AU2018357499A2 - Method for constructing ataxia animal model and application thereof - Google Patents
Method for constructing ataxia animal model and application thereof Download PDFInfo
- Publication number
- AU2018357499A2 AU2018357499A2 AU2018357499A AU2018357499A AU2018357499A2 AU 2018357499 A2 AU2018357499 A2 AU 2018357499A2 AU 2018357499 A AU2018357499 A AU 2018357499A AU 2018357499 A AU2018357499 A AU 2018357499A AU 2018357499 A2 AU2018357499 A2 AU 2018357499A2
- Authority
- AU
- Australia
- Prior art keywords
- ataxia
- animal model
- tmem30a
- cerebellar
- mouse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010003591 Ataxia Diseases 0.000 title claims abstract description 77
- 238000010171 animal model Methods 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 25
- 101150028405 TMEM30A gene Proteins 0.000 claims abstract description 73
- 210000000449 purkinje cell Anatomy 0.000 claims abstract description 55
- 230000002490 cerebral effect Effects 0.000 claims abstract description 37
- 241001465754 Metazoa Species 0.000 claims abstract description 18
- 238000003209 gene knockout Methods 0.000 claims description 17
- 238000011830 transgenic mouse model Methods 0.000 claims description 4
- 241000282693 Cercopithecidae Species 0.000 claims description 3
- 206010008025 Cerebellar ataxia Diseases 0.000 abstract description 25
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 8
- 238000012216 screening Methods 0.000 abstract description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 32
- 210000001638 cerebellum Anatomy 0.000 description 26
- 239000003153 chemical reaction reagent Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 16
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 238000011813 knockout mouse model Methods 0.000 description 11
- 239000012528 membrane Substances 0.000 description 9
- 108010054624 red fluorescent protein Proteins 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- 230000005021 gait Effects 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000008506 pathogenesis Effects 0.000 description 7
- 230000000750 progressive effect Effects 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- 206010003694 Atrophy Diseases 0.000 description 6
- 102100029145 DNA damage-inducible transcript 3 protein Human genes 0.000 description 6
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 description 6
- 208000010112 Spinocerebellar Degenerations Diseases 0.000 description 6
- 230000037444 atrophy Effects 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 201000003636 hereditary ataxia Diseases 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 5
- 239000002052 molecular layer Substances 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 108091033409 CRISPR Proteins 0.000 description 4
- 206010008027 Cerebellar atrophy Diseases 0.000 description 4
- 101150017921 DDIT3 gene Proteins 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000005252 bulbus oculi Anatomy 0.000 description 3
- 210000004720 cerebrum Anatomy 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 210000001525 retina Anatomy 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 238000010354 CRISPR gene editing Methods 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 208000024556 Mendelian disease Diseases 0.000 description 2
- 241000009328 Perro Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 101001010097 Shigella phage SfV Bactoprenol-linked glucose translocase Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 102000014823 calbindin Human genes 0.000 description 2
- 108060001061 calbindin Proteins 0.000 description 2
- 210000004087 cornea Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000003950 pathogenic mechanism Effects 0.000 description 2
- 230000007425 progressive decline Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 102000016838 Calbindin 1 Human genes 0.000 description 1
- 108010028310 Calbindin 1 Proteins 0.000 description 1
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- 102100026772 Cell cycle control protein 50A Human genes 0.000 description 1
- 206010050389 Cerebral ataxia Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 101710156077 DNA damage-inducible transcript 3 protein Proteins 0.000 description 1
- 241001492222 Epicoccum Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 208000028782 Hereditary disease Diseases 0.000 description 1
- 101000910814 Homo sapiens Cell cycle control protein 50A Proteins 0.000 description 1
- 101000701366 Homo sapiens Phospholipid-transporting ATPase IB Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028372 Muscular weakness Diseases 0.000 description 1
- 102100030447 Phospholipid-transporting ATPase IB Human genes 0.000 description 1
- 102000006010 Protein Disulfide-Isomerase Human genes 0.000 description 1
- 206010040026 Sensory disturbance Diseases 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000003198 cerebellar cortex Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004973 motor coordination Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000007121 neuropathological change Effects 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108020003519 protein disulfide isomerase Proteins 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 210000001213 vestibule labyrinth Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Mycology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Provided is a method for constructing an ataxia animal model, which comprises knocking out a target sequence on a genome of the cerebellar Purkinje cells of a target animal; the target sequence is the Tmem30a gene. Also provided are an animal model of the cerebellar ataxia constructed by the described method and a method for screening a medicament used for preventing or treating an ataxia disease using the animal model.
