CN106434748A - Development and applications of heat shock induced Cas9 enzyme transgene danio rerio - Google Patents
Development and applications of heat shock induced Cas9 enzyme transgene danio rerio Download PDFInfo
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Abstract
The present invention relates to the technical field of biology, particularly to development and applications of a heat shock induced Cas9 enzyme transgene danio rerio. The present invention firstly provides a Cas9 enzyme expression vector, which utilizes a heat shock induced promoter HSP70 to drive the expression of a downstream Cas9 gene. According to the present invention, the Cas9 enzyme expression vector and Tol2 mRNA are co-injected into wild type danio rerio single cell fertilized egg, and selection is performed to obtain the heat shock induced Cas9 enzyme transgene danio rerio, such that the gene editing research of the CRISPR-Cas9 system in the danio rerio is successfully achieved, and the know-out of the MC4R gene in the transgene danio rerio is firstly achieved; and the Cas9 enzyme expression vector is further suitable for heat shock induced gene knockout, gene knock-in, gene expression modification and other applications of other fishes.
Description
Technical field
The invention belongs to biological technical field is and in particular to a kind of development of heat-shock inducible Cas9 enzyme transgenic zebrafish
And application.
Background technology
Brachydanio rerio (Danio rerio) is belonging to Actinopterygii (Actinopterygii) Cyprinidae (Cyprinidae) short hundredweight
Buddhist nun fish belongs to a kind of bony fish of (Danio), because its side has the equally longitudinal dark blue of an image patch horse striped alternate with silver color
Gain the name.Brachydanio rerio and human gene have 87% high homology it means that with Brachydanio rerio as laboratory animal, the knot obtaining
Fruit in most cases goes for human body, and therefore it is very prominent as the advantage of model organism.Its juvenile fish has breeding soon,
Ovum and the transparent feature of juvenile fish whole body, are commonly used for the monitoring of water environment pollution thing, making of relevant disease model etc..Wherein
The maturation of transgenic technology also gives the bigger prospect that the application of Brachydanio rerio brings.The preparation of transgenic zebrafish generally include with
Under several steps：Build transgenic recombinant expression carrier first, will be with genes of interest using classical microinjection technique
Or the recombinant expression carrier of fluorogene imports in the unicellular germ cell of Brachydanio rerio, enable external source genes of interest or fluorogene
It is incorporated on zebrafish embryo chromosome, and make genes of interest or fluorescent marker gene expression, by PCR or fluorescence microscope
To detect whether exogenous gene is integrated and expressed successfully, thus the chimera of screening and identification transgenic progeny or transgenic.
CRISPR-Cas9 system is successfully transformed into third generation artificial endonucleases, with zinc finger Cobra venom endonuclease (ZFN)
The editor that can be used for various complex genomes the same with class activating transcription factor effector nuclease (TALEN).Its major function
Part includes identifying gRNA the and Cas9 albumen of target site, and gRNA is responsible for identifying target site, Cas9 albumen exercises shearing function, then
Using the nonhomologous end reparation (NHEJ) of body itself, during repairing, introduce mutation to genome.This technology at present
It has been successfully applied to human cell, the genome of Brachydanio rerio and mice and antibacterial is accurately modified, modified types include insertion and lack
Lose mutation, gene site-directed knock in, the disappearance of large fragment.Because its mutation efficiency is high, make simple and low cost, recognized
For being a kind of genome fixed point transformation molecular tool with broad prospect of application.MC4R belongs to the family of G- G-protein linked receptor
Race, is class peptide matters of ventromedial nucleus of hypothalamus secretion, has weight on adjusting homeostasis energy and obesity generation
Act on.By the research of more than ten years, functional study in mammal for the MC4R gene has been compared thoroughly.However, because
Efficient gene edit is lacked, the research of the MC4R gene function of Fish is just at the early-stage in Fish.
Content of the invention
In order to overcome the problem in the presence of prior art, it is an object of the invention to provide a kind of heat-shock inducible Cas9
The manufacture method of enzyme transgenic zebrafish and application.
To achieve these goals and other related purposes, the present invention adopts the following technical scheme that：
A first aspect of the present invention, provides a kind of Cas9 expression of enzymes carrier, including：Cas9 expression of enzymes element and be located at institute
State the transposon Tol2 at Cas9 expression of enzymes element two ends, described Cas9 expression of enzymes element includes heat-shock inducible promoter HSP70
With Cas9 enzyme gene coded sequence.
