CN109112125A - A kind of method for building up and application thereof of dyskinesia animal - Google Patents
A kind of method for building up and application thereof of dyskinesia animal Download PDFInfo
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Abstract
The present invention relates to a kind of method for building up and application thereof of dyskinesia animal.The present invention reduces the animal of expression or inactivation using Prrt2 gene, in conjunction with specific experimental implementation tool and operating method, obtains dyskinesia animal model.The animal model that method of the invention obtains is a kind of dyskinesia animal model stable, that typical character is presented, can be used for study movement disorder disease, and can be used for screening and the testing experiment of certain drug.
Description
Technical field
The present invention relates to field of biotechnology；More particularly, to a kind of foundation side of dyskinesia mouse model
Method and application thereof.
Background technique
The transmembrane protein 2 (proline-rich transmembrane protein 2, PRRT2) of people's Pro-rich
(Entrez Gene:112476) gene is located at human chromosome 16p11.2, includes four exons, wherein the 1st exon is not
Encode albumen.PRRT2 may encode three kinds of different isomer proteins by different mRNA montages.Wherein main isomers
1 includes 340 amino acid residues, is guarded relatively (about 80% compared with mammal, compared with zebra fish about 30%) between species.
Isomers 1 is mainly comprising rich proline structure domain and two transmembrane domains.The isomers 2 comprising 394 amino acid of prediction
Have longer c-terminus, and include 299 amino acid residues isomers 3 be a kind of isomers 1 truncated-type (Valtorta,
F.,et al.,PRRT2:from Paroxysmal Disorders to Regulation of Synaptic
Function.Trends in Neurosciences 2016Sep 10,39(10),668-679.)。
The evidence of the mankind and rodent all shows that PRRT2 is the albumen of nervous specific expression, and molecular function may
Be related to: PRRT2, which can be positioned at cynapse, may influence the transmitting of nerve signal；PRRT2 may influence the migration of neuron；PRRT2
It may be the important member of neurotransmitter regulator related protein complex.
PRRT2 be initially subjected to concern be because its be noted may be episodic ataxia Disease-causing gene, including
Clinical symptoms have: (1) dyskinesia of paroxysmal exercise induced (paroxysmal kinesigenic dyskinesia,
PKD)；(2) paroxysmal exercise induced choreoathetosis (paroxysmal kinesigenic
Choreoathetosis, PKC)；(3) with the paroxysmal exercise induced dyskinesia (paroxysmal of eclampsia infantum
Kinesigenic dyskinesia with infantileconvulsions, PKD/IC).
Deeper into research discovery PRRT2 be also possible to have relationship with other diseases symptom, including but not limited to following symptom:
The non-athletic induction property dyskinesia of paroxysmal (paroxysmal nonkinesigenic dyskinesia, PNKD)；Progressive
Incoordination (progressive ataxia)；Benign familial baby epilepsy (benign familial infantile
Epilepsy, BFIE)；Migraine (migraine)；Hemiplegia type migraine (hemiplegic migraine)；Paroxysmal torticollis
(paroxysmal torticollis)；The dyskinesia of sleep period paroxysmal (paroxysmal hypnogenic dystonia,
PHD)；Periodical incoordination (episodic ataxia, EA)；Feeblemindedness (mental retardation).
The heterozygous mutant of people PRRT2 does not only result in the clinical symptoms of multiplicity, is also embodied by non-fully dominant, that is, have heterozygosis
Carriers of mutation will not fall ill.The homozygous mutation of people PRRT2 may result in serious consequence, and the same patient might have
Several symptoms stated, especially feeblemindedness.The expression of Prrt2 gene is sensitive to the mutation of gene, and clipped form albumen can not
So as to cause Prrt2 shortage, this may be caused by mRNA unstable for expression.
It has been reported that Prrt2 knock out mice shows some dyskinesia performances, if disequilibrium number increases, retreats secondary
Number increases, but these faint dyskinesias and the relationship of human motion obstacle also need to further confirm that.The inventor have observed that
The ataxia of extremely low ratio (< 1%) can be observed in the ataxia phenomenon become apparent, Prrt2 knock out mice, transports
Dynamic imbalance shows as losing walking ability, with abnormal posture show as limbs it is improper lift, tail it is improper stiff,
Incoordination etc., and same mouse ataxia frequency is few, Symptomatic mouse all only observes 1 time that the time is lasting substantially
Several minutes to two hours.Therefore Prrt2 gene (stem cell methods/Cas9 method) simply are knocked out no on rat/mouse
It can provide phenomenon apparent animal model for research human motion obstacle.The spontaneous rate of extremely low dyskinesia limits the disease
The exploitation of research and new drug.This field also needs further to seek, the dyskinesia animal model of Improvement.