Description
METHOD FOR CONSTRUCTING ATAXIA ANIMAL MODEL AND APPLICATION
The application claims priority to Chinese Patent Application No. 201711012105.9 filed on October 26, 2017 and entitled METHOD FOR CONSTRUCTING ATAXIA ANIMAL MODEL AND APPLICATION, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The disclosure relates to the technical field of medical engineering, and in particular to a method for constructing an ataxia animal model and an application.
BACKGROUND
Ataxia is a kind of pathological state that patients are not able to maintain fine gait and complete precise movement in a certain form, and it is possibly caused by any lesion related to cerebellum afferent and efferent ways. Most ataxia are caused by genetic factors, therefore, it is collectively called Hereditary ataxia (HA). HA is a kind of hereditary disease featured by chronic progressive cerebella ataxia. HA exhibits major three characteristics of pathological changes, namely, hereditary background, ataxia representation and cerebellar damage. HA classification has not been clear. Although there are more than 60 reports up to now, there is no uniform and accepted method. Based on pathogenic sites, it is divided into four types: (1) deep sensory disturbance ataxia; (2) cerebellar ataxia; (3) vestibular labyrinth ataxia; (4) cerebral ataxia. Cerebellar ataxia is a major type of HA and belongs to a broad class of nervous system degenerative disease with genetic heterogeneity. Its major symptoms are instability of gait and weakness of limbs with cerebellar symptoms as its outstanding feature, and accompanied with cognitive disorder. There are diversified genetic models of cerebellar ataxia, including autosomal dominant, recessive heredity, X-linkage heredity and other various modes as well as some sporadic cases. At present, more than 30 types of cerebellar ataxia have been found, accounting for about 10%-15% of nervous system hereditary diseases.
The clinical symptom of cerebellar ataxia varies in a wide range, and even shows high heterogeneity in the same family. Moreover, clinical symptom of cerebellar ataxia caused by different disease genes often overlapped. Therefore, it is very difficult to conduct precise clinical classification due to largely overlapping clinical phenotypes. Therefore, to achieve correct diagnosis, cerebellar ataxia must rely on genetic diagnosis. Complicated relationship between the diversified phenotypes and numerous disease genes causes serious problems for clinical treatment. In addition, there is no specific effective therapeutic method to cerebellar ataxia. The main reason is lack of detailed pathogenesis studies. In the future, the diagnosis and treatment of cerebellar ataxia depends on discovery of disease genes and mechanistic studies.
Suitable animal models for ataxia research are vital to mechanistic studies and therapeutic development. However, currently available animal models cannot meet the rising demand.
In view of this, this disclosure is proposed herein.
i
SUMMARY
An objective of the disclosure lies in including but not limited to provide a method for constructing an ataxia animal model, namely, a cerebellar ataxia animal model of specifically knocking out Tmem30a gene from cerebellar Purkinje cells is constructed by the construction method, thus studying functions thereof in cerebellar Purkinje cells.
Another objective of the disclosure lies in including but not limited to provide an ataxia animal model obtained by the above construction method.
Another objective of the disclosure lies in including but not limited to provide an application of the ataxia animal model obtained by the above construction method in ataxia study.
The disclosure is achieved below:
A method for constructing an ataxia animal model, knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal;
The target sequence is a Tmem30a gene.
An application of the ataxia animal model obtained by the above method for constructing an ataxia animal model is applied in ataxia study.
The ataxia animal model obtained by the above method for constructing an ataxia animal model is applied in screening drugs for preventing or treating ataxia diseases.
Based on an ataxia animal model, the Tmem30a gene on the genome of the cerebellar Purkinje cells of the target animal is knocked out.
The disclosure has the following beneficial effects:
Based upon the method for constructing an ataxia animal model provided by the disclosure, a cerebellar ataxia animal model may be constructed by specifically knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal. The animal model shows typical characteristics of cerebellar ataxia: instability of gait, cerebellar atrophy, progressive apoptosis of Purkinje cells, etc. The model may be used for studying cerebellar ataxia to provide basis for the discovery of cerebellar ataxia genes and the exploration of pathogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
To describe the technical solution of embodiments of the disclosure, the drawings required in the embodiments are described briefly, it should be understood that the following drawings merely show some embodiments of the disclosure, therefore, these are not considered to define the scope, other related drawings may be obtained by those skilled in the art based on those drawings without creative efforts.
FIG. 1 is a diagram showing a route for constructing a specific cerebellar Purkinje cell Tmem30a knockout model provided by embodiments of the disclosure and identification of the knocked-out mouse genotype.
FIG. 2 is a schematic diagram showing the construction of tdTomato reporter genes in cerebellar Purkinje cells provided by embodiments of the disclosure (in the figure: tdTomato is a reporter gene Tomato of a red transgenic fluorescent protein. There is a terminator sequence in front of an initial codon of the red fluorescent protein; both ends of the terminator carry aligned LoxP loci, and when Cre enzyme is present, the terminator is deleted, the red fluorescent protein may be expressed and Cre positive cells are labelled red).