Preferably, described heat-shock inducible promoter HSP70 contains the sequence as shown in SEQ ID NO.7.
Preferably, described Cas9 enzyme gene coded sequence contains the sequence as shown in SEQ ID NO.8.
The transposon Tol2 being preferably located at described Cas9 expression of enzymes element two ends is included positioned at described Cas9 expression of enzymes unit
Transposon Tol2 and the transposon Tol2 holding positioned at described Cas9 expression of enzymes element 3 ' that part 5 ' is held.
It is preferably located at the transposon Tol2 that described Cas9 expression of enzymes element 5 ' holds to contain as shown in SEQ ID NO.9
Sequence.
It is preferably located at the transposon Tol2 that described Cas9 expression of enzymes element 3 ' holds to contain as shown in SEQ ID NO.10
Sequence.
Preferably, described Cas9 expression of enzymes element also includes fluorescent marker protein sequence.
Preferably, described fluorescent marker protein sequence is connected with Cas9 enzyme gene coded sequence by 2A sequence.
It is further preferred that described 2A sequence contains the sequence as shown in SEQ ID NO.11.
Preferably, described fluorescent marker protein is green fluorescent protein eGFP.
Preferably, described Cas9 expression of enzymes carrier contains the sequence as shown in SEQ ID NO.12.
A second aspect of the present invention, provides use in cultivating Cas9 enzyme transgenic animal for the aforementioned Cas9 expression of enzymes carrier
On the way.
A third aspect of the present invention, provides a kind of Cas9 enzyme transgenic zebrafish cultural method, including：By aforementioned Cas9 enzyme
Expression vector and Tol2mRNA co-injection enter the unicellular germ cell of wild-type zebrafish, and selection-breeding obtains Cas9 enzyme transgenic zebra
Fish.
Preferably, described Tol2mRNA contains the sequence as shown in SEQ ID NO.13.
Preferably, described injection adopts microinjection.
Preferably, described selection includes：Heat-inducible is carried out to the germ cell after injection.
It is further preferred that described selection specifically includes：Germ cell after injection cultivates 24h under the conditions of 28 DEG C,
Then it is incubated 1h under the conditions of 37 DEG C, screening is with fluorescently-labeled fish roe incubation culture to adult fish.
Preferably, described selection also includes being sheerly the screening of Cas9 enzyme transgenic zebrafish,
Express the chimera P0 of fluorescence first in selective fertilization ovum and wild-type zebrafish carries out hybridization and produces heterozygote F1
Generation, the individuality fluorescing in screening offspring；By the single individual and wild type fluorescing in described F1 generation carry out test cross obtain miscellaneous
In fit F2 generation, fluoresce in screening offspring individuality；By the raun fluorescing in heterozygote F2 generation and milter selfing, obtain F3 generation；
Gained F3 with wild type test cross and is counted the situation that fluoresces after the incubation of F4 fish roe for single individual continuation；If F4 fish roe is all
Fluoresce it is determined that its parent F3 is pure lines Cas9 enzyme transgenic zebrafish.
A fourth aspect of the present invention, provides a kind of Cas9 enzyme transgenic zebrafish being obtained by preceding method.
A fifth aspect of the present invention, there is provided described Cas9 enzyme transgenic zebrafish is used in the research of fish gene function
Purposes.
Described fish gene includes but is not limited to MC4R gene.
A kind of a sixth aspect of the present invention, there is provided method of researching fish gene function, including：Will be for fish gene
SgRNA be expelled to pure lines Cas9 enzyme transgenic zebrafish unicellular germ cell in, to knock out fish gene.
A seventh aspect of the present invention, provides a kind of CRISPR-Cas9 gene knockout test kit, and described test kit includes：Before
State Cas9 expression of enzymes carrier and sgRNA.
