CN107287243A - A kind of humanized's retinoschisis transgene mouse model and its construction method - Google Patents
A kind of humanized's retinoschisis transgene mouse model and its construction method Download PDFInfo
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Abstract
The present invention provides a kind of construction method of humanized's retinoschisis transgene mouse model, with humanizedRS1Based on gene, screening human disease's gene is knocked in mouse by gene editing means, obtains having as patients clinical signRS1hKI mouse disease models.
Description
Technical field
The present invention relates to a kind of humanized's retinoschisis transgene mouse model and its construction method.
Background technology
Hereditary retinoschisis is a kind of X chromosome disorders, therefore also known as chain retinoschisis of X（X-
linked retinoschisis, XLRS）.The disease has generation in white man, Black people and yellow race crowd, and its incidence of disease is 1:
5000-1:25000.XLRS is mainly male's morbidity, and women morbidity is more rare, and heredity person is more common in children and young people,
It is one of Main Hereditary Diseases of teenager's blinding.XLRS clinical symptoms are generally macula area retinoschisis, strabismus, nystagmus,
Weak-eyed even total blindness, ERG is presented abnormal, and b wave amplitudes are obviously reduced, and OCT scan shows significantly " nest wheel shape " blister cavities splitting,
It is serious to show as each thickness degree of retina and gross thickness is thinning.Even to this day, hereditary retinoschisis also having without row
The treatment method of effect.
RS1The mutation of gene is considered as the pathogenic genetic mechanism of congenital retinoschisis,RS1The assignment of genes gene mapping in
Xp22.2, is made up of six extrons, encodes 224 amino acid.At present, there is more than 230RS1Gene mutation site
Report, the mutation of these genes is considered as the pathogenic genetic mechanism of congenital retinoschisis, and theseRS1Gene mutation
Site cause the missing of retina R S1 albumen or the forfeiture of RS1 protein functions, it is considered to be XLRS morbidity key factor,
The site of these single gene mutations also becomes the important grappling target spot during gene therapy.
It is based on to the clear and definite of the chain retinoschisis genetic mechanisms of X, people utilizeRS1Gene has carried out correlation
The structure of disease model.At present, the whole world has 3 kindsRS1 Gene knock-out mice model（RS1-KO）Used in succession in last decade
In XLRS research.The first beRS1 The 3rd extron on insertion LacZ/Neor form unloaded mouse model.Second
Kind beRS1The 1st extron on insertion one neomycin gene result in the mouse model that RS1 albumen is not expressed.3rd
It is to use nitroso ureas to plant（N-ethyl-N-nitrosurea, ENU) mouse model for obtaining random mutation is handled, mainly obtain
It is the mouse model of point mutation on the 2nd extron and 26 base deletions.So far, theseRS1- KO mouse are
Endogenous mouse disease model, the mutation of its mutational site and patient's gene is simultaneously different, so this evaluation to later phase clinical
It may be deviated.Research also confirms that theseRS1Knock-out mice part shows as the feature of XLRS patient, including b wave amplitudes
Significantly reduce, there is splitting in retina, photoreceptor is degenerated and ganglion-cell layer and inner nuclear layer it is thinning, but retina neural
Cellular layer and inner nuclear layer are thinning, and the retinoschisis clinical symptoms of the sign such as cleaving occurs in inner plexiform layer and people have difference.Have
Pass humanized's mouse model is international and domestic all to have no report, humanized's mutationRS1Gene is transferred to the XLRS diseases of mouse formation
Whether model has and clinical patient is similar or identical characterizes and also has no report.Carry out humanizedRS1h- KI disease models
Research, this more preferably can more accurately simulate human diseases pathogenic process, for research XLRS provides it is more accurate with enrich
Experimental animal object.
The content of the invention
There is provided a kind of humanized's retinoschisis transgene mouse model and its structure side for problem above by the present invention
Method.
Goal of the invention is realized by following scheme：A kind of structure side of humanized's retinoschisis transgene mouse model
Method, with humanizedRS1Based on gene, screening human disease's gene is knocked in mouse by gene editing means, is had
Have as patients clinical signRS1h- KI mouse disease models.
Further, with TALENs technology, obtained according to the screening of XLRS sufferersRS1The mutational site of gene, design
With XLRS sufferer identical Oligo sequences, the carrier built is transferred in cell, and screening is hadRS1Gene mutation it is steady
Determine cell line, vivoexpression, isolated mRNA, and carry out embryonated egg microinjection and embryo transfer to the pregnant mouse uterus in F0 generations
In, breeding obtains stablizing the F1 generation of phenotypeRS1h- KI disease model mouse.
Further, Oligo sequences are：TGGGTGTTTGAGTGTGTGCTGTTTTTCCTCCCCAGAATGCCCATATCA
CAAGCCCCTGGGTTTCGAGTCAGGGGAGGTCACGCCAGATCA。
Further, saltant typeRS1The protein sequence of gene translation：MPHKIEGFFLLLLFGYEATLGLSSTEDEGE
DPWYQKACKCDCQVGANALWSAGATSLDCIPECP。
Further, gene knock-in mouse is built with TALEN, design is directed toRS1On 4th extron of gene c.195
T>G mutational site, allows TALEN to be combined in cell with the target site of genome using TALEN two hand cradle, forms FokI
Dimer plays endonuclease activity, causes left and right TALEN spacer regions to occur double-strand DNA cleavage, so that radiation-indued DNA damage
Repair mechanism, cell reaches gene knock-in into the gene of mouse, TALEN by nonhomologous end engagement mechanisms DNA plerosis
Two hand cradle：TALEN_Left primers are：TGACTCGAAACCCAGGGG；TALEN_Right primers are：
TGCCCATATCACAAG；Spacer regions are：TGCCCATATCACAAG.
