stem cell line E14TG2a, were genetically engineered to oversecrete the glial cell-derived neurotrophic factor ( GDNF ). Cell suspensions, containing approximately 200,000 cells and expressing approximately 35ng/10(6) cells /24 h GDNF, were injected into the vitreous cavity of TgN S334ter rat eyes at postnatal day 21 (P21) without immunosuppression. Histological and immunofluorescence imaging was used to evaluate photoreceptor survival up to P90. Local (vitreous) and systemic ( serum ) concentrations of GDNF were determined and ocular side effects were monitored. RESULTS: Green fluorescent protein ( GFP )-expressing mES cells were observed on the inner limiting membrane of the retina in retinal flatmounts up to P90. In cryostat sections at P45, some GFP -expressing cells had integrated into the inner retina, but did not migrate into the outer nuclear layer. After an initial lag period, the photoreceptor cell counts were significantly higher (p or =0.05) in animals treated with GDNF-secreting mES cells than in untreated animals, principally in the peripheral retina. Several adverse side effects such as tractional detachments and areas of hyperplasia were seen in a minimal number of treated eyes. Abnormally high levels of GDNF in the peripheral circulation were also observed. CONCLUSIONS: ES cells engineered to secrete GDNF exerted a neuroprotective effect for at least three months on retinal structure in the TgN S334ter rat model of retinal degeneration. Immunosuppression was not required for this. Several adverse effects were identified which require further investigation to make cell-based delivery of neuroprotection a viable clinical strategy. Affiliation: Department of Clinical Neuroscience, Faculty of Medicine, Imperial College London, London , UK . k.gregory-evans@imperial.ac.uk Pubmed MeSH: Cell Differentiation , Disease Models, Animal , Embryonic Stem Cells , Histocytochemistry , Rats , Statistics, Nonparametric , Stem Cell Transplantation , Transfection , Vitreous Body Wikipedia: Alien , Animal , Animalia , Blood Serum , Blood circulation , Border Crossing , Brain Drain , Cell Membrane , Cell count , Cell line , Chain migration , Cistron , Critique , Cytoplasmic membrane , Destination , Drugs , Emigrant , Emigration , Evaluation , Evaluation research , Fluorescence , Fluorescent antibody technique , Foreigner , GDNF , Gene , Gene Therapy , Genetic Engineering , Genetic material , Glial cell line-derived neurotrophic factor , House Mouse , House mice , Hyperplasia , Immigrant , Immigration , Immunofluorescence , Immunosuppression , Injectable , Injection , International migration , Laboratory mice , Laboratory mouse , Medicine , Membrane , Mice , Migration , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Nerve growth factors , Neuroprotection , Neurotrophic factors , Neurotrophic protein , Neurotrophins , Out-migration , Photoreceptor , Pigmentary retinopathy , Plasma membrane , Plasmalemma , Proteins , Retina , Retinal degeneration , Retinal disease , Retinitis , Retinitis Pigmentosa , Return migration , Rhodopsin , Serum , Somatic gene therapy , Stem cell differentiation , Suspensions , Tissue , Visual purple Title: Decreased levels of the RNA splicing factor Prpf3 in mice and zebrafish do not cause photoreceptor degeneration. PMID: 18552388 Related Articles Authors: Graziotto, J J , Inglehearn, C F , Pack, M A , Pierce, E A Journal: Invest Ophthalmol Vis Sci , Vol. 49 (9): 3830-8 , 2008 Abstract: PURPOSE: Pre-mRNA processing factor 3 ( PRPF3 ) is a spliceosomal component essential for pre-mRNA processing. Mutations in PRPF3 have been implicated in retinitis pigmentosa (RP) 18 through an unknown mechanism. The authors created and characterized Prpf3 knockout mice and zebrafish to determine whether RP18 is a result of haploinsufficiency. METHODS: Mice were produced from a Prpf3 gene trap cell line, and parameters of retinal function, structure, and RNA splicing were analyzed. The retinas of prpf3 insertional mutant zebrafish were also analyzed histologically. RESULTS: Homozygous Prpf3 knockout mice do not survive to 14 