Compared with prior art, the present invention has the advantages that：
The present invention constructs a kind of Cas9 expression of enzymes carrier first, and described carrier utilizes heat-shock inducible promoter HSP70
To drive downstream Cas9 gene expression.Wild-type zebrafish is entered using described Cas9 expression of enzymes carrier and Tol2mRNA co-injection
Unicellular germ cell, selection-breeding obtains heat-shock inducible Cas9 enzyme transgenic zebrafish, is successfully realized and carries out in Brachydanio rerio
The gene editing research of CRISPR-Cas9 system, and achieve knockout in this transgenic zebrafish for the MC4R gene first.Institute
State that Cas9 expression of enzymes carrier is equally applicable to the heat-shock inducible gene knockout of other Fish, gene knock-in, gene expression are modified
Deng application.
Brief description
Fig. 1 is the Cas9 expression vector pTol2-HSP70-Cas9-2A-eGFP collection of illustrative plates of the present invention.
Fig. 2 is plasmid pTol2-LoxP-CMV-eCFP collection of illustrative plates.
Fig. 3 is plasmid pISceI-HSP70-Cas9-2A-eGFP collection of illustrative plates.
Fig. 4 is MC4R gene insertion and deletion sequencer map, and in Fig. 4, involved each sequence is respectively as SEQ ID NO.14～17 institute
Show.
Fig. 5 is MC4R gene T7E1 testing result electrophoretogram.
Specific embodiment
Before further describing the specific embodiment of the invention it should be appreciated that under protection scope of the present invention is not limited to
State specific specific embodiments；It is also understood that term is specifically concrete in order to describe used in the embodiment of the present invention
Embodiment, rather than in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example,
Generally according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides numerical range it should be appreciated that except non-invention is otherwise noted, holding for two of each numerical range
Between point and two end points, any one numerical value all can be selected for.Unless otherwise defined, used in the present invention all technology and
The same meaning that scientific terminology and those skilled in the art of the present technique are generally understood that.Except concrete grammar used in embodiment, equipment,
Outside material, according to the record of the grasp to prior art for the those skilled in the art and the present invention, can also use and this
Any method of the similar or equivalent prior art of the method described in inventive embodiments, equipment, material, equipment and material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method all using this technology lead
The conventional molecular biology in domain, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniquess of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1 obtains Cas9 expression of enzymes carrier pTol2-HSP70-Cas9-2A-eGFP
Using BamHI and SalI double digestion plasmid vector pTol2-CMV-eGFP (as shown in Figure 2), enzyme action system：10x
Green Buffer 5ul, plasmid vector pTol2-CMV-eGFP 8.8ul (0.284ug/ul), BamHI 2.5ul, SalI
2.5ul, add water and supply 50ul.Reclaim the fragment of 7993bp by agarose gel electrophoresiies, obtain skeleton fragment pTol2.
By BamHI and SalI double digestion plasmid vector pISceI-HSP70-Cas9-2A-eGFP (as shown in Figure 3), enzyme
Cut system：10x Green Buffer 5ul, plasmid vector pISceI-HSP70-Cas9-2A-eGFP 7.7ul (0.321ug/
Ul), BamHI2.5ul, SalI 2.5ul, add water and supply 50ul.After the completion of enzyme action, digestion products are carried out agarose gel electricity
6969bp fragment is reclaimed in swimming, obtains Insert Fragment HSP70-Cas9-2A-eGFP.
Skeleton fragment pTol2 and Insert Fragment HSP70-Cas9-2A-eGFP two fragment are attached, coupled reaction body
System：HSP70-Cas9-2A-eGFP 3ul, pTol2 2ul, 10xBuffer 2ul, T4 ligase 0.2ul, aquesterilisa 12.8ul.
With T4DNA ligase, 16 DEG C connect overnight.
Take connection product 10ul conversion 90ul competent escherichia coli cell DH5 α, gently inhaled with pipettor and beat mixing, ice
After upper incubation 30min, 42 DEG C of thermal shock 90s, it is immediately placed on 2min on ice, add 37 DEG C of LB culture medium 900ul, in 37 DEG C of 180rpm
Shaking table activates 1h, activation bacterium solution 5000rpm centrifugation 3min enrichment, sops up 900ul supernatant, 100ul bacterium solution is applied to ammonia after mixing
On benzyl resistance culture ware, it is inverted overnight incubation in 37 DEG C of incubators.10 single bacterium colonies of random choose are examined using PCR method
Survey, by positive colony amplification culture in 6ml LB fluid medium, 37 DEG C of 250rpm overnight incubation, collect bacterium solution and extract matter
Grain.Plasmid order-checking checking will be obtained, sequencing result is consistent with expection, obtains Cas9 expression of enzymes carrier pTol2-HSP70-Cas9-
2A-eGFP.