Further, forRS1C.195 T on 4th extron of gene>G mutational site, designs gene knock-in
Oligo sequences be：TGGGTGTTTGAGTGTGTGCTGTTTTTCCTCCCCAGAATGCCCATATCACAAGCCCCTGGGTTT
CGAGTCAGGGGAGGTCACGCCAGATCA。
Further, existRS1One Bpu10I restriction enzyme site is set at the 215bp of gene.
Further, the good humanized of design constructionRS1Gene Oligo sequences, are carried out in-vitro transcription, using aobvious
The mRNA sequence of in-vitro transcription is injected into the ripe egg cells of C57BL/6J by the mode of microinjection, and injecting successful embryonated egg can
Culture of being become pregnant in replace-conceive dams uterus is stayed overnight or be transferred to immediately in vitro culture.
Further, after the farrowing of replace-conceive dams, the tail and toe of F1 generation mouse is taken, full-length genome is extracted, enter performing PCR expansion
Increase, Bpu10I digestion digestion verifications, sequencing identification.Identified for genes result shows that we successfully constructRS1h- KI mouse diseases
Model.
A kind of humanized's retinoschisis transgene mouse model,RS1h- KI murine genes sequencing sequences:
CATTATCTCTGTTGAGTTCTGGGATAGGAGGGGTTCATTCCTTCTTTTGTATTTTGGGATGAGGTCTCAATACTTTC
TCCGGACAGCCCAGAGAAGCTGGGACTCCACACTGTGAACATATGCTACCACTCTGCCTGGAGATTTGGGTGTTTGA
GTGTGTGCTGTTTTTCCTCCCCAGAATGCCCTTAGCACAAGCCCCTGGGTTTCGAGTCAGGGGAGGTCACGCCAGAT
CAGATCACTTGCTCCAACCCAGAGCAGTATGTGGGCGGTATTCCTCCA。
The beneficial effects of the present invention are：Visual performance and retinal structure detection can illustrateRS1h- KI mouse regard
In nethike embrane retina impaired cause is linked mainly due to the cynapse between photosensory cell and Beale's ganglion cellsRS1h- KI mouse visions
Dysfunction, this is exactlyRS1h- KI mouse occur caused by the splitting in outer plexiform layer of retina, and this is characterized with clinical patient
It is identical；Human diseases pathogenic process more preferably can be more accurately simulated, more accurate and abundant experiment is provided for research XLRS
Animal target.
Brief description of the drawings
Fig. 1 builds figure for the present invention.
Fig. 2 is humanizedRS1h- KI mouse qualification result figures.
Fig. 3 is humanizedRS1h- KI Mouse Retina Structural Identifications result figure one.
Fig. 4 is humanizedRS1h- KI Mouse Retina Structural Identifications result figure two.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described：
With reference to accompanying drawing 1-4, embodiment 1, a kind of structure side of humanized's retinoschisis transgene mouse model of the invention
Method, with humanizedRS1Based on gene, screening human disease's gene is knocked in mouse by gene editing means, is had
Have as patients clinical signRS1h- KI mouse disease models.
A kind of construction method of humanized's retinoschisis transgene mouse model, with TALENs technology, according to
The screening of XLRS sufferers is obtainedRS1The mutational site of gene, design and XLRS sufferer identical Oligo sequences, the carrier built,
It is transferred in cell, screening is hadRS1The stable cell lines of gene mutation.Vivoexpression, isolated mRNA, and carry out by
Smart ovum microinjection and embryo transfer are into the uterus in F0 generations, and breeding obtains stablizing the F1 generation of phenotypeRS1h- KI disease models are small
Mouse.
A kind of construction method of humanized's retinoschisis transgene mouse model, Oligo sequences are：
TGGGTGTTTGAGTGTGTGCTGTTTTTCCTCCCCAGAATGCCCATATCACAAGCCCCTGGGTTTCGAGTCAGGGGAGG
TCACGCCAGATCA。
A kind of construction method of humanized's retinoschisis transgene mouse model, saltant typeRS1The egg of gene translation
White matter sequence：MPHKIEGFFLLLLFGYEATLGLSSTEDEGEDPWYQKACKCDCQVGANALWSAGATSLDCIPECP.
A kind of construction method of humanized's retinoschisis transgene mouse model, the hand cradle refers to recognition sequence.
A kind of construction method of humanized's retinoschisis transgene mouse model, with TALEN (transcription
activator-like （TAL) effector nucleases, activating transcription factor sample effector nucleic acid zymotechnic）Build base
Because knocking in mouse（RS1h-KI）, design is directed toRS1C.195 T on 4th extron of gene>G mutational site, is utilized
TALEN two hand cradle allow TALEN to be combined in cell with the target site of genome, form FokI dimers and play inscribe enzyme activity
Property, cause left and right TALEN spacer regions to occur double-strand DNA cleavage（DSB, Double-Strand Breaks）, so as to lure
DNA damage repair mechanism is sent out, cell passes through nonhomologous end engagement mechanisms（NHEJ, Non-homologous End
Joining）DNA plerosis, reach gene knock-in into the gene of mouse.
A kind of construction method of humanized's retinoschisis transgene mouse model, forRS1The 4th of gene is outer
C.195 T on aobvious son>G mutational site, design gene knock-in Oligo sequences be：TGGGTGTTTGAGTGTGTGCTGTTT
TTCCTCCCCAGAATGCCCATATCACAAGCCCCTGGGTTTCGAGTCAGGGGAGGTCACGCCAGATCA（The albumen of translation
Matter sequence：MPHKIEGFFLLLLFGYEATLGLSSTEDEGE DPWYQKACKC DCQVGANALW SAGATSLDCI PECP）.