days postfertilization (dpf), implying that this allele is required for early embryonic development. Homozygous Prpf3 knockout zebrafish die by 4dpf, well beyond the mid-blastula transition at which transcription activates. Zebrafish knockout embryos reveal abnormally high levels of cell death in the developing eye. Heterozygous Prpf3 knockout mice have less than the expected 50% reduction in Prpf3 at the mRNA and protein levels, implying compensatory expression from the wild-type allele. The heterozygous mice develop normally, with no changes in retinal function, no evidence for photoreceptor degeneration at up to 23 months of age, and no decrease in pre-mRNA splicing of transcripts mutated in other forms of RP in the retina. Similarly, heterozygous prpf3 knockout zebrafish develop normally and show no retinal degeneration up to 12 months of age. CONCLUSIONS: These models suggest that RP18 is not a result of haploinsufficiency but instead arises from a toxic gain of function caused by missense mutations in PRPF3 . Affiliation: F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Philadelphia , Pennsylvania , USA . Pubmed MeSH: Animals , Chimera , DNA Primers , Electroretinography , Embryonic Stem Cells , RNA Precursors , Ribonucleoprotein, U4-U6 Small Nuclear Wikipedia: Allele , Allelomorph , Brachydanio rerio , Cell death , Cell line , Cistron , Danio rerio , Embryo development , Embryogenesis , Embryonic development , Gene , Genetic material , House Mouse , House mice , Knock-out mice , Knockout mice , Knockout mouse , Laboratory mice , Laboratory mouse , Messenger RNA , Mice , Missense mutation , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Mutation , Photoreceptor , Pigmentary retinopathy , Poly(A) tail , Proteins , RNA , RNA splicing , Retina , Retinal degeneration , Retinitis , Retinitis Pigmentosa , RiboNucleic Acid , Spliceosome , Transactivation , Zebra Danio , Zebra Fish , Zebrafish Title: Ush1c216A knock-in mouse survives Katrina. PMID: 17174357 Related Articles Authors: Lentz, J J , Pan, F , Ng, S S , Deininger, P L , Keats, B J Journal: Mutat Res , Vol. 616 (1-2): 139-44 , 2007 Abstract: Usher syndrome is the most common cause of inherited deafness found in combination with blindness. All Usher patients suffer progressive retinitis pigmentosa , with the degree of hearing impairment and the presence or absence of vestibular function differing among subtypes. A cryptic splice site mutation (216G--A) in exon 3 of the USH1C gene on chromosome 11p, which encodes a PDZ-domain protein, harmonin, was found in Acadian Usher type IC patients in south Louisiana. In vitro analysis using constructs containing the mutant 216A and subsequent analysis of patient cell lines revealed a deletion of 35 bases in the transcript. In order to analyze the impact of this frame-shift mutation, we created a knock-in mouse model containing the human 216G--A mutation. A targeting construct was made containing 5' and 3' homology arms, each 4kb in length, and a 650 base pair fragment containing exons 3 and 4 of human USH1C cloned from an Acadian patient homozygous for the 216A mutation. W4/129S6 embryonic stem (ES) cells were electroporated with the targeting construct, and after 10 days of neomycin selection, clones were picked and screened by polymerase chain reaction ( PCR ) and Southern blot analysis for homologous recombination. Two positive clones for targeted insertion were microinjected into C57BL/6 blastocysts which were then transplanted into pseudo-pregnant females. Chimeras were bred with Cre recombinase -expressing mice for simultaneous deletion of the neomycin gene and germline transmission of the 216A allele. Homozygous Ush1c216A (216AA) mice are hyperactive, display circling and head tossing behavior, and do not have a Preyer reflex at 21-25 days old. RT-PCR analysis of the cochlea and retina from 216AA mice shows the same 35 base deletion characteristic of Usher IC patients. Affiliation: Department of Genetics, Louisiana State University Health Sciences Center, New Orleans , LA 70112, USA . Pubmed MeSH: Adaptor Proteins, Signal