As shown in figure 1, Cas9 expression of enzymes carrier pTol2-HSP70-Cas9-2A-eGFP, including：Cas9 expression of enzymes element
And it is located at the transposon Tol2 at described Cas9 expression of enzymes element two ends, described Cas9 expression of enzymes element includes heat-shock inducible and opens
Mover HSP70 and Cas9 enzyme gene coded sequence.
Wherein, heat-shock inducible promoter HSP70 contains the sequence as shown in SEQ ID NO.7.As SEQ ID NO.7 institute
The sequence shown specifically refers to Sequence Listing Part.
Wherein, Cas9 enzyme gene coded sequence contains the sequence as shown in SEQ ID NO.8.As shown in SEQ ID NO.8
Sequence specifically refer to Sequence Listing Part.
Include holding positioned at described Cas9 expression of enzymes element 5 ' positioned at the transposon Tol2 at described Cas9 expression of enzymes element two ends
Transposon Tol2 and the transposon Tol2 holding positioned at described Cas9 expression of enzymes element 3 '.
Wherein, the transposon Tol2 holding positioned at described Cas9 expression of enzymes element 5 ' contains the sequence as shown in SEQ ID NO.9
Row.Sequence as shown in SEQ ID NO.9 specifically refers to Sequence Listing Part.
The transposon Tol2 holding positioned at described Cas9 expression of enzymes element 3 ' contains the sequence as shown in SEQ ID NO.10.As
Sequence shown in SEQ ID NO.10 specifically refers to Sequence Listing Part.
Described Cas9 expression of enzymes element also includes fluorescent marker protein sequence.Described fluorescent marker protein sequence passes through 2A sequence
Row are connected with Cas9 enzyme gene coded sequence.Described 2A sequence contains the sequence as shown in SEQ ID NO.11.As SEQ ID
Sequence shown in NO.11 specifically refers to Sequence Listing Part.
Cas9 expression of enzymes carrier pTol2-HSP70-Cas9-2A-eGFP contains the sequence as shown in SEQ ID NO.12.As
Sequence shown in SEQ ID NO.12 specifically refers to Sequence Listing Part.
Cas9 expression of enzymes vector injection is entered zebra fish fertilized egg by embodiment 2
(1) acquisition of zebra fish fertilized egg
Male and female parent fish is separately pressed 1 by prefecundation:1-2 ratio is put into and is carried out isolation culture in spawning box.Spawning box is placed in 26
Carry out dark incubated overnight, the photoperiod is daytime 14h, dark 10h in DEG C -29 DEG C of isoperibols.Culture terminates, and detaches dividing plate simultaneously
By fenced in for bottom internal layer slant setting on outer layer copulation box, and it is placed in isoperibol, obtain zebra fish fertilized egg.
(2) Cas9 expression of enzymes vector injection is entered zebra fish fertilized egg
Cas9 expression of enzymes carrier pTol2- embodiment 1 built using microinjection instrument and with reference to microinjection
HSP70-Cas9-2A-eGFP (as shown in Figure 1) and Tol2mRNA co-injection enter the unicellular germ cell of Brachydanio rerio of acquisition.Note
Beam system is 10ul, wherein Cas9 expression vector 50ng/ul, and Tol2mRNA 1ul, phenol red 2ul add water and supply 10ul.Take
In 0.6ul injection injection capillary tube, it is loaded in injection instrument, is injected into the animal pole of zebra fish fertilized egg in order.Whole
Individual operating process completes in 45min, is completed with guaranteeing to inject before the First cleavage of cell.
Wherein, described Tol2mRNA contains the sequence as shown in SEQ ID NO.13.Sequence as shown in SEQ ID NO.13
Row specifically refer to Sequence Listing Part.