FIG. 3 shows immunofluorescent staining (Immunohistochemistry, IHC) provided by the embodiments of the disclosure, indicating that Pcp2-Cre is specifically expressed in mouse cerebellar Purkinje cells (in the figure: GCL: Granular cell layer, granular cell layer; PCL: Purkinje cell layer, Purkinje cell layer: ML: Molecular layer, molecular layer. Calbindin is a protein expressed in Purkinje cells).
FIG. 4 verifies the reduced expression level of Tmem30a protein in knocked-out mouse cerebellum by Western blot (Western blot, WB) provided by the embodiments of the disclosure (in the figure: WT: Wildtype, wild-type control: KO: Knockout, gene knockout mutation; Cerebrum: cerebrum; Cerebellum: cerebellum).
FIG. 5 shows a picture of the Purkinje cell specific Tmem30a knockout mouse provided by the embodiments of the disclosure when its tail is lifted to curl up hind legs (in the figure: 6mo refers to 6 month-age.)
FIG. 6 shows a picture of cerebellar atrophy of the Tmem30a KO mouse provided by the embodiments of the disclosure (in the figure: lOmo refers to 10 month-age.)
FIG. 7 shows a result of paraffin section staining provided by the embodiments of the disclosure.
FIG. 8 shows progressive decrease of cerebellar Purkinje cells of the Tmem30a KO mouse provided by the embodiments of the disclosure.
FIG. 9 is a diagram showing immunohistochemical staining of endoplasmic reticulum stress marker proteins Chop and Pdi in the Tmem30a KO cerebellum provided by the embodiments of the disclosure; (In the figure, A shows the expression of the endoplasmic reticulum stress marker proteins C/EBP homologous protein (Chop) in Tmem30a KO cerebellum slices. The arrowhead indicates cells occurring endoplasmic reticulum stress. It can be seen that Chop protein expressing cells in the Tmem30a KO cerebellum increase significantly. In the figure, B shows the expression of the endoplasmic reticulum stress marker proteins Protein Disulfide Isomerase (Pdi) in Tmem30a KO cerebellum slices. The arrowhead indicates cells occurring endoplasmic reticulum stress. It can be seen that Pdi expressing cells in the Tmem30a KO cerebellum increase significantly. In the figure, C is quantification of Chop and Pdi protein expression levels in panel A and B).
DETAILED DESCRIPTION
To describe the objectives, technical solutions and advantages of the embodiments of the disclosure more clearly, the technical solutions in the embodiments of the disclosure will be described more clearly and integrally here. Any non-indicated in the embodiments shall be performed in accordance with conventional conditions or the conditions recommended by the manufacturer. Any reagent or instrument without manufacturer shall be conventional products commercially purchased available.
The method for constructing an ataxia animal model and application of the embodiments of the disclosure will be described in detail.
The typical neuropathologic change of cerebellar ataxia is cerebellar atrophy, degenerative change and loss of Purkinje cells. Purkinje cell (PC) is a neuron capable of achieving efferent impulse sent from cerebellar cortex. PC axon reaches to deep cerebellum nucleus after through a granular layer and white matter, and it plays an important role in motor coordination.
ATP8A2 (a P4-ATP enzyme) gene mutations may cause cerebellum dysfunction, and patients suffer a symptom of cerebellar ataxia, indicating that the activity of the P4-ATP enzyme is closely related to cerebellar ataxia. P4-ATP enzyme is a kind of enzyme with phospholipid flippase activity in cytomembrane, also called phospholipid flippase, and it plays an important role in keeping the distribution of bilateral phospholipids in cytomembrane asymmetry. The asymmetrical distribution of the phospholipids at both sides of the cytomembrane is crucial to maintain stable membrane micro environment, exertion of membrane protein functions, transport of cell vesicae, cell polarity, cell apoptosis, etc. Some of the phospholipids are collectively distributed on one side of cytoplasm, e.g., PE and PS, and such asymmetrical distribution depends on the activity of the P4-ATP enzyme in cytomembrane. The genome of mammals encodes 14 different P4-ATP enzymes, and the defect of the P4-ATP enzyme may cause various diseases, indicating its great importance in vivo.
Cdc50 family (Cdc50a, b, c, also called Tmem30a, b, c) may interact with P4-ATP enzymes, and it is essential to the correct folding and subcellular localization of P4-ATP enzymes, therefore, it is regarded as β subunits of P4-ATP enzymes. There are 14 kinds of P4-ATP enzymes in animal's body, indicating that the enzymes have redundant functions and interact with each other in tissues; therefore, it is difficult to research the functions.