A kind of construction method of humanized's retinoschisis transgene mouse model,RS1Set at the 215bp of gene
One Bpu10I restriction enzyme site.
A kind of construction method of humanized's retinoschisis transgene mouse model, the good humanized of design constructionRS1
Gene Oligo sequences, are carried out in-vitro transcription, are injected into the mRNA sequence of in-vitro transcription by the way of microinjection
In C57BL/6J maturation egg cells, inject successful embryonated egg can in vitro culture stay overnight or be transferred to immediately in replace-conceive dams uterus by
Pregnant culture.
A kind of construction method of humanized's retinoschisis transgene mouse model, after the farrowing of replace-conceive dams, takes F1 generation
The tail and toe of mouse, extract full-length genome, enter performing PCR amplification, Bpu10I digestion digestion verifications, sequencing identification.Gene reflects
Determine result and show that we successfully constructRS1h- KI mouse disease models.
A kind of humanized's retinoschisis transgene mouse model,RS1h- KI murine genes sequencing sequences:
CATTATCTCTGTTGAGTTCTGGGATAGGAGGGGTTCATTCCTTCTTTTGTATTTTGGGATGAGGTCTCAATACTTTC
TCCGGACAGCCCAGAGAAGCTGGGACTCCACACTGTGAACATATGCTACCACTCTGCCTGGAGATTTGGGTGTTTGA
GTGTGTGCTGTTTTTCCTCCCCAGAATGCCCTTAGCACAAGCCCCTGGGTTTCGAGTCAGGGGAGGTCACGCCAGAT
CAGATCACTTGCTCCAACCCAGAGCAGTATGTGGGCGGTATTCCTCCA。
A kind of construction method of humanized's retinoschisis transgene mouse model, A,RS1h- KI disease model mouse.
B、RS1h- KI disease model mouse digestion qualification results.1 representsRS1hWhen-KI disease models mouse is without digestionRS1hGene is
309bp, 2 represent with obvious two band after Bpu10I digestions：215bp and 94bp；There is digestion condition in Control groups（4th
Swimming lane）With without digestion（3rd swimming lane）Under the conditions of be showed no digestion band.C, Control mouse andRS1h- KI disease model mouse
Gene sequencing result.RS1C.195 T on 4th extron of gene>G mutational site, illustrates successfully to buildRS1h-KI
Disease model mouse.
A kind of construction method of humanized's retinoschisis transgene mouse model, uses optical coherence tomography first
Technology scans wild-type mice（wide type,WT）WithRS1h- KI Mouse Retinas E, it has been found thatRS1h- KI mouse regard
There is obvious splitting chamber in nethike embrane（Blueness circle mark）, outer nuclear layer and photoreceptor layer are thinning.Further do immuning tissue
Detection is learned, F is HE coloration results, compared with WT mouse,RS1h- KI Mouse Retinas structure change is abnormal, and it is outer from shape layer（It is blue
Color arrow）There is contact between obvious cavity, cell to tail off.RS1hThe outer nuclear layer of-KI Mouse Retinas（At yellow arrows）
With section layer outside photosensory cell（Cyan arrow）Thinning, statistical analysis is shown（G、H）, difference is extremely notable（n=24, P<
0.0001）.
A kind of construction method of humanized's retinoschisis transgene mouse model, OCT scan result, WT mouse views
Membrane structure is clear,RS1h- KI mouse significantly cleave chamber in the outer plexiform layer of retina.Immunohistochemical method compares wild type
Mouse andRS1hThe retinal structure difference of-KI mouse, compared with WT mouse,RS1hThe outer plexiform layer of-KI Mouse Retinas occurs
The contact obviously cleaved between chamber, cell tails off.RS1hThe outer section thickness of-KI Mouse Retinas outer nuclear layer, photosensory cell
Statistic analysis result is spent, compared with WT mouse,RS1hThe outer plexiform layer and photoreceptor layer thickness of-KI Mouse Retinas are thinning,
Significant difference.
A kind of construction method of humanized's retinoschisis transgene mouse model,RS1h- KI mouse are in institutional framework
The notable difference of upper presentation, also shows the Clinical signs similar to patient in its physiological function.We are rightRS1h- KI mouse
The retina that be recorded when electroretinogram detection electroretinogram is light stimulation based retinal from cornea or corresponding site is carried out
Total electricity reaction, the function of main reflection first and second grade of neuron of retina.Normal wild-type mice flash of light FERG (fash-
ERG) there are the less a ripples of a negative and a larger positive b ripple successively（J、M、P）.AndRS1hThe FERG of-KI mouse is obvious
It is abnormal：In dark adaptation electroretinogram detection rod cell reaction（K、O）, compared with WT, b ripples are obviously reduced（n=12, P<
0.0001）, unobvious, the indifference of a ripples change（n=12, P>0.5）；In dark adaptation hybrid reaction（N）When, compared with WT, a ripples
（L）With b ripples（O）It is obviously reduced, significant difference（n=12, P<0.0001）；When checking cone cell scintillation response（Q）, with WT
Compare, a ripples（n=12, P<0.001）Almost disappear, b ripples are obviously reduced（n=12, P<0.01）.Statistical analysis a/b ratios are found
（R）, the rod cell of dark adaptation in wild-type mice andRS1hChanged greatly between-KI mouse, significant difference；And cone cell
Do not change but with the a/b of the recombination reaction of rod cell；But the difference of cone cell is obvious（n=12, P<0.05）.