Summary of the invention
The purpose of the present invention is to provide a kind of animal model of dyskinesia, the models coupling PRRT2 mutation and it is specific
Examination on experimental operation, particular experiment operating method be selected from following one kind: light genetic technique, cerebral injury.
A kind of preparation method of mouse movement obstacle animal model is provided, this method, which is related to Prrt2 gene, reduces expression
Or inactivate animal and specific Examination on experimental operation.
The another object of the invention is to provide the dyskinesia mouse model of preparation in scientific research and drug test side
The application in face.
In the first aspect of the present invention, the kit for being used to prepare dyskinesia animal is provided, includes: in the kit
(1) make the reagent that Prrt2 gene reduces expression or inactivates in Animal genome；
(2) for carrying out the reagent or device of blue laser stimulation to animal brain；Or for carrying out brain to animal brain
The device of damage.
In a preferred embodiment, in (2), the reagent or device for being used to carry out animal brain blue laser stimulation
Include: the expression construct for expressing light-operated cationic channel gene, fiber stub, blue laser, stereotaxic apparatus and/or
Micro-injection pump.
In another preferred example, the light-operated cationic channel gene is ChR2 gene.
In another preferred example, in (2), the device for carrying out cerebral injury to animal brain includes:
Thin rodlike instrument, preferably: syringe needle, sticking plaster, glass bar；Or
Surgical catheters and the conduit inner core to match with the conduit (conduit of outer diameter 0.1mm~10mm and can such as be set
In this to the conduit inner core in pipe).
In another preferred example, in (1), described makes the examination that Prrt2 gene reduces expression or inactivates in Animal genome
Agent is selected from: realizing that Prrt2 gene reduces the reagent of expression or inactivation by gene knockout；Or pass through insertion foreign gene or gene
Segment realizes that Prrt2 gene reduces the reagent of expression or inactivation.
In another preferred example, making the reagent that Prrt2 gene reduces expression or inactivates in Animal genome is cas9 gene
Edit reagent.
In another preferred example, the animal is mouse, volume of the cas9 gene editing reagent in Prrt2 gene
Terminator codon is knocked in after the C666 of code sequence.
In another preferred example, the gene editing reagent includes: the sgRNA for mouse Prrt2, and sequence is such as
Shown in SEQ ID NO:2；Donor plasmid, it includes the sequences as shown in SEQ ID NO:3；Preferably, the gene editing
Reagent further include: form the reagent of Cas9mRNA in the cell.
In another preferred example, making the reagent that Prrt2 gene reduces expression or inactivates in Animal genome is TALEN targeting
Gene knockout reagent.
In another preferred example, the animal is rat, and the TALEN targeted gene disruption reagent is in Prrt2 base
10bp base sequence is deleted after the translation initiation codon ATG of cause.
In another preferred example, the targeting sequence such as SEQ ID NO:4 of the TALEN targeted gene disruption reagent.
In another preferred example, the fiber stub diameter is 0.2mm, and blue laser is BL473T3-050
(Shanghai Laser&Optics Century Co.,Ltd.)。
In another preferred example, the animal is rodent；Preferably, the animal is mouse (such as rat, mouse).
In another aspect of this invention, a kind of method preparing dyskinesia animal is provided, which comprises
(a) Prrt2 gene in Animal genome is reduced by expression or inactivation；
(b) animal obtained to (a) carries out the stimulation of laser blue light or cerebral injury, to obtain dyskinesia animal.
In a preferred embodiment, in step (b), carrying out the stimulation of laser blue light includes: the cerebellum or neighbouring mind in animal
Through being placed in fiber stub in member tissue (preferably cerebellar granule cell layer)；It is stimulated 2~20 times using blue laser；Preferably
Ground, 6~15 seconds/time, 6~15 (preferably 8~12 seconds) of interval carry out the 2nd stimulation.
In another preferred example, when stimulation, the terminal light intensity of the blue laser is 10~20mW；Preferably 13~
17mW。
In another preferred example, in step (a), make Prrt2 gene in Animal genome using cas9 gene editing reagent
Reduce expression or inactivation；Or reduce Prrt2 gene in Animal genome by expression or inactivation using Cre and loxp method.
In another aspect of this invention, the application of the animal for the dyskinesia that the method obtains is provided, conduct is used for
The drug candidate of screening treatment neurological disease or the animal model of therapeutic agent are (preferably, the application is nondiagnostic, non-therapeutic
Application)；Or for the animal model as study movement obstacle.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
The construction strategy and protein expression detection for the Prrt2STOP mouse that Fig. 1, the present invention use；
A, the strategy of the construction of strategy Prrt2STOP mouse of the mouse is constructed using cas9 technology；
B, the gene order of the mouse of sequencing confirmation transformation is consistent with expection；
C, the result of protein immunization analysis detection PRRT2 albumen presence or absence.