But there are only three β subunits, and most of P4-ATP enzymes interact with Tmem30a, therefore, to study the functions of P4-ATP enzymes and specify the pathogenesis of cerebellar ataxia, studying β-subunit Tmem30a is the best choice.
Based on the above facts, the discloser of the disclosure constructs a specific cerebellar Purkinje cell Tmem30a knockout mouse model, thus providing a basis for the intensive study on its pathogenesis of cerebellar ataxia.
In one aspect, embodiments of the disclosure provide a method for constructing an ataxia animal model, knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal; where the target sequence is the Tmem30a gene.
Further, in some embodiments of the disclosure, the target sequence is an exon sequence of the Tmem30a gene.
The Tmem30a gene has 4 exons (i.e., No.l exon, No.2 exon, No.3 exon, No.4 exon), therefore, any one or more of the exons can be knocked out to make the Tmem30a gene inactive, thus achieving knockout.
Further, in some embodiments of the disclosure, the target animal is selected from any of mouse, rat, dog, monkey and ape.
Further, in some embodiments of the disclosure, the target animal is a mouse, and the sequence of the exon is the sequence of No. 3 exon.
Certainly, in other embodiments of the disclosure, the sequence of No. 1 exon, the sequence of No. 2 exon, the sequence of No. 4 exon, or a combination thereof may be optionally knocked out, which belongs to the protection scope of the disclosure.
Further, in some embodiments of the disclosure, the target sequence is knocked out by Cre-loxP knockout technology.
Generally, there are lots of gene knockout technologies, such as CRISPR/Cas9. Therefore, CRISPR/Cas9 or other technological means can be taken to knock out Tmem30a gene in other embodiments, belonging to the protection scope of the disclosure as well.
Certainly, in some embodiments of the disclosure, a conditional Tmem30a gene knockout homozygote mouse mates with a Pcp2-Cre transgenic mouse to obtain a conditional cerebellar Purkinje cell Tmem30a gene knockout mouse.
It should be noted that the obtaining of the conditional Tmem30a gene knockout homozygote mouse can be referred to a Chinese patent application 2017103803265, titled construction method of a conditional pancreatic β cell Tmem30a gene knockout mouse model and application.
In another aspect, embodiments of the disclosure further provide an application of the ataxia animal model obtained by the above method for constructing an ataxia animal model in ataxia study.
Further, in some embodiments of the disclosure, the study is a study on the pathogenesis or pathogenic mechanism of human ataxia.
In a further aspect, the disclosure provides an application of the ataxia animal model obtained by the above method for constructing an ataxia animal model in screening drugs for preventing or treating ataxia diseases.
Still further, in some embodiments of the disclosure, the application includes: a candidate reagent is applied in the ataxia animal model;
if it is detected that one or more of the following phenomena occurs in the ataxia animal model after applying the candidate reagent, it indicates that the candidate reagent may serve as a drug for preventing or treating ataxia diseases:
phenomenon (1): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: normal or more stable gait, or hind legs do not curl up when its tail is lifted or curling degree is lower;
phenomenon (2): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: no progressive atrophy occurs in cerebellum or atrophy degree is lower with age;
phenomenon (3): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: no decrease of Purkinje cells occurs in cerebellum or the decrease scope of Purkinje cells is lower with age;
phenomenon (4): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: the expression quantity of endoplasmic reticulum stress marker protein CHOP is lower.
In a further aspect, the disclosure provides an ataxia animal model, and the Tmem30a gene in the genome of a cerebellar Purkinje cell of the animal model is knocked out.
Further, in some embodiments of the disclosure, the target animal is mouse, rat, dog, monkey and ape.
Further, in some embodiments of the disclosure, any of No. 1 exon, No. 2 exon, No. 3 exon and No. 4 exon of the Tmem30a gene in the genome of the cerebellar Purkinje cells of the animal model is knocked out or multiple exons are knocked out at the same time.
Further, in some embodiments of the disclosure, the animal model is a mouse, No. 3 exon of the Tmem30a gene in the genome of the cerebellar Purkinje cells of the animal model is knocked out.
In a further aspect, the disclosure provides an application of the above ataxia animal model in ataxia study.
Further, in some embodiments of the disclosure, the study is a study on the pathogenesis or pathogenic mechanism of human ataxia.
In a further aspect, the disclosure provides a method for screening drugs for preventing or treating ataxia disease, including: applying a candidate reagent to the above ataxia animal model.