Transducing , Animals , Cloning, Molecular , DNA, Recombinant , Disease Models, Animal , Mice, Inbred C57BL , Mice, Transgenic Wikipedia: Allele , Allelomorph , Base pair , Base pairing , Blastocyst , Blotting, southern , Cell line , Cistron , Client , Cloning , Cochlea , Cryptic splice site , Deaf-mutism , Deaf mutism , Deafness , Electroporation , Exon , Frame shift mutation , Frameshift mutation , Gene , Genetic Recombination , Genetic material , Hearing Impairment , Hearing Loss , House Mouse , House mice , Human Cloning , Hypoacusis , Inverse PCR , Inverse polymerase chain reaction , Laboratory mice , Laboratory mouse , Mice , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Mutation , Neomycin , Neomycin sulfate , Nested PCR , Nested polymerase chain reaction , PCR , Patient , Pigmentary retinopathy , Polymerase Chain Reaction , Prelingual deafness , Proteins , RT-PCR , RTPCR , Recombination , Recombination, genetic , Reflex , Retina , Retinitis , Retinitis Pigmentosa , Reverse Transcriptase Polymerase Chain Reaction , Southern Blot , Southern Blotting , Syndrome , Usher Syndrome , Usher syndromes Title: Differentiation of embryonic stem cells to retinal cells in vitro. PMID: 16846039 Related Articles Authors: Zhao, X , Liu, J , Ahmad, I Journal: Methods Mol Biol , Vol. 330 , 2006 Abstract: Currently, there is no effective treatment for photoreceptor degeneration, the most common cause of blindness caused by diseases like retinitis pigmentosa , age-related macular degeneration, and diabetic retinopathy. Two promising approaches include cell therapy to replace degenerating cells and neuroprotection to rescue affected cells from premature death. Determination of the potential of embryonic stem (ES) cells to differentiate into photoreceptors will provide reagents for both approaches. First, neural progenitors with retinal potential will be available in unlimited supply to test the efficacy of cell therapy; second, the controlled differentiation of ES cells into photoreceptors, in addition to providing cells to replace degenerating photoreceptors, will offer a robust in vitro model of photoreceptor differentiation for better understanding of degenerative processes and screening of neuroprotective drugs/reagents. In addition, it will allow the identification of genes ( gene discovery) that play critical roles in photoreceptor differentiation and degeneration. Here, we describe the protocol to promote differentiation of the mouse ES cell-derived neural progenitors into retinal cells , specifically the rod photoreceptors. Affiliation: Department of Ophthalmology, University of Nebraska Medical Center, Omaha , NE , USA . Pubmed MeSH: Animals , Antineoplastic Agents , Biological Markers , Cell Culture Techniques , Embryo, Mammalian , Retina , Stem Cells , Tretinoin Wikipedia: Age-related maculopathy , Cell differentiation , Cell therapy , Cistron , Device , Diabetic retinopathy , Equipment , Gene , Genetic material , House Mouse , House mice , Indicators , Inventories , Inventory , Laboratory mice , Laboratory mouse , Macular Degeneration , Medical device , Mice , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Neuroprotection , Photoreceptor , Pigmentary retinopathy , Reagents , Retinitis , Retinitis Pigmentosa , Rod (retina) , Rods (retina) , Stem cell differentiation , Supplies Title: A single, abbreviated RPGR - ORF15 variant reconstitutes RPGR function in vivo. PMID: 15671266 Related Articles Authors: Hong, D H , Pawlyk, B S , Adamian, M , Sandberg, M A , Li, T Journal: Invest Ophthalmol Vis Sci , Vol. 46 (2): 435-41 , 2005 Abstract: PURPOSE: The retinitis pigmentosa GTPase regulator (RPGR) is essential for the maintenance of photoreceptor viability. RPGR is expressed as constitutive and ORF15 variants because of alternative splicing. This study was designed to examine whether the retina-specific ORF15 variant alone could substantially substitute for RPGR function. A further objective was to test whether the highly repetitive purine-rich region of ORF15 could be abbreviated without ablating the function, so as to accommodate