In a word,RS1h- KI electroretinographicresponses responses, which are detected, to be shown,RS1h- KI Mouse Retinas visual performance is damaged, and is retina the
The dysfunction of I and II neuron.Above allRS1h- KI Mouse Retina structure detections result is foundRS1h- KI is small
There is obvious splitting chamber in the external plexiform layer of Rat retina, and this is prominent exactly between photosensory cell cell and bipolar retinal cells
Link variation abnormality is touched, this is probablyRS1hThe abnormal major reason of-KI Mouse Retina b ripples.Secondly,RS1h- KI mouse exist
Photoreceptor layer is shown as in structure thinning, but a ripples are unchanged under dark adaptation, explanationRS1h- KI Mouse Retina light sensations are thin
The visual performance influence of rod cell is little in born of the same parents.Although showing b ripples in cone cell to reduce, cone cell number in mouse
Amount is less, therefore the visual performance influence on mouse cone cell is not obvious.Therefore, visual performance and retinal structure detection all may be used
With explanationRS1hLink and be damaged mainly due to the cynapse between photosensory cell and Beale's ganglion cells in-KI Mouse Retina retinas
CauseRS1h- KI mouse visual performance is abnormal, and this is exactlyRS1h- KI mouse occur caused by the splitting in outer plexiform layer of retina
, this is identical with clinical patient sign, illustrates that we build and has obtained humanized'sRS1h- KI mouse disease models.
Although the present invention is described by reference to preferred embodiment, those of ordinary skill in the art should
Work as understanding, the description of above-described embodiment can be not limited to, in the range of claims, can make each in form and details
Plant change.
Sequence table
<110>Wenzhou Medical University
<120>A kind of humanized's retinoschisis transgene mouse model and its construction method
<160> 2
<210> 1
<211> 5855bp
<212> RNA
<213> Mus musculus
<400> 1
aacaatacct ccccagactg cttgttgagg caggggacta tgtggcttaa ttggatgggg 60
gctgagtgaa agacctaaga actaaatgaa ataagatgct taagttaatc gcctgctcct 120
atgccagctc tccacttcac ttagatcttg ctgtgaccaa ggacaaggag aaaatgccac 180
acaagattga aggcttcttc ttgttacttc tctttggcta tgaagccaca ttgggattgt 240
catcgacaga ggatgagggt gaggacccct ggtaccagaa agcatgcaag tgtgattgcc 300
aggtaggagc caatgctctg tggtctgctg gagctacctc cttagactgt attccagaat 360
gcccatatca caagcccctg ggtttcgagt caggggaggt cacgccagat cagatcactt 420
gctccaaccc agagcagtat gtgggctggt attcctcatg gacagcaaac aaggcccgac 480
tcaacagtca aggttttggg tgtgcttggc tttccaagta tcaggacagc agccagtggt 540
tacagataga tttgaaggag atcaaggtga tttcggggat cctgacccaa ggacgctgtg 600
acatagacga gtgggtgacc aagtacagtg tgcagtatag gactgatgag cgcctgaact 660
ggatctacta taaggatcag accggaaaca atcgggtctt ctatggaaac tcagaccgga 720
gttctacagt tcagaactta ctcaggcccc ccatcatttc ccgcttcatc cgactgatcc 780
ctctaggctg gcatgtccga attgccatcc ggatggagct gcttgagtgt gccagcaagt 840
gtgcctgatg tctatttcag ctcagttctg tcacttgcag ggagagcttt ggggggaggg 900
caccctgaaa taccactggg atgaatgggg ctatatctta taagcacttt acaatgtaga 960
tctgggtaga gaatatttca ctttttaaaa gatagtttca aatttcaaag ggagagagag 1020
agagagagag agagagagag agagagagag agaagctttt aagcttttct tcaactcagg 1080
gccaaagaaa ataagaacaa agaaagtatc tctcaaacca attttcttac acaaacccaa 1140
atagcagggg taatttgctg ctctggctcc ccatcttttt ctctttgtct cagtgacacc 1200
gtcaaaggtg caggcccagg ggaacacaaa gcagctctga taatttgaaa attctttttg 1260
gctcttagca cattcagaag aggagcgtac tctttggcaa agcctcacat gaacacttgc 1320
atgaaatcct cactatagag gcctggtgac aagtacatag gtgtgaagaa agcagaagga 1380
agcctggcgt atggcccacg agtctggagc cagtaaccaa ccaaccaact aaccaactaa 1440
ccaaccagtc aaggcttccc cctccctgga attgcttttg cctggaaggg gtggagctcc 1500
ttgtgcagaa actccattct acagcccttt tggcaatgtg tccttctgtg attgcctgga 1560
gatgaatgca aggggcaggc tatatgtgtg tgtggatagt gaggtttccc ttaaactgag 1620
gaagcccaaa taactagaat acatcacacc ttctttatcc atggctacag tttcccagaa 1680
attgaacagg cttagaaaac agctgagata aaccatatct ctatctactg ggaagagatc 1740
atgctttggg ggaaggggtt aggactccta tcatagagga ctgtacacag atgatctgtc 1800