Further, in some embodiments of the disclosure, if it is detected that one or more of the following phenomena occurs in the ataxia animal model after applying the candidate reagent, it indicates that the candidate reagent may serve as a drug for preventing or treating ataxia diseases:
phenomenon (1): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: normal or more stable gait, or hind legs do not curl up when its tail is lifted or curling degree is lower;
phenomenon (2): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: no progressive atrophy occurs in cerebellum or atrophy degree is lower with age;
phenomenon (3): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: no decrease of Purkinje cells occurs in cerebellum or the decrease scope of Purkinje cells is lower with age;
phenomenon (4): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: the expression quantity of endoplasmic reticulum stress marker protein CHOP is lower.
Features and performance of the disclosure will be further described in detail with reference to the embodiments.
Embodiment 1
In this embodiment, mouse served as a target animal, set as an example to describe the method for constructing an ataxia animal model provided by the disclosure, specifically as follows.
1. A Tmem30a gene (No. 3 exon) conditional knockout homozygote mouse was obtained by reference to a Chinese patent application 2017103803265, titled construction method of pancreatic β cell Tmem30a gene conditional knockout mouse model and application;
2. The conditional Tmem30a gene knockout homozygote mouse mated a Pcp2-Cre transgenic mouse to obtain a conditional cerebellar Purkinje cell Tmem30a gene knockout mouse (FIG. 1A). As shown in FIG. 1A, half of the descendants of the Pcp2-Cre transgenic mouse and conditional Tmem30a gene knockout homozygote mouse carried Pcp2-Cre and Tmem30a conditional knockout at the same time. The animal mated again with the conditional Tmem30a gene knockout homozygote mouse to obtain a specific cerebellar Purkinje cell Tmem30a knockout mutation animal (represented by Tmem30a KO).
Cre enzyme was expressed in cerebellar Purkinje cells of Pcp2-Cre gene to achieve the effect of conditional Tmem30a gene knockout of cerebellar Purkinje cells.
3. The Tmem30a conditional knockout mouse was identified by primers 2: 5’-TGGTTAGGAACACATAGAACAAA-3’ and R2: 5’-CTAAGGGGCTGGTATGGGAAT-3’. Cre genotype was identified by primers CreF: 5’-ATTTGCCTGCATTACCGGTC-3’; Cre-R: 5’-ATCAACTGGTTCTTTTCGG-3’, and results were shown in FIG. IB. Results of FIG. 1 show that (in the figure: WT denotes wildtype mouse, Tmem30a loxp/loxp, Pcp2-Cre denotes conditional cerebellar purkinje cell Tmem30a gene knockout homozygote mouse (abbreviated for Tmem30a KO) to obtain conventional cerebellar Purkinje cell Tmem30a gene knockout homozygote mouse.
Embodiment 2
To determine recombination efficiency of Cre-mediated loxP loci, tdTomato reporter gene was constructed in this embodiment (FIG. 2). Without Cre expression, a STOP gene cassette carrying loxP recombination loci at both sides can prevent the expression of the downstream red fluorescent protein tdTomato. In the presence of Cre, the STOP gene cassette is removed from the Cre specifically-expressed tissues, resulting in the expression of tdTomato. Therefore, the expression pattern of a Cre recombinase may be determined by observing the fluorescence brought by tdTomato expression. An immunohistochemistry was used to observe the specific expression of Cre in Tmem30a KO animal in cerebellar Purkinje cells (as shown in FIG. 3). In FIG. 3: GCL: Granular cell layer, granular cell layer; PCL: Purkinje cell layer; ML: Molecular layer.
Immunohistochemistry:
A mouse was killed by cervical dislocation, then eyeball was taken and fast put into 4% PFA on ice to fix for 15 min, and an incision was cut on the cornea, then fixation was performed continuously on ice. After 2 h, it was washed by a PBS buffer solution, then the eyeball was put into a 30% sucrose solution for dehydration for 2 h, then cornea and lens were cut out under an anatomical lens, embedded by OCT, and rapidly put into a -80°C refrigerator for freezing. OCT embedded eyeball was taken out about 10 min later, and put into a freezing microtome at -25°C for balancing for about 30 min, then sliced. Slice thickness was 12 pm.
After slicing, higher-quality slices were selected and put into an oven for 30 min at 37°C, then an immunohistochemical pen was used to draw a circle on the site with retina tissues, it was washed by PBS for three times to remove OCT, then sealed by 5% NDS (containing 0.25% Triton) for 2 h, primary antibodies were incubated for staying overnight at 4°C. It was washed by PBS for twice in the following day, corresponding fluorescent second antibodies were incubated, then washed by PBS for twice, and slices were sealed for observation.
Embodiment 3
Western blot (WB) was applied to observe the expression of TMEM30A protein in Tmem30a KO mouse.