RPGR replacement genes in adenoassociated virus (AAV) vectors. METHODS: A cDNA representing RPGR-ORF15 but shortened by 654 bp in the repetitive region was placed under the control of a chicken beta-actin (CBA) hybrid promoter. The resultant construct was transfected into mouse embryonic stem cells . Clones expressing the transgene were selected and injected into mouse blastocysts. Transgenic chimeras were crossed with RPGR knockout (KO) mice . Mice expressing the transgene but null for endogenous RPGR ( Tg /KO) were studied from 1 month to 18 months of age by light and electron microscopy, immunofluorescence, and electroretinography (ERG). The results were compared with those of wild-type (WT) and RPGR-null control mice . RESULTS: Transgenic RPGR- ORF15 was found in the connecting cilia of rod and cone photoreceptors, at approximately 20% of the WT level. Photoreceptor morphology, cone opsin localization, expression of GFAP (a marker for retinal degeneration ) and ERGs were consistent with the transgene exerting substantial rescue of retinal degeneration due to loss of endogenous RPGR. CONCLUSIONS: RPGR- ORF15 is the functionally significant variant in photoreceptors. The length of its repetitive region can be reduced while preserving its function. The current findings should facilitate the design of gene replacement therapy for RPGR-null mutations. Affiliation: Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston , Massachusetts 02114, USA . Pubmed MeSH: Animals , Carrier Proteins , Exons , Eye Proteins , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation , Genetic Vectors , Glial Fibrillary Acidic Protein , Guanine Nucleotide Exchange Factors , Immunoblotting , Mice, Inbred C57BL , Mice, Transgenic , Open Reading Frames , Proteins , Recombinant Fusion Proteins , Transfection Wikipedia: Adeno-Associated Virus , Alternate splicing , Alternative splicing , Animal virus , Blastocyst , Cilia , Cilium , Cistron , Clone , Clone cells , Complementary DNA , Cone (retina) , Cones (retina) , Dependovirus , Electron , Electron Microscopy , Electronic , Electroretinography , Fluorescent antibody technique , GTPase , Gene , Gene Therapy , Genetic material , House Mouse , House mice , Immunofluorescence , Injectable , Injection , Knock-out mice , Knockout mice , Knockout mouse , Laboratory mice , Laboratory mouse , Maintenance , Mice , Microscopy , Microscopy, electron , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Mutation , Negatron , Opsin , Photoreceptor , Pigmentary retinopathy , Positron , Retinal degeneration , Retinitis , Retinitis Pigmentosa , Rods and cones , Somatic gene therapy , Transgene , Vertebrate photoreceptor , Virus Title: Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. PMID: 11818560 Related Articles Authors: Kawasaki, H , Suemori, H , Mizuseki, K , Watanabe, K , Urano, F , Ichinose, H , Haruta, M , Takahashi, M , Yoshikawa, K , Nishikawa, S I , Nakatsuji, N , Sasai, Y Journal: Proc Natl Acad Sci U S A , Vol. 99 (3): 1580-5 , 2002 Abstract: We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells , including midbrain tyrosine hydroxylase-positive ( TH (+)) dopaminergic neurons, from mouse embryonic stem cells . We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells . Induced neurons contain TH (+) neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH (+) neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (approximately 5 weeks). In addition, 8% of the colonies contain large patches of Pax6 (+)-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa . Affiliation: Department of Medical Embryology and Neurobiology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan . Pubmed MeSH: Animals , Biological Markers , Cell Differentiation , Cell Transplantation , Cells, Cultured , Epithelial Cells , Macaca fascicularis , Mice, SCID , Neural Cell Adhesion Molecules , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells , Stromal Cells , Time Factors , Transplantation, Heterologous , Tyrosine 3-Monooxygenase Wikipedia: 3,4-dihydroxyphenethylamine , Dopamine , Dopamine hydrochloride , Drugs , Epithelium , House Mouse , House mice , Intropin , L-Tyrosine , Laboratory mice , Laboratory mouse , Mesencephalon , Mesothelium , Mice , Midbrain , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Nerve cell , Neuron , Paralysis agitans , Parkinson's Disease , Parkinson disease , Pigmentary retinopathy , Pigmentation , Primate , Retina , Retinitis , Retinitis Pigmentosa , Tyrosine , Tyrosine metabolism Title: Na,K-ATPase subunit beta1 knock-in prevents lethality of beta2 deficiency in mice . PMID: 9801359 Related Articles Authors: Weber, P , Bartsch, U , Schachner, M , Montag, D Journal: J Neurosci , Vol. 18 (22): 9192-203 , 1998 Abstract: The beta2 subunit of the Na,K-ATPase displays functional properties of both an integral constituent of an ion pump and an adhesion and neurite outgrowth-promoting molecule in vitro. To investigate whether the beta1 subunit of the Na,K-ATPase can functionally substitute for the beta2 isoform in vivo, we have generated beta2 / beta1 knock-in mice by homologous recombination in embryonic stem cells . In beta2 / beta1 knock-in mice , expression of beta2 was abolished, whereas beta1 mRNA expression from the mutated gene amounted to approximately 15% of the normal expression of beta2 in the adult mouse brain and prevented the juvenile lethality observed for beta2 null mutant mice . In contrast to beta2 null mutant mice , the overall morphological structure of all analyzed brain regions was normal. By immunohistochemical analysis, beta1 expression was detected in photoreceptor cells in the retina of knock-in mice at an age when expression of beta1 and beta2 , respectively, is downregulated and persisting in the wild-type mice . Morphological analysis by light and electron microscopy revealed a progressive degeneration of photoreceptor cells . Apoptotic death of photoreceptor cells determined quantitatively by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis increased in beta2 / beta1 knock-in mice with age. These observations suggest that the beta1 subunit of the Na,K-ATPase can substitute sufficiently, at least in certain cell types, for the role of the beta2 subunit as a component of a functional Na,K-ATPase, but they do not allow us to determine the possible role of the beta2 subunit as an adhesion molecule in vivo. Affiliation: Department of Neurobiology, Swiss Federal Institute of Technology, CH-8093 Zrich , Switzerland . Pubmed MeSH: Amino Acid Sequence , Animals , Apoptosis , Cell Adhesion Molecules , Homeostasis , Immunohistochemistry , In Situ Nick-End Labeling , Mice, Mutant Strains , Mutagenesis , Neuroglia , Phenotype , Retinitis Pigmentosa , Sodium-Potassium-Exchanging ATPase , Stem Cells Wikipedia: Adult , Bylaw , Cistron , ConstitutioN , Constitutions , Down-regulation , Downregulation , Electron , Electron Microscopy , Electronic , Gene , Genetic Recombination , Genetic material , House Mouse , House mice , Ion Pump , Ion pumps , Ions , Isoforms , Laboratory mice , Laboratory mouse , Messenger RNA , Mice , Microscopy , Microscopy, electron , Mouse , Mus , Mus domesticus , Mus musculus , Mus musculus domesticus , Mutation , Negatron , Neurite , Photoreceptor , Poly(A) tail , Positron , Protein isoforms , Receptor down-regulation , Recombination , Recombination, genetic , Retina http://news.sciencenet.cn//htmlnews/2010/3/228904.shtm 美科学家移植胚胎干细胞助患病实验鼠恢复视力 该研究成果有望应用于治疗人类视网膜疾病 美国哥伦比亚大学医学中心科学家所领导的国际研究小组表示，他们利用实验鼠胚胎干细胞取代受损视网膜细胞，成功地帮助患色素性视网膜炎的实验鼠恢复了视力。该研究成果有望用于开发治疗人类色素性视网膜炎的新方法。 色素性视网膜炎在人类中的发病率为1/3000至1/4000，每年全球新增患者约150万人。人体视网膜色素上皮细胞是维持视觉能力的特殊细胞，色素性视网膜炎可导致视网膜周边的视觉细胞死亡，出现视觉变狭窄和四周模糊的隧道视觉病症。 该研究项目负责人、哥伦比亚大学医学中心眼科、病理学和细胞生物学副教授斯蒂芬曾表示，新的研究具有光明的前景，因为他们将干细胞转化成视觉细胞，帮助患病的实验鼠恢复了视力。他说，移植的细胞不仅外表像视觉细胞，而且其功能也与视觉细胞相当。 研究中，有1/4患病实验鼠在接受干细胞移植后视力得到恢复，但有些实验鼠出现了良性肿瘤和视网膜脱离的并发症。对此，斯蒂芬曾和同事表示，他们将进一步优化技术，以减少人类胚胎干细胞移植用于人体试验时并发症的发病率。 斯蒂芬曾表示，一旦并发症的问题得到解决，这将成为新的治疗方法，不仅能够医治色素性视网膜炎，而且还能治疗与年龄变老相关的视网膜黄斑变性、斯特格病变以及其他种类的视网膜疾病，它们均具有视网膜细胞损伤的特征。 老年化视网膜黄斑变性是视网膜中心的视网膜细胞出现退化并导致视觉中心部分出现模糊的疾病。有研究表明，在美国，2010年将有900万人患上此种疾病，而2020年的发病率将是现在的两倍。年龄达75岁时，30%的人将患上某种类型的视网膜黄斑变性。 更多阅读 美国哥伦比亚大学医学中心相关报道（英文） 干细胞移植中国之旅 领跑背后的监管难题
标签: 胚胎干细胞