The building of the Prrt2-D10 rat of Fig. 2, Prrt2 gene inactivation and Testing and appraisal；
A, the construction of strategy Prrt2-D10 of the mouse is constructed compared with the sequence of wild type using cas9 technology；
B, the gene order of the mouse of sequencing confirmation transformation is consistent with expection；
C, the protein expression of the cerebral cortex of Prrt2-D10 and cerebellum changes.
The position of Fig. 3, virus injection, and slice confirmation expressing viral range.
The light heredity of the homozygote mouse of Fig. 4, Prrt2STOP induces the process of phenotype.
Fig. 5, Prrt2 wild-type mice are compared with Prrt2STOP mouse movement.
The light heredity of the homozygote rat of Fig. 6, Prrt2STOP induces the process of phenotype.
Fig. 7, Prrt2-D10 rat are compared with the movement of Prrt2 wild-type rats.
Specific embodiment
The present inventor passes through in-depth study, discloses a kind of foundation side of dyskinesia (Dyskinesia) animal model
Method and application thereof.The preparation method of dyskinesia animal model of the invention reduces expression using Prrt2 gene or inactivates dynamic
Object is implemented in conjunction with specific experimental implementation tool and operating method.The animal model that method of the invention obtains is a kind of
Effectively, typical dyskinesia animal model, can be used for study movement disorder disease, and can be used for the screening of certain drug
And testing experiment.
Prrt2 gene
Transmembrane protein 2 (proline-rich transmembrane protein 2, PRRT2) base of people's Pro-rich
It include four exons, wherein the 1st exon is not compiled because (Entrez Gene:112476) is located at human chromosome 16p11.2
Code albumen.Three kinds of different isomer proteins may be encoded by different mRNA montages.Wherein main isomers 1 includes
340 amino acid residues, between species relatively it is conservative (about 80% compared with mammal, compared with zebra fish about 30).Isomers 1
It is main to include rich proline structure domain and two transmembrane domains.The isomers 2 comprising 394 amino acid of prediction has longer
C-terminus, and include the isomers 3 of 299 amino acid residues be a kind of truncated-type of isomers 1.
The evidence of the mankind and rodent all shows that PRRT2 is the albumen of nervous specific expression, and molecular function may
Be related to: PRRT2, which can be positioned at cynapse, may influence the transmitting of nerve signal；PRRT2 may influence the migration of neuron；PRRT2
It may be the important member of neurotransmitter regulator related protein complex.
As described in the background section, diseases range caused by the mutation of mankind PRRT2 is very wide, including clinical symptoms
Have: (1) paroxysmal exercise induced dyskinesia (paroxysmal kinesigenic dyskinesia, PKD)；(2) paroxysm
Property exercise induced choreoathetosis (paroxysmal kinesigenic choreoathetosis, PKC)；(3) companion
With paroxysmal exercise induced dyskinesia (the paroxysmal kinesigenic dyskinesia with of eclampsia infantum
Infantileconvulsions, PKD/IC)；The non-athletic induction property dyskinesia (paroxysmal of paroxysmal
Nonkinesigenic dyskinesia, PNKD)；Progressive incoordination (progressive ataxia)；Benign familial
Baby's epilepsy (benign familial infantile epilepsy, BFIE)；Migraine (migraine)；The inclined head of hemiplegia type
(hemiplegic migraine) bitterly；Paroxysmal torticollis (paroxysmal torticollis)；Paroxysmal movement barrier in sleep
Hinder (paroxysmal hypnogenic dystonia, PHD)；Periodical incoordination (episodic ataxia, EA)；Intelligence
Power low (mental retardation) etc..
Although known PRRT2 is related with the non-athletic induction property this kind of dyskinesia of dyskinesia of paroxysmal, this field
Personnel's discovery, knocks out the gene and is only capable of causing rodent faint dyskinesia, can not reach typical movement barrier
Hinder state.Therefore, the difficult point that the typical dyskinesia model of rodent is still this field is obtained.
It should be understood that term " PRRT2 " further includes the variant form of various naturally occurring Prrt2 genes.Representative example
Son includes: the nucleotide sequence that PRRT2 albumen identical with wild type is encoded because of the degeneracy of codon, encoding wild type
The nucleotide sequence of the conservative variation's polypeptides of PRRT2 albumen.
The preparation of animal model
In the present invention, a kind of very effective dyskinesia animal model is provided.The described method includes: (a) makes
Prrt2 gene reduces expression or inactivation in object genome；(b) animal obtained to (a) carries out the stimulation of laser blue light or brain damage
Wound, to obtain dyskinesia animal model.
As used herein, term " animal " is preferably rodent；E.g., including (but being not limited to): rat, small
Mouse.