WB:
(1) Retina tissues of wildtype and mutant mice were respectively separated, placed into a 1.5 ml centrifugal tube, and added 200 μΐ protein lysate RIPA;
(2) Retina tissues were broken by ultrasonication and disintegrated on ice for 20 min;
(3) 16000 g were centrifuged for 10 min at 4°C, supernatant was taken and transferred to another clean centrifugal tube, 50 μΐ protein loading solution was added for mixing well, then heated for 5 min at 95°C;
(4) After cooling, 20 μΐ samples were respectively taken for polyacrylamide gel electrophoresis (SDS-PAGE) at 160V to separate the protein;
(5) At the end of SDS-PAGE, proper-size nitrocellulose membrane was trimmed as required, and filter paper, glue, nitrocellulose membrane and filter paper were assembled in sequence. Air bubbles were removed, membrane transfer was conducted by 0.28 A current for 2 h in a membrane groove in an ice-water bath;
(6) At the end of membrane transfer, the nitrocellulose membrane was washed by pure water, aired and labeled. Then 8% skim milk was used for blocking for 2 h;
(7) At the end of blocking, a certain amount of primary antibodies were diluted in a blocking buffer according to certain proportion (operation manual of the antibody), incubated at 4°C overnight.
(8) Primary antibodies were removed, the membrane was washed by a IxTBST buffer solution for 4 times 10 min each time. Proper secondary antibodies were selected according to the source of the primary antibodies. IxTBST was used to dilute the HRP-labeled secondary antibodies, and secondary antibody was incubated on a shaker for 2 h at room temperature.
(9) At the end of secondary antibodies incubation, the membrane was washed by IxTBST for 3 times 10 min each time. Protein expression was detected by a Thermo ELC kit. The instrument used was a chemiluminiscence gel-imaging system from Bio-Rad.
Results were shown in FIG. 4. In the figure, WT denotes wildtype mouse, KO denotes Tmem30a KO mouse. Cerebrum denotes brain tissues, Cerebellum denotes cerebellar cells. WB has proved that Tmem30a protein is knocked out in cerebellar Purkinje cells of the Tmem30a KO mouse (FIG. 4). Due to the existence of granular cells and glial cells in cerebellum, Tmem30a in those two cells were not knocked out. Tthe expression quantity of Tmem30a in cerebellum decreased partially.
Based upon the above results, the specific cerebellar Purkinje cell Tmem30a knockout mouse model Tmem30a KO may be provided.
Embodiment 4
Study on cerebellar ataxia by the constructed Tmem30a gene knockout mouse model
Tmem30a KO mouse suffered form cerebellar ataxia
By an observation of Tmem30a KO behaviors and Hind-limb clasping test, it indicated that Tmem30a KO knockout mouse suffered the symptom of cerebellar ataxia. Specific performance was instability of gait and curled hind legs after its tail was lifted (FIG. 5). 6mo in FIG. 5 denoted 6 month-age.
Tmem30a KO mouse suffered cerebellar progressive atrophy
Cerebellum of the knockout mouse was dissected to find that the knockout mouse suffered cerebellar progressive atrophy with age (FIG. 6, in the figure: WT denoted a wildtype control, KO denoted a Purkinje cell knockout animal; lOmo denoted 10 month-age; Cross-sectional area of cerebellum denoted vertical cross-sectional area of cerebellum, and the vertical cross-sectional area of cerebellum was merely 50% of the WT mouse at 10 month-age. Paraffin section was performed to the cerebellum of the mouse at different ages, H&E staining was conducted to the section to find that the molecular layer of cerebellum thinned gradually and granular cells decreased. In FIG. 7, WT denoted a wildtype control, KO denoted a Tmem30a Purkinje cell knockout animal; P42 denoted 42 lOmo denoted 10 days of age; 5mo denoted 5 month-age, 12mo denoted 12 days of age.
Tmem30a KO mouse suffered progressive decrease of cerebellar Purkinje cells
An analysis on the immunohistochemical staining by frozen section were conducted to the knockout mouse, Purkinje cell marker protein Calbindin-D28K was stained to find that Purkinje cells in cerebellum decreased gradually, and almost all Purkinje cells in the knockout mouse died on the 42nd day after birth (FIG. 8). In FIG. 8: Calbindin denoted calcium binding protein, a marker protein of Purkinje cells, DAPI denoted 4', 6-diamidino-2-phenylindole, and a fluorochrome capable of binding DNA strongly, usually used in fluorescent microscope observation. P16 denoted 16 days of age, P25 denoted 25 days of age, P30 denoted 30 days of age and P42 denoted 42 days of age.