agtaaagggc ttgctgtaca gcataggaac ttgagattat atttctagaa tgcatgtaaa 1860
aagctaggta tggacaggta tggtggaaca tgccattagt cttagtattt ggaagacaga 1920
ggcaagcaga tctgagttca gggccagcct ggtctacata gcagttccaa gctagccaag 1980
gctacacagt aaggccctgt ttcaagaaaa aaagaccagg catggagggg cacacctgca 2040
aatccaggac taggaggcag gcagagacag gatgatgcct agagcttgtc tgccaatcag 2100
tacactccag gttcatggtc agaccctgtc tcaaaactaa agtgaagagc tattgaggaa 2160
gataccagat gtcaacctct gtccaacatg cacacataca catacactgg taggtttcaa 2220
acctggggca aatggcagag gtgcctagtt aaggttctcc cagcatctct cagcccctac 2280
caggtcctcc tagttggcat accccgcccc ctaccccgaa actcttcagc ccagggggtt 2340
gggcttcttc cctatataat ccaactattt tggtcaccct ttctctttgt acctttgggc 2400
ctcctggttg ctgcatctgg ttcctctgtt ctcccttatc tcttttcctc cctcctctcc 2460
tcacaaggcc cagcttagag tgttcatgtc tattctggac tctctcccgg atgcccctgc 2520
ctctggctat gctctcccac atatctacca taaactctct cctctaccat acctaggaca 2580
agtcatgttc ctttcctttt cctttttgtt tcccattcaa acatgtaaat aggaaaaaca 2640
aaatacaaca acaacaaaca aaaataaaac cacaaaagcc ttctctccga gcggcttcct 2700
cccgtcctgc ccacccaaac atgcttactc agacatgaca tttgaaactt ctttcggtag 2760
tcattctctg gcaggaatag ctcatttaat caggagcttt ggagcctgag agaatgacta 2820
ctggtctcta gctcatttcc atctgtagct caagctgggt ttgaggctct tctctgggag 2880
aggctgttaa ctggagtgag ggtcaaaaag gcatgcgggg agaagctgag aatgagaact 2940
gcccagagac aaggtcttgg cctcacacac acacaataca gccttggtta gtacttttct 3000
ctgagctatt ttctatttcc aggtattaaa ggagattaca tggattcaca ccaaaataac 3060
tttggatggc cagacatggt atcttccatc tttaatccta gcactgggaa aacacagaca 3120
ggtggatctc tgtgagttca aggccaggtt tgcatagcaa gctccagacc agccaaggct 3180
atgtagtgag actgtctcaa aaatcaatcc atcaatcaaa tgatcttgga tgtcacattc 3240
ttctcagtga atagaacaac attttaatag catcaaattt cctacctagt attttggagt 3300
tccacaggtg tgtgtgtatg tgtggcaatg tacaacaata ttcctggtat gactacatat 3360
gtacaaattc attttggata atattcctga cagcagacct ttatttggaa aattgagtta 3420
cctttctcac cacactgagt aattgttaga gaaaaatggc aataacttaa gaatactgat 3480
cagggagaaa aaaacccaaa tggtacaagg catgggtaaa tagtcatcaa attaaaaaat 3540
catgccataa atttgttttc taatagtttc attaacttca cataaacagt cctaattcat 3600
ttaaggtgtg taattcaata tagtatattc aaacttgaac aatcatcact acaagtaact 3660
ttagactatt cttatcaccc ccaagagaat cctctaacaa tgggccatca gccaccacag 3720
ctctagatta ctcgtcatcc attttctatt tgtgtgtgga ttttttttcc attctgggca 3780
tttctccaca caaatggaat cttagaatat atagtagttt atgacttgtt tcttgtactt 3840
atcatagtgt ttttaaagtc catgtgttgg agaagtacat cctccttttt tgttattaac 3900
attctgtgta gggtattgta ttattattta tccatgcatt tgggtggggc tagtatgaat 3960
aatgctgctt catgaaaatt tatatgtaag ttattgcgtg gacatgtttt atcttgggca 4020
tactcacact gagaatagag aattgttggg tctcttggga actatttctc caggtggctg 4080
taccatcttc tgttcccacc agcaatgtct aatgcttcca gcctctcatc tttagcaatg 4140
cttcttgtat ttcattagat ctgtcctagt atgtatgaag tagcattcga tgatgaattt 4200
gatttgcatt tctctaataa ccaatgtttt ttgagcatct tttcaagtca ccagtgacat 4260
atttttggag aaagttctat tcagaccctt gcccatataa agatttcatt tcacggattc 4320
attaagagtt ctttgggtgt tctacataca attctcttat gcaaacattt cttccatcct 4380
gtttttcttc ttgtcattgt cttggtttag ttttgcacca ggatttcact atgtagctca 4440
ggctggcctg gaacttgcaa agatccttct gtctctgcct cccaggttct gagattctgg 4500
gtatttgcca ccacacctgg cctttctctg aagcacaaca aataccaaat tttaatgaag 4560
tccaaccttc cggtttcctt ttttggtgta ttatctgaga aactactgtc aaaccatggt 4620
caagaagatt cactcttgtg ttttcttcta aatgttttga aaaaaaattt ttaaaaagtt 4680
ttaaagtttt aactcttact ttgtgaacag ttataaagta agggtgtaat ttcattcttt 4740
ggatatcaat atgcagctgt cttggtagct tgtgttgtct tcaccacagt caatggtctt 4800
tattcttcca aaaacaactg accaagaata ttagagtata tgctaggcct cataattcta 4860
ttaccacata gtcttcatta ctgtagcatt gtagtgagtt gtggaattag gacatataaa 4920
tcttccaatt ctggttatgt ttcaagactg aactaactat tctgggttcc ttaaacttct 4980
aagtaagtat atctgtaaat aagccagcca ggggtctcac aatggaaaat gttttcaatt 5040
tcaaaattaa ataaacaatg agatattaag atctgcaaga tatttatctg tagttaattt 5100
tctttcaata gttaatgaaa atgttagcat tttcttcctt ttgtgttagg atatatatat 5160
aaaagaaacc ctattattaa agtaaacctg aaaaagaatc ttcctattaa acaaaaaata 5220
tgtttgcaat attatttctt ttagggggca actaattcaa taaagattat ataatggagg 5280