As used herein, " animal of dyskinesia " or " animal model of dyskinesia " refer to generation selected from
Under it is one or more (preferably at least two kinds of；It is more preferably at least three kinds of or 3 kinds or more) animal of the situation of motor disorder or disorder:
Can not normal gait, lose walking ability, forelimb trembles, and centre of body weight is decreased obviously, abdomen patch ground, abdomen perineum patch ground,
Abnormal posture, acral to tremble, trunk, four limbs, tail are stiff.
As used herein, term " Prrt2 gene reduces expression or inactivation " includes that one or two Prrt2 gene is lowered
The case where expression or inactivation, including Prrt2 genetic heterozygosis and homozygously reduces expression or inactivation.For example, Prrt2 gene drops
The animal of low expression or inactivation can be the animal of heterozygosis or homozygosis, most preferably be homozygous animal.
As used herein, " Prrt2 gene reduces expression " refers to compared with the animal of wild type, the animal of transformation
The expression quantity conspicuousness of the Prrt2 gene of (whole body or portion of tissue) reduces, and is such as reduced to 50% or less wild type；Preferably
It is reduced to the 30% or less of wild type；More preferably it is reduced to the 10% or 5% or less of wild type.Also, " the Prrt2 base
Expressed because reducing " also including the situation of " Prrt2 gene is not expressed ".
As used herein, term " Prrt2 gene reduce expression or inactivation " include whole body or particular cell types, or
The Prrt2 gene of specific organization's range reduces expression or inactivation.
A variety of methods can be used so that " Prrt2 gene reduces expression or inactivation ", comprising: gene knockout is transferred to external source
Gene (or segment) and make Prrt2 gene reduce expression or inactivation；Gene interference or gene silencing are carried out with RNAi reagent；With anti-
Prrt2 antibody is handled so that Prrt2 albumen loses activity.
In a preferred embodiment of the present invention, the reduction expression of Prrt2 gene or inactivation are by conditional gene knockout reality
Existing；Or realized by being inserted into foreign gene (or segment) in Prrt2 gene.
As preferred embodiment of the invention, by knocking out Prrt2 gene, to lower the expression of Prrt2 gene.Preferably
Ground carries out gene editing using CRISPR/Cas9 system.Suitable sgRNA target site, can bring higher gene editing to imitate
Rate, the present inventor design and have found preferred target site, devise sgRNA based on this.By sgRNA or described in capable of being formed
The nucleic acid of sgRNA, Cas9mRNA can form the nucleic acid corotation of the Cas9mRNA and enter in fertilised non-human eggs, and acquisition is compiled through gene
The animal collected.As a kind of selection, the nucleic acid that can form the sgRNA is nucleic acid construct or expression vector or institute
The nucleic acid that can form the Cas9mRNA stated is nucleic acid construct or expression vector, these expression vectors are imported into cell
It is interior, to form active sgRNA and Cas9mRNA in the cell.Furthermore, it is possible to be transcribed in vitro obtain Cas9mRNA and
sgRNA。
As another selection mode of the invention, reduce Prrt2 gene in Animal genome using Cre and loxp method
Expression or inactivation.
It further include further being operated to animal after carrying out Prrt2 gene to animal and reducing expression or inactivation,
To obtain ideal dyskinesia animal, the dyskinesia phenomenon of the animal can be observed steadily.Described is further
Operation is selected from following one kind: targeting the stimulation of laser blue light, the targeting cerebral injury of brain.
As one embodiment of the present invention, carrying out the stimulation of laser blue light includes: the cerebellum or neighbouring mind in animal
It is organized through member, is preferably placed in fiber stub in cerebellar granule cell layer；Several times using blue laser stimulation.
The animal model that the present invention constructs can be used for screening and the testing experiment of certain drug.
The animal model that the present invention constructs can be used as the powerful of scientific research and new drug evaluation.
The method for preparing animal model of this hair is easy to operate, and the animal model stability of acquisition is good.
In the present invention, a kind of drug candidate using animal model screening treatment neurological disease of the invention is additionally provided
Or the method for therapeutic agent.
In the present invention, drug candidate or therapeutic agent refer to it is known with certain pharmacological activity or be detected can
Can have the substance of certain pharmacological activity, including but not limited to nucleic acid, albumen, carbohydrate, chemically synthesized small molecule or big point
Sub- compound, cell etc..The administration mode of drug candidate or therapeutic agent can be oral, intravenous injection, intraperitoneal injection, subcutaneous note
It penetrates, canalis spinalis is administered or direct intracerebral injection.
Kit
It is described the present invention also provides the kit for being used to prepare dyskinesia animal model based on method of the invention
It include: the reagent that (1) makes that Prrt2 gene reduces expression or inactivates in Animal genome in kit；(2) for animal brain
Carry out the device of blue laser stimulation；Or the device for carrying out cerebral injury to animal brain.