Tmem30a KO appeared endoplasmic reticulum stress (ER stress) in cerebellar Purkinje cells
To explore the death cause of Purkinje cells, frozen section was performed to the knockout mouse for immunohistochemical staining analysis. Tmem30a is associated with intracellular transport, therefore, its deletion may cause the disorder of intracellular vesicle, resulting in endoplasmic reticulum stress. Therefore, endoplasmic reticulum stress marker protein CHOP and PDI staining was conducted to cerebellum freezing slices to find that the expression quantity of Purkinje cells CHOP and PDI of the Tmem30a KO mouse increased obviously on the 20th day after birth (FIG. 9). In FIG. 9: GCL: Granular cell layer; PCL: Purkinje cell layer; ML: Molecular layer; P20 denoted 20 days of age. Compared with the wildtype control, the expression of endoplasmic reticulum stress protein marker CHOP in Tmem30a KO cerebellar cells increased by nearly 6 times, and PDI increased by nearly 4 times (FIG. 9). Tmem30a knockout would cause endoplasmic reticulum stress. After appearing endoplasmic reticulum stress, related pathway of cells apoptosis was activated, resulting in final apoptosis.
To sum up, in the embodiments of the disclosure, the specific cerebellar Purkinje cell Tmem30a gene knockout mouse model is constructed, and the animal model shows typical characteristics of cerebellar ataxia: instability of gait, cerebellar atrophy, progressive apoptosis of Purkinje cells, etc. The model may be used in the study of cerebellar ataxia.
The mentioned above are merely preferred embodiments of the disclosure, not employed to limit the disclosure, various modifications and changes of the disclosure are available to those io skilled in the art. Any modification, equivalent replacement, improvement, etc. made according to the spirit and principle of the disclosure shall be included within the protection scope of the disclosure.
Industrial applicability: By the method for constructing an ataxia animal model provided by the disclosure, a specific Tmem30a knockout ataxia animal model of pancreatic β cells in cerebellar Purkinje cells may be constructed, and the model shows typical ataxia features; the mouse model may be used in the study of ataxia or screening drugs for treating or preventing ataxia and other fields, thus providing a model basis to further know the pathogenesis of ataxia and screen ataxia drugs.
Claims (5)
- What is claimed is:1. A method for constructing an ataxia animal model, characterized by knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal; wherein the target sequence is the Tmem30a gene.
- 2. The method for constructing an ataxia animal model according to claim 1, wherein the target sequence is an exon sequence in the Tmem30a gene.
- 3. The method for constructing an ataxia animal model according to claim 2, wherein the target animal is selected from any of mouse, rat, dog, monkey and ape.
- 4. The method for constructing an ataxia animal model according to claim 3, wherein the target animal is mouse, and the exon sequence is a No. 3 exon sequence, preferably, wherein a conditional Tmem30a gene knockout homozygote mouse mates a Pcp2-Cre transgenic mouse to obtain a conditional cerebellar Purkinje cell Tmem30a gene knockout mouse.
- 5. An application of the ataxia animal model obtained by the method for constructing an ataxia animal model according to any one of claims 1-4 in the study of ataxia.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711012105.9A CN107586791B (en) | 2017-10-26 | 2017-10-26 | A kind of construction method of incoordination animal model and application |
CN201711012105.9 | 2017-10-26 | ||
PCT/CN2018/106132 WO2019080667A1 (en) | 2017-10-26 | 2018-09-18 | Method for constructing ataxia animal model and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2018357499A1 AU2018357499A1 (en) | 2019-11-21 |
AU2018357499A2 true AU2018357499A2 (en) | 2019-12-19 |
Family
ID=61043889
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2018102151A Ceased AU2018102151A4 (en) | 2017-10-26 | 2018-09-18 | Method for constructing ataxia animal model and application thereof |
AU2018357499A Pending AU2018357499A1 (en) | 2017-10-26 | 2018-09-18 | Method for constructing ataxia animal model and application thereof |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2018102151A Ceased AU2018102151A4 (en) | 2017-10-26 | 2018-09-18 | Method for constructing ataxia animal model and application thereof |
Country Status (3)
Country | Link |
---|---|
CN (1) | CN107586791B (en) |
AU (2) | AU2018102151A4 (en) |
WO (1) | WO2019080667A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107586791B (en) * | 2017-10-26 | 2018-09-21 | 四川省人民医院 | A kind of construction method of incoordination animal model and application |
CN109182355B (en) * | 2018-09-17 | 2019-07-12 | 四川省人民医院 | The construction method of retinal neovascularization disease model and application |