aaagtcattc ctatcttaac acattttgtt tatttttacc actagacttt gggttattaa 5340
ggattttatt ttttatggtt ttgaggacat aacccaggtt tttcatacat ggtaggcaaa 5400
tactcttcaa ttgagttctc tctgcagcct gcagatcaaa acaaaacaaa acattatggt 5460
aaagcataac aatttacata cttttagtta actgtgaagc attaaataat gatattaaga 5520
aaaaaatgga agtaatcctg gtagttgatg tgggaggtta tatagtgtgg tggtttggat 5580
atgcttggcc tattaggagg tgtggctttg ttggagtagg tgtggccttg ttggaagaag 5640
tacatcattg tgggagtggg ctttgacacc ctcctcctcg ctgcccgaaa gacagtattc 5700
tggctacctt tggatcaaga tatagaactc ttggctcctt ctccagcacc atgtctgcct 5760
gcatgctgtt ggctaggatg ataattgact aaacatctga aactgtaagc cagccccaat 5820
taaatgttgt ctttctaaga aaaaaaaaaa aaaaa 5855
<210> 2
<211> 279bp
<212> RNA
<213> Mus musculus
<400> 2
CATTATCTCT GTTGAGTTCT GGGATAGGAG GGGTTCATTC CTTCTTTTGT ATTTTGGGAT 60
GAGGTCTCAA TACTTTCTCC GGACAGCCCA GAGAAGCTGG GACTCCACAC TGTGAACATA 120
TGCTACCACT CTGCCTGGAG ATTTGGGTGT TTGAGTGTGT GCTGTTTTTC CTCCCCAGAA 180
TGCCCTTAGC ACAAGCCCCT GGGTTTCGAG TCAGGGGAGG TCACGCCAGA TCAGATCACT 240
TGCTCCAACC CAGAGCAGTA TGTGGGCGGT ATTCCTCCA 279
Sequence table
<110>Wenzhou Medical University
<120>A kind of humanized's retinoschisis transgene mouse model and its construction method
<160> 2
<210> 1
<211> 5855bp
<212> RNA
<213> Mus musculus
<400> 1
aacaatacct ccccagactg cttgttgagg caggggacta tgtggcttaa ttggatgggg 60
gctgagtgaa agacctaaga actaaatgaa ataagatgct taagttaatc gcctgctcct 120
atgccagctc tccacttcac ttagatcttg ctgtgaccaa ggacaaggag aaaatgccac 180
acaagattga aggcttcttc ttgttacttc tctttggcta tgaagccaca ttgggattgt 240
catcgacaga ggatgagggt gaggacccct ggtaccagaa agcatgcaag tgtgattgcc 300
aggtaggagc caatgctctg tggtctgctg gagctacctc cttagactgt attccagaat 360
gcccatatca caagcccctg ggtttcgagt caggggaggt cacgccagat cagatcactt 420
gctccaaccc agagcagtat gtgggctggt attcctcatg gacagcaaac aaggcccgac 480
tcaacagtca aggttttggg tgtgcttggc tttccaagta tcaggacagc agccagtggt 540
tacagataga tttgaaggag atcaaggtga tttcggggat cctgacccaa ggacgctgtg 600
acatagacga gtgggtgacc aagtacagtg tgcagtatag gactgatgag cgcctgaact 660
ggatctacta taaggatcag accggaaaca atcgggtctt ctatggaaac tcagaccgga 720
gttctacagt tcagaactta ctcaggcccc ccatcatttc ccgcttcatc cgactgatcc 780
ctctaggctg gcatgtccga attgccatcc ggatggagct gcttgagtgt gccagcaagt 840
gtgcctgatg tctatttcag ctcagttctg tcacttgcag ggagagcttt ggggggaggg 900
caccctgaaa taccactggg atgaatgggg ctatatctta taagcacttt acaatgtaga 960
tctgggtaga gaatatttca ctttttaaaa gatagtttca aatttcaaag ggagagagag 1020
agagagagag agagagagag agagagagag agaagctttt aagcttttct tcaactcagg 1080
gccaaagaaa ataagaacaa agaaagtatc tctcaaacca attttcttac acaaacccaa 1140
atagcagggg taatttgctg ctctggctcc ccatcttttt ctctttgtct cagtgacacc 1200
gtcaaaggtg caggcccagg ggaacacaaa gcagctctga taatttgaaa attctttttg 1260
gctcttagca cattcagaag aggagcgtac tctttggcaa agcctcacat gaacacttgc 1320
atgaaatcct cactatagag gcctggtgac aagtacatag gtgtgaagaa agcagaagga 1380
agcctggcgt atggcccacg agtctggagc cagtaaccaa ccaaccaact aaccaactaa 1440
ccaaccagtc aaggcttccc cctccctgga attgcttttg cctggaaggg gtggagctcc 1500
ttgtgcagaa actccattct acagcccttt tggcaatgtg tccttctgtg attgcctgga 1560
gatgaatgca aggggcaggc tatatgtgtg tgtggatagt gaggtttccc ttaaactgag 1620
gaagcccaaa taactagaat acatcacacc ttctttatcc atggctacag tttcccagaa 1680
attgaacagg cttagaaaac agctgagata aaccatatct ctatctactg ggaagagatc 1740
atgctttggg ggaaggggtt aggactccta tcatagagga ctgtacacag atgatctgtc 1800
agtaaagggc ttgctgtaca gcataggaac ttgagattat atttctagaa tgcatgtaaa 1860
aagctaggta tggacaggta tggtggaaca tgccattagt cttagtattt ggaagacaga 1920
ggcaagcaga tctgagttca gggccagcct ggtctacata gcagttccaa gctagccaag 1980
gctacacagt aaggccctgt ttcaagaaaa aaagaccagg catggagggg cacacctgca 2040
aatccaggac taggaggcag gcagagacag gatgatgcct agagcttgtc tgccaatcag 2100
tacactccag gttcatggtc agaccctgtc tcaaaactaa agtgaagagc tattgaggaa 2160
gataccagat gtcaacctct gtccaacatg cacacataca catacactgg taggtttcaa 2220
acctggggca aatggcagag gtgcctagtt aaggttctcc cagcatctct cagcccctac 2280
caggtcctcc tagttggcat accccgcccc ctaccccgaa actcttcagc ccagggggtt 2340
gggcttcttc cctatataat ccaactattt tggtcaccct ttctctttgt acctttgggc 2400
ctcctggttg ctgcatctgg ttcctctgtt ctcccttatc tcttttcctc cctcctctcc 2460