As preferred embodiment of the invention, the device for carrying out blue laser stimulation to animal brain includes:
Fiber stub, blue laser etc..
As preferred embodiment of the invention, described is used to carry out animal brain cerebral injury (preferably, damage intracerebral mind
Through member) device include: syringe needle；Or surgical catheters and the conduit inner core to match with the conduit, such as outer diameter 0.1mm
The conduit of~10mm and this can be placed in the conduit inner core in pipe)；Etc..
In the kit, it may also include making for the method for illustrating to implement preparation dyskinesia animal model of the invention
With specification, in order to those skilled in the art's application.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
Embodiment 1, the Prrt2STOP mouse for preparing Prrt2 gene inactivation
In the present embodiment, Prrt2 gene inactivated mice, i.e. Prrt2STOP mouse are prepared.The mouse simulates mankind Prrt2
Hot spot mutation.
Construct the construction of strategy Prrt2STOP mouse of the mouse using cas9 technology, Figure 1A show wild type and
The alignment of Prrt2stop mouse, (the i.e. sequence 5 '-behind the C666 of the coded sequence of mouse Prrt2 gene
After acctcactcaccaccctcaactaaaacacccccagccaatggggctcccccc-3 ' (SEQ ID NO:1)) it knocks in
Termination codon TAGTAA.Arrow indicates the position that insertion stops sequence.Rectangle shading is highlighted the sequence knocked in
TAGTAA。
The sgRNA target sequence of use are as follows:
5'-CGAGTTTCTGCAGCACACGGGGG-3'(SEQ ID NO:2)；
Donor oligomerization single-stranded DNA sequence is as follows, the sequence knocked in frame:
5’-CTCACCACCCTCAACTAAAACACCCCCAGCCAATGGGGCTCCCCCC
CGTGTGCTGCAGAAACTCGTTGAGGAAGACAGAATAGGAAGGGCAC-3 ' (SEQ ID NO:3) (is synthesized from Invitrogen
(Shanghai) trade Co., Ltd).
Cas9mRNA, sgRNA and donor oligomerization single stranded DNA are injected into mouse fertilized egg.Wherein, Cas9mRNA is used
mMESSAGET7Ultra Kit is transcribed in vitro；SgRNA uses MEGAshortscriptTMKit into
Row is transcribed in vitro, and product uses MEGAclearTM Kit to purify.
For zygote transplation to false pregnancy female rat uterus, the head that can get gene knockout later builds mouse.Extracting genome DNA is certainly
Newborn mice rat-tail carries out sequence verification.Head comprising expected mutation is built mouse and wild-type mice mating, is obtained comprising pre-
The F1 generation hybrid mice of phase mutation.F1 generation hybrid mice, which carries out mating, can be obtained F2 for homozygote mouse.Figure 1B is shown
The peak figure that one F2 is sequenced for homozygote mouse confirms that the gene order of the mouse of preparation is consistent with expection.
The forebrain and cerebellar tissue of newborn mice are obtained, protein immunization analysis is carried out.Fig. 1 C shows that protein immunization is analyzed
As a result, show that the homozygote of Prrt2Stop mouse results in PRRT2 albumen and can not be detected, that is, cause Prrt2 gene to lose
It is living.
Therefore, a kind of Prrt2 gene inactivated mice, i.e. Prrt2STOP mouse has successfully been obtained.
The Prrt2-D10 rat of embodiment 2, Prrt2 gene inactivation
In the present embodiment, the Prrt2-D10 rat of Prrt2 gene inactivation is prepared.
Utilize TALEN (Transcription Activator-Like Effector Nuclease) targeted gene disruption
Technology building.The Prrt2-D10 mouse of gene knockout is as shown in Figure 2 A compared with the sequence of wild type, in the Prrt2 gene of rat
The gene of 10bp is eliminated after translation initiation codon ATG.Rectangle shading is highlighted the sequence of rejecting
AGCCAGTAGC, arrow indicate the position for rejecting sequence.
The TALEN of use targets sequence are as follows:
5’-CTCCCATCTCTCTTCTCTAAGATGGCAGCCAGTAGCTCTGAGGTCTCTGAGATG-3’(SEQ ID
NO:4)；
The identification region corresponding sequence of TALEN are as follows:
5 '-CTCCCATCTCTCTTCTCTA (SEQ ID NO:5) and GCTCTGAGGTCTCTG AGATG-3 ' (SEQ ID
NO:6)；
Spacer regional sequence:
5’-AGATG(wherein, underscore section indicates translation initiation password to GCAGCCAGTA-3 ' (SEQ ID NO:7)
Son).