CN110100788B (en) * | 2019-05-14 | 2021-07-16 | 电子科技大学附属医院·四川省人民医院 | Method for constructing disease model based on gene operation strategy and application |
CN111154809B (en) * | 2020-01-09 | 2020-11-27 | 电子科技大学附属医院·四川省人民医院 | Method for constructing glomerular disease model by using gene manipulation technology and application |
CN113957074B (en) * | 2021-10-25 | 2023-08-04 | 电子科技大学 | Construction method and application of cerebellar ataxia disease model |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030014769A1 (en) * | 2001-03-29 | 2003-01-16 | Allen Keith D. | Transgenic mice containing GPCR-like transmembrane protein disruptions |
WO2008153743A2 (en) * | 2007-05-21 | 2008-12-18 | Dana Farber Cancer Institute | Compositions and methods for cancer gene discovery |
CA2773109A1 (en) * | 2009-09-02 | 2011-03-10 | Brian Popko | Methods and systems for inducible ablation of neural cells |
US20160143255A1 (en) * | 2013-03-15 | 2016-05-26 | Exemplar Genetics, Llc | Animal Models of Ataxia-Telangiectasia (A-T) |
RU2744831C2 (en) * | 2015-03-16 | 2021-03-16 | Регенерон Фармасьютикалз, Инк. | Non-human animal exhibiting diminished upper and lower motor neuron function and sensory perception |
CN107164406B (en) * | 2017-05-25 | 2020-03-31 | 朱献军 | Construction method and application of mouse model for conditional knockout of Tmem30a gene in pancreatic islet β cells |
CN107586791B (en) * | 2017-10-26 | 2018-09-21 | 四川省人民医院 | A kind of construction method of incoordination animal model and application |
-
2017
- 2017-10-26 CN CN201711012105.9A patent/CN107586791B/en active Active
-
2018
- 2018-09-18 AU AU2018102151A patent/AU2018102151A4/en not_active Ceased
- 2018-09-18 AU AU2018357499A patent/AU2018357499A1/en active Pending
- 2018-09-18 WO PCT/CN2018/106132 patent/WO2019080667A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
CN107586791B (en) | 2018-09-21 |
CN107586791A (en) | 2018-01-16 |
WO2019080667A1 (en) | 2019-05-02 |
AU2018357499A1 (en) | 2019-11-21 |
AU2018102151A4 (en) | 2020-05-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2018102151A4 (en) | Method for constructing ataxia animal model and application thereof | |
Mattapallil et al. | The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes | |
Lu et al. | Ablation of EYS in zebrafish causes mislocalisation of outer segment proteins, F-actin disruption and cone-rod dystrophy | |
Riazuddin et al. | Tricellulin is a tight-junction protein necessary for hearing | |
Vasireddy et al. | Genetics and molecular pathology of Stargardt-like macular degeneration | |
Hansen et al. | The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract | |
Song et al. | Arl13b interacts with Vangl2 to regulate cilia and photoreceptor outer segment length in zebrafish | |
Kong et al. | Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein | |
Duan et al. | Rabl2 GTP hydrolysis licenses BBSome‐mediated export to fine‐tune ciliary signaling | |
Ju et al. | Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1 | |
Chekuri et al. | IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis | |
Yu et al. | A novel MIP gene mutation associated with autosomal dominant congenital cataracts in a Chinese family | |
Qu et al. | Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy | |
CN110100788A (en) | Methods and applications based on gene manipulation strategy building disease model | |
Marko et al. | The ocular surface phenotype of Muc5ac and Muc5b null mice | |
Beryozkin et al. | A new mouse model for retinal degeneration due to Fam161a deficiency | |
Xia et al. | Arginine 54 and tyrosine 118 residues of αA-crystallin are crucial for lens formation and transparency | |
Hanada et al. | Fibroblast growth factor 12 is expressed in spiral and vestibular ganglia and necessary for auditory and equilibrium function | |
Chrystal et al. | The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision | |
Kuny et al. | Long-term retinal cone survival and delayed alteration of the cone mosaic in a transgenic mouse model of Stargardt-like dystrophy (STGD3) | |
Subramanian et al. | Loss of Raf-1 kinase inhibitory protein delays early-onset severe retinal ciliopathy in Cep290rd16 mouse | |
Mechaussier et al. | TUBB4B variants specifically impact ciliary function, causing a ciliopathic spectrum | |
Biswas et al. | A mutation in IFT43 causes non-syndromic recessive retinal degeneration | |
Faber et al. | Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme | |
Jones et al. | Neurl4 contributes to germ cell formation and integrity in Drosophila |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
DA3 | Amendments made section 104 |
Free format text: THE NATURE OF THE AMENDMENT IS AS SHOWN IN THE STATEMENT FILED 01 NOV 2019 |
|
DA3 | Amendments made section 104 |
Free format text: THE NATURE OF THE AMENDMENT IS: APPLICATION IS TO PROCEED UNDER THE NUMBER 2018102151 |