tcacaaggcc cagcttagag tgttcatgtc tattctggac tctctcccgg atgcccctgc 2520
ctctggctat gctctcccac atatctacca taaactctct cctctaccat acctaggaca 2580
agtcatgttc ctttcctttt cctttttgtt tcccattcaa acatgtaaat aggaaaaaca 2640
aaatacaaca acaacaaaca aaaataaaac cacaaaagcc ttctctccga gcggcttcct 2700
cccgtcctgc ccacccaaac atgcttactc agacatgaca tttgaaactt ctttcggtag 2760
tcattctctg gcaggaatag ctcatttaat caggagcttt ggagcctgag agaatgacta 2820
ctggtctcta gctcatttcc atctgtagct caagctgggt ttgaggctct tctctgggag 2880
aggctgttaa ctggagtgag ggtcaaaaag gcatgcgggg agaagctgag aatgagaact 2940
gcccagagac aaggtcttgg cctcacacac acacaataca gccttggtta gtacttttct 3000
ctgagctatt ttctatttcc aggtattaaa ggagattaca tggattcaca ccaaaataac 3060
tttggatggc cagacatggt atcttccatc tttaatccta gcactgggaa aacacagaca 3120
ggtggatctc tgtgagttca aggccaggtt tgcatagcaa gctccagacc agccaaggct 3180
atgtagtgag actgtctcaa aaatcaatcc atcaatcaaa tgatcttgga tgtcacattc 3240
ttctcagtga atagaacaac attttaatag catcaaattt cctacctagt attttggagt 3300
tccacaggtg tgtgtgtatg tgtggcaatg tacaacaata ttcctggtat gactacatat 3360
gtacaaattc attttggata atattcctga cagcagacct ttatttggaa aattgagtta 3420
cctttctcac cacactgagt aattgttaga gaaaaatggc aataacttaa gaatactgat 3480
cagggagaaa aaaacccaaa tggtacaagg catgggtaaa tagtcatcaa attaaaaaat 3540
catgccataa atttgttttc taatagtttc attaacttca cataaacagt cctaattcat 3600
ttaaggtgtg taattcaata tagtatattc aaacttgaac aatcatcact acaagtaact 3660
ttagactatt cttatcaccc ccaagagaat cctctaacaa tgggccatca gccaccacag 3720
ctctagatta ctcgtcatcc attttctatt tgtgtgtgga ttttttttcc attctgggca 3780
tttctccaca caaatggaat cttagaatat atagtagttt atgacttgtt tcttgtactt 3840
atcatagtgt ttttaaagtc catgtgttgg agaagtacat cctccttttt tgttattaac 3900
attctgtgta gggtattgta ttattattta tccatgcatt tgggtggggc tagtatgaat 3960
aatgctgctt catgaaaatt tatatgtaag ttattgcgtg gacatgtttt atcttgggca 4020
tactcacact gagaatagag aattgttggg tctcttggga actatttctc caggtggctg 4080
taccatcttc tgttcccacc agcaatgtct aatgcttcca gcctctcatc tttagcaatg 4140
cttcttgtat ttcattagat ctgtcctagt atgtatgaag tagcattcga tgatgaattt 4200
gatttgcatt tctctaataa ccaatgtttt ttgagcatct tttcaagtca ccagtgacat 4260
atttttggag aaagttctat tcagaccctt gcccatataa agatttcatt tcacggattc 4320
attaagagtt ctttgggtgt tctacataca attctcttat gcaaacattt cttccatcct 4380
gtttttcttc ttgtcattgt cttggtttag ttttgcacca ggatttcact atgtagctca 4440
ggctggcctg gaacttgcaa agatccttct gtctctgcct cccaggttct gagattctgg 4500
gtatttgcca ccacacctgg cctttctctg aagcacaaca aataccaaat tttaatgaag 4560
tccaaccttc cggtttcctt ttttggtgta ttatctgaga aactactgtc aaaccatggt 4620
caagaagatt cactcttgtg ttttcttcta aatgttttga aaaaaaattt ttaaaaagtt 4680
ttaaagtttt aactcttact ttgtgaacag ttataaagta agggtgtaat ttcattcttt 4740
ggatatcaat atgcagctgt cttggtagct tgtgttgtct tcaccacagt caatggtctt 4800
tattcttcca aaaacaactg accaagaata ttagagtata tgctaggcct cataattcta 4860
ttaccacata gtcttcatta ctgtagcatt gtagtgagtt gtggaattag gacatataaa 4920
tcttccaatt ctggttatgt ttcaagactg aactaactat tctgggttcc ttaaacttct 4980
aagtaagtat atctgtaaat aagccagcca ggggtctcac aatggaaaat gttttcaatt 5040
tcaaaattaa ataaacaatg agatattaag atctgcaaga tatttatctg tagttaattt 5100
tctttcaata gttaatgaaa atgttagcat tttcttcctt ttgtgttagg atatatatat 5160
aaaagaaacc ctattattaa agtaaacctg aaaaagaatc ttcctattaa acaaaaaata 5220
tgtttgcaat attatttctt ttagggggca actaattcaa taaagattat ataatggagg 5280
aaagtcattc ctatcttaac acattttgtt tatttttacc actagacttt gggttattaa 5340
ggattttatt ttttatggtt ttgaggacat aacccaggtt tttcatacat ggtaggcaaa 5400
tactcttcaa ttgagttctc tctgcagcct gcagatcaaa acaaaacaaa acattatggt 5460
aaagcataac aatttacata cttttagtta actgtgaagc attaaataat gatattaaga 5520
aaaaaatgga agtaatcctg gtagttgatg tgggaggtta tatagtgtgg tggtttggat 5580
atgcttggcc tattaggagg tgtggctttg ttggagtagg tgtggccttg ttggaagaag 5640
tacatcattg tgggagtggg ctttgacacc ctcctcctcg ctgcccgaaa gacagtattc 5700
tggctacctt tggatcaaga tatagaactc ttggctcctt ctccagcacc atgtctgcct 5760
gcatgctgtt ggctaggatg ataattgact aaacatctga aactgtaagc cagccccaat 5820
taaatgttgt ctttctaaga aaaaaaaaaa aaaaa 5855
<210> 2
<211> 279bp
<212> RNA
<213> Mus musculus
<400> 2
CATTATCTCT GTTGAGTTCT GGGATAGGAG GGGTTCATTC CTTCTTTTGT ATTTTGGGAT 60