It will be transcribed in vitro by the TALEN plasmid of above-mentioned construction of strategy, the TALEN mRNA transcribed is injected to greatly
Mouse fertilized eggs.Zygote transplation can get the neonate rat of gene knockout to false pregnancy female rat uterus later.Extracting genome DNA
From rat ear samples, sequence verification is carried out.Head comprising expected mutation is built rat and wild-type rats mating, is included
It is expected that the F1 generation heterozygote rat of mutation.F1 generation heterozygote rat, which carries out mating, can be obtained F2 for homozygote rat.
Fig. 2 B shows a F2 for a part of homozygote rat sequencing peak figure, and arrow indicates the position for rejecting sequence
It sets, rectangle frame shows the GCAGCCAGTA of rejecting, it is shown that the gene order of the mouse of sequencing confirmation preparation is consistent with expection.
The mutation leads to frameshit, the translation termination codon after there is frameshit after 16bp after deleting site.
Fig. 2 C shows the protein expression variation of the cerebral cortex and cerebellum of Prrt2-D10.This visible mutation causes in figure
Wild type Prrt2 gene inactivation, additional band may be the mistake that Prrt2 remaining ATG is translated as translation initiation codon
Accidentally truncated-type albumen.
Embodiment 3, light genetic technique stimulation Prrt2 gene inactivated mice construct dyskinesia animal model
1, material
Viral pAAV-hSyn-ChR2 (the H134R)-mcherry of neuron expression ChR2-mCherry by with first biological skill
Art (Shanghai) limited liability company provides.
Standard type stereotaxic apparatus is purchased from RWD Life Science Co., Ltd. Shenzhen.
Virus injection system uses U.S. Parker Picospritzer pressure injection (administration) system.
Blue light 50mW laser is purchased from An Lai software Science and Technology Ltd..
2, virus injection and fiber stub are placed
The Prrt2 gene inactivated mice obtained using embodiment 1 is objective for implementation.
Anesthetized mice (70mg/kg yellow Jackets), mouse is fixed on stereotaxic instrument, is adjusted orientation of head, is cut
It cuts the opening skin, cotton swab wipes bone surface soft tissue, and positioning to coordinates of targets is bored using cranium and answers bone surface to drill in purpose coordinate pair.
In addition, drilling through two holes in forebrain, light heredity fiber stub is auxiliarily fixed using screw.Screw on two screw (specification M1.2
×3mm).PAAV-hSyn-ChR2 (H134R)-mcherry virus injection to cerebellar granule cell layer (such as coordinate AP:-
6.0；ML:0；DV:2.8), 1 μ l of injection volume.Virus makes target cell express PROTEIN C hR2, is that can be activated to open by blue light
Cationic channel protein, such target nerve member can be excited when by blue light illumination, reaches that intervene neuron living
Dynamic purpose.MCherry is coupled above this ChR2, mCherry is fluorescin, can be inspired under the irradiation of green light red
Color fluorescence can indicate the position of ChR2-mCherry expression).
Then fiber stub is placed to virus injection position, smears biogum (3MTMVetbondTMTissue
Adhesive), dental cement (curing liquid denture acrylic self-solidifying artificial tooth base resin II type in Shanghai two) is placed after biogum is dry, etc.
It is solidified to dental cement, operation is completed.
Fig. 3 shows the position of virus injection, and the expression of slice confirmation ChR2-mcherry.
3, blue light stimulates
After injecting virus 3~4 weeks, blue laser is connected, terminal light intensity is adjusted to about 15mW, gives 10s every time
Stimulation, the interval 10s.6 times with internal stimulus can induce Prrt2 inactivated mice lose walking with abnormal posture typical motion hinder
Hinder phenotype.The pattern of 10s stimulation standard-sized sheet or can be stimulated, such as 40Hz, the stimulation of each pulse persistance 10ms with pulse type.
Fig. 4 shows that the light heredity of the homozygote mouse of Prrt2STOP induces the process of phenotype, stimulates preceding mouse normally
Walking and activity, visible apparent ambulatory status is abnormal during stimulation, and hind leg is raised extremely, considerable after stopping stimulation about 1 minute
Mouse movement obstacle is observed, phenotype is to lose walking ability, with abnormal posture.
Embodiment 4 implements cerebral injury building dyskinesia animal model to Prrt2 gene inactivated mice
The Prrt2 gene inactivated mice obtained using embodiment 1 is objective for implementation, not carry out the same of Prrt2 gene inactivation
Kind mouse is as control.
1, it performs the operation
Anesthetized mice (70mg/kg yellow Jackets), mouse is fixed on stereotaxic instrument, is adjusted orientation of head, is cut
It cuts the opening skin, cotton swab wipes bone surface soft tissue, drills above target brain area, and the position of this example drilling is AP:-6.0ML:0.
2, damage stimulation
It is spare that the syringe needle of 1ml specification is truncated to 2mm.Handle postoperative 24 hours or more mouse put behavior observation case
Own activity 1-2 minutes, above-mentioned 2mm needle point piercing cerebellar tissue is then caused into slight brain tissue impairment, puts back to behavior immediately
It is observed in observation case.
As shown in figure 5, Prrt2 wild-type mice does not have obvious abnormal response, but Prrt2STOP mouse goes out in 30 seconds
Now apparent dyskinesia, show as can not normal gait, abdomen patch ground, trunk, four limbs, tail are stiff, acral to tremble
Deng.
Embodiment 5, light genetic technique induction Prrt2 gene inactivation rat construct dyskinesia animal model
1, virus injection and fiber stub are placed
Anesthetized rat (50mg/kg yellow Jackets), rat is fixed on stereotaxic instrument, is adjusted orientation of head, is cut
It cuts the opening skin, cotton swab wipes bone surface soft tissue, and positioning to coordinates of targets is bored using cranium and answers bone surface to drill in purpose coordinate pair.
In addition four holes are drilled through in forebrain in order to use screw that light heredity fiber stub is auxiliarily fixed.Screw on fixed screw (specification
M1.4×3mm).PAAV-hSyn-ChR2-mcherry virus injection to cerebellar granule cell layer (such as coordinate AP:-
11.6ML:0DV:2.7 unit mm), 1 μ l of injection volume.Then fiber stub is placed to virus injection position, smears biogum
(3MTMVetbondTMTissue Adhesive), dental cement, which is placed, after biogum is dry (cures liquid denture acrylic self-solidifying in Shanghai two
Artificial tooth base resin II type), dental cement solidification is waited, operation is completed.
2, blue light stimulates
Annotation virus connected blue laser after 3~4 weeks, adjusts terminal light intensity to about 15mW, gives 10s every time
Stimulation, the interval 10s.6 times with internal stimulus can induce Prrt2 inactivation rat lose walking with abnormal posture typical motion hinder
Hinder phenotype.The pattern of 10s stimulation standard-sized sheet or can be stimulated, such as 40Hz, the stimulation of each pulse persistance 10ms with pulse type.
Fig. 6 shows that the light heredity of the homozygote rat of Prrt2STOP induces the process of phenotype, stimulates preceding rat normally
Rat motor obstacle can be observed in walking and activity, stimulation after about 1 minute, phenotype is to lose walking ability, and forelimb trembles, body
Center of gravity is decreased obviously, abdomen perineum patch ground, with abnormal posture.
Embodiment 6, traumatic brain injury induction Prrt2 gene inactivate the dyskinesia animal model of rat
1, operation and conduit are placed
Anesthetized rat (50mg/kg yellow Jackets), rat is fixed on stereotaxic instrument, is adjusted orientation of head, is cut
It cuts the opening skin, cotton swab wipes bone surface soft tissue, and positioning to coordinates of targets is bored using cranium and answers bone surface to drill in purpose coordinate pair.
In addition four holes are drilled through in forebrain in order to use screw that conduit is auxiliarily fixed.Screw on fixed screw (specification M1.4 × 3mm).?
Conduit is placed in cerebellar cortex (such as coordinate AP:-11.6ML:0DV:2.5 unit mm), stainless steel pipe outer diameter 0.64mm, interior
Diameter 0.45mm.Smear biogum (3MTMVetbondTMTissue Adhesive), placed after biogum is dry dental cement (on
Cure liquid denture acrylic self-solidifying artificial tooth base resin II type in sea two), dental cement solidification is waited, operation is completed.
2, damage stimulation
It is spare that the stainless steel pipe inner core of internal diameter 0.4mm is truncated to and sharpening 3mm longer than conduit.Handle postoperative 24 hours
Above rat puts behavior observation case own activity 1-2 minutes, and above-mentioned conduit inner core is then pierced into cerebellar tissue along conduit and is made
At slight brain tissue impairment, puts back in behavior observation case and observe immediately.As shown in fig. 7, Prrt2 wild-type rats are not obviously transported
Move obstacle, but Prrt2-D10 rat apparent dyskinesia occurred in 1 minute, show as can not normal gait, abdomen
Portion's perineum patch ground, it is acral to tremble.
Embodiment of above is one of preferable embodiment, not limitation of the present invention.It is other any
Made changes, modifications, substitutions, combinations, simplifications without departing from the spirit and principles of the present invention should be equivalent displacement
Mode is included within the scope of the present invention.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
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Claims (17)
1. being used to prepare the kit of dyskinesia animal, which is characterized in that include: in the kit
(1) make the reagent that Prrt2 gene reduces expression or inactivates in Animal genome；
(2) for carrying out the reagent or device of blue laser stimulation to animal brain；Or for carrying out cerebral injury to animal brain
Device.