GAGGTCTCAA TACTTTCTCC GGACAGCCCA GAGAAGCTGG GACTCCACAC TGTGAACATA 120
TGCTACCACT CTGCCTGGAG ATTTGGGTGT TTGAGTGTGT GCTGTTTTTC CTCCCCAGAA 180
TGCCCTTAGC ACAAGCCCCT GGGTTTCGAG TCAGGGGAGG TCACGCCAGA TCAGATCACT 240
TGCTCCAACC CAGAGCAGTA TGTGGGCGGT ATTCCTCCA 279
Claims (10)
1. a kind of construction method of humanized's retinoschisis transgene mouse model, it is characterised in that：With humanizedRS1Base
Because basic, screening human disease's gene is knocked in mouse by gene editing means, obtains having and patients clinical is characterized
The sameRS1h- KI mouse disease models.
2. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 1, it is special
Levy and be：With TALENs technology, obtained according to the screening of XLRS sufferersRS1The mutational site of gene, design and XLRS sufferers
Identical Oligo sequences, the carrier built is transferred in cell, and screening obtains the stable cell lines with RS1 gene mutations,
Vivoexpression, isolated mRNA, and embryonated egg microinjection and embryo transfer are carried out into the pregnant mouse uterus in F0 generations, breed
To the F1 generation for stablizing phenotypeRS1h- KI disease model mouse.
3. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 2, it is special
Levy and be：Oligo sequences are：TGGGTGTTTGAGTGTGTGCTGTTTTTCCTCCCCAGAATGCCCATATCACAAGCCCCTG
GGTTTCGAGTCAGGGGAGGTCACGCCAGATCA。
4. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 2, it is special
Levy and be：The protein sequence of saltant type RS1 gene translations：MPHKIEGFFLLLLFGYEATLGLSSTEDEGEDPWYQKACKC
DCQVGANALWSAGATSLDCIPECP。
5. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 2, it is special
Levy and be：Gene knock-in mouse is built with TALEN, design is directed toRS1C.195 T on 4th extron of gene>G mutation
Site, allows TALEN to be combined in cell with the target site of genome using TALEN two hand cradle, forms FokI dimers hair
Endonuclease activity is waved, causes left and right TALEN spacer regions to occur double-strand DNA cleavage, so that radiation-indued DNA damage repair machine
System, cell reaches gene knock-in into the gene of mouse by nonhomologous end engagement mechanisms DNA plerosis, two of TALEN
Hand cradle：TALEN_Left primers are：TGACTCGAAACCCAGGGG；TALEN_Right primers are：TGCCCATATCACAAG；
Spacer regions are：TGCCCATATCACAAG.
6. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 5, it is special
Levy and be：ForRS1C.195 T on 4th extron of gene>G mutational site, designs the Oligo sequences of gene knock-in
It is classified as：TGGGTGTTTGAGTGTGTGCTGTTTTTCCTCCCCAGAATGCCCATATCACAAGCCCCTGGGTTTCGAGTCAGG
GGAGGTCACGCCAGATCA。
7. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 5, it is special
Levy and be：RS1One Bpu10I restriction enzyme site is set at the 215bp of gene.
8. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 2, it is special
Levy and be：The good humanized of design constructionRS1Gene Oligo sequences, are carried out in-vitro transcription, using the side of microinjection
The mRNA of in-vitro transcription is injected into the ripe egg cells of C57BL/6J by formula, inject successful embryonated egg can in vitro culture stay overnight or
The culture of becoming pregnant is transferred in replace-conceive dams uterus immediately.
9. a kind of construction method of humanized's retinoschisis transgene mouse model according to claim 2, it is special
Levy and be：After the farrowing of replace-conceive dams, the tail and toe of F1 generation mouse are taken, full-length genome is extracted, enters performing PCR amplification, Bpu10I
Digestion digestion verification, sequencing identification.
10. a kind of humanized's retinoschisis transgene mouse model according to claim 1, it is characterised in that：RS1h- KI murine genes sequencing sequences:CATTATCTCTGTTGAGTTCTGGGATAGGAGGGGTTCATTCCTTCTTTTGTATT
TTGGGATGAGGTCTCAATACTTTCTCCGGACAGCCCAGAGAAGCTGGGACTCCACACTGTGAACATATGCTACCACT
CTGCCTGGAGATTTGGGTGTTTGAGTGTGTGCTGTTTTTCCTCCCCAGAATGCCCTTAGCACAAGCCCCTGGGTTTC
GAGTCAGGGGAGGTCACGCCAGATCAGATCACTTGCTCCAACCCAGAGCAGTATGTGGGCGGTATTCCTCCA。
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