2. kit as described in claim 1, which is characterized in that (2) in, described swashs for carrying out blue light to animal brain
The reagent or device of light stimulus include: the expression construct for expressing light-operated cationic channel gene, fiber stub, blue laser
Device, stereotaxic apparatus and/or micro-injection pump.
3. kit as described in claim 1, which is characterized in that (2) in, for carrying out the device of cerebral injury to animal brain
Include:
Thin rodlike instrument, preferably: syringe needle, sticking plaster, glass bar；Or
Surgical catheters and the conduit inner core to match with the conduit.
4. kit as described in claim 1, which is characterized in that (1) in, described makes Prrt2 gene in Animal genome
The reagent for reducing expression or inactivation is selected from:
Realize that Prrt2 gene reduces the reagent of expression or inactivation by gene knockout；Or
Realize that Prrt2 gene reduces the reagent of expression or inactivation by insertion foreign gene or genetic fragment.
5. kit as claimed in claim 4, which is characterized in that Prrt2 gene in Animal genome is made to reduce expression or lose
Reagent living is cas9 gene editing reagent.
6. kit as claimed in claim 5, which is characterized in that the animal is mouse, the cas9 gene editing
Reagent knocks in terminator codon after the C666 of the coded sequence of Prrt2 gene.
7. kit as claimed in claim 6, which is characterized in that the gene editing reagent includes:
For the sgRNA of mouse Prrt2, sequence is as shown in SEQ ID NO:2；
Donor plasmid, it includes the sequences as shown in SEQ ID NO:3；
Preferably, the gene editing reagent further include: form the reagent of Cas9mRNA in the cell.
8. kit as claimed in claim 4, which is characterized in that Prrt2 gene in Animal genome is made to reduce expression or lose
Reagent living is TALEN targeted gene disruption reagent.
9. kit as claimed in claim 8, which is characterized in that the animal is rat, the TALEN target gene
It knocks out reagent and deletes 10bp base sequence after the translation initiation codon ATG of Prrt2 gene.
10. kit as claimed in claim 9, which is characterized in that the targeting sequence of the TALEN targeted gene disruption reagent
Column such as SEQ ID NO:4.
11. kit as described in claim 1, which is characterized in that the fiber stub diameter is 0.2 ± 0.1mm, blue light
Laser is BL473T3-050.
12. kit as described in claim 1, which is characterized in that the animal is rodent；Preferably, described is dynamic
Object is mouse.
13. a kind of method for preparing dyskinesia animal, which is characterized in that the described method includes:
(a) Prrt2 gene in Animal genome is reduced by expression or inactivation；
(b) animal obtained to (a) carries out the stimulation of laser blue light or cerebral injury, to obtain dyskinesia animal.
14. method as claimed in claim 13, which is characterized in that in step (b), carrying out the stimulation of laser blue light includes: dynamic
Fiber stub is placed in the cerebellum of object or neighbouring neuronal tissue；It is stimulated 2~20 times using blue laser；Preferably, 6~
15 seconds/time, interval 6~15 carries out the 2nd stimulation.
15. method as claimed in claim 13, which is characterized in that when stimulation, the terminal light intensity of the blue laser is
10~20mW；Preferably 13~17mW.
16. method as claimed in claim 13, which is characterized in that in step (a), make animal using cas9 gene editing reagent
Prrt2 gene reduces expression or inactivation in genome；Or drop Prrt2 gene in Animal genome using Cre and loxp method
Low expression or inactivation.
17. the application of the animal for the dyskinesia that any method of claim 13-16 obtains, for as screening treatment
The drug candidate of neurological disease or the animal model of therapeutic agent；Or for the animal model as study movement obstacle.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107955818A (en) * | 2016-10-17 | 2018-04-24 | 中国科学院上海生命科学研究院 | A kind of method for building up of non-human primate neurological disease animal model and application thereof |
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| CN105112446A (en) * | 2015-06-25 | 2015-12-02 | 中国医学科学院基础医学研究所 | Method for high-efficiency establishment of genetically modified animal model through haploid stem cells |
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| CN105112446A (en) * | 2015-06-25 | 2015-12-02 | 中国医学科学院基础医学研究所 | Method for high-efficiency establishment of genetically modified animal model through haploid stem cells |
| CN106119284A (en) * | 2016-06-27 | 2016-11-16 | 北京维通达生物技术有限公司 | A kind of product for building immunodeficient animals model and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955818A (en) * | 2016-10-17 | 2018-04-24 | 中国科学院上海生命科学研究院 | A kind of method for building up of non-human primate neurological disease animal model and application thereof |
| CN107955818B (en) * | 2016-10-17 | 2021-08-13 | 中国科学院脑科学与智能技术卓越创新中心 | Establishing method and application of non-human primate animal model with neurological diseases |
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