(3) fluorescent screening and filial generation are cultivated
Germ cell after injection is incubated at 28 DEG C, is incubated 1h in 37 DEG C of thermostat water baths after 24h, then utilizes stereoscopic micro-
Mirror is observed, and the fish roe with green fluorescence for the screening carries out incubation culture to adult fish.1d-4d after screening, described Brachydanio rerio
Embryo cultivates in 26 DEG C of -29 DEG C of isoperibols, obtains juvenile fish；5d-9d juvenile fish feeding yolk water, daily feeding 2 times；10d-
16d, the yolk water that juvenile fish 2 feeding fairy shrimp is ground with filter screen, concrete grammar is first to put into a small amount of fairy shrimp in raising container,
The yolk water that filter screen grinds is added, daily feeding 2 times after 25min-35min；After 17d, juvenile fish feeding fairy shrimp, daily
Feeding 2 times, daily replacing is once breeded fish water.
(4) screen the pure lines Cas9 enzyme transgenic zebrafish of stable heredity
2 monthly age of clip P0 chimera individuality tail fin extracts genomic DNA first, and PCR method detects Cas9 gene order,
PCR program：94℃5min；94 DEG C of 5s, 55 DEG C of 15s, 72 DEG C of 20s totally 35 circulations, 72 DEG C of 5min.Screening PCR result is the positive
P0 Brachydanio rerio hybridized with wild-type zebrafish, fish roe is placed in 37 DEG C of water-baths incubation 1h, then shows in stereoscopic fluorescence
Micro- Microscopic observation fluorescence, the ovum picking out green fluorescence is cultivated to sexual maturity, obtains heterozygote F1 generation.PCR detects F1 generation, sieve
Select the Brachydanio rerio that PCR result is positive to cultivate to sexual maturity, the single individual and wild type in this heterozygote F1 carried out test cross,
After two monthly ages, PCR filters out positive Brachydanio rerio, cultivates to sexual maturity, and this is heterozygote F2.Because heterozygote F2 is from same
Parent's (heterozygote F1 and wild type), therefore all F2 heterozygote genotype are consistent.By the raun in heterozygote F2 with milter certainly
Hand over, in its offspring F3, have 1/4 probability to obtain the homozygote F3 containing Cas9 transgenic zebrafish.After two monthly ages, PCR cuts tail detection sieve
Select all of homozygote and heterozygote, for verifying homozygote F3 therein, the F3 filtering out is continued and wild type test cross PCR
Detection F4 fish roe DNA, if all PCR of F4 fish roe are positive, after 37 DEG C of incubations of fish roe, then Stereo fluorescence microscope observation has entirely
Green fluorescence, then can determine that its parent F3 is pure lines.
The knockout of embodiment 3 Brachydanio rerio MC4R gene
(1) preparation of the determination in MC4R gene targeting site and gRNA
Design MC4R gene targeting site, according to website (http://zifit.partners.org/favicon.ico)
The result that is given selects suitable target practice sequence, the target practice sequence that the present invention selects as shown in SEQ ID NO.1, specially：
GGGGGTGTTTGTGGTGTGCT.
The present invention amplifies the transcription templates of gRNA using the method for PCR.Drawn according to the target practice sequential design selected first
Thing, upstream primer sequence such as SEQ ID NO.2, specially：
TAATACGACTCACTATAGGGGGTGTTTGTGGTGTGCTGTTTTAGAGCTAGAAATAGC；Downstream primer sequence
Arrange as shown in SEQ ID NO.3, specially：AGCACCGACTCGGTGCCAC.
With the plasmid containing gRNA skeleton as template, frame sequence as shown in SEQ ID NO.4, specially：
AGCTTGAAATTAATACGACTCACTATAGGGAGTCTTCAGATCTAACACACAACTCGAGGAAGACATGTTTTAGAGCT
AGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCG.
PCR amplification program is：98℃5min；98 DEG C of 10s, 58 DEG C of 30s, 72 DEG C of 20s totally 35 circulations, 72 DEG C of 5min.PCR
After product detects through 1% agarose gel electrophoresiies, by PCR primer purification kit recovery product.With product after purification it is
Template carries out in vitro transcription, and transcription system is 20ul, product 600ng wherein after purification, T7 transcriptase 1ul, reaction buffer
2ul, dNTP mixture 1ul, plus sterilized water supplies 20ul.37 DEG C of incubation 3h.Transcription product detects through 1% agarose gel electrophoresiies
Afterwards, reclaimed by RNA Purification Kit, obtain gRNA, in -80 DEG C of Refrigerator stores.
(2) external microinjection is verified with knocking out
Prepare germ cell：Select the pure lines Cas9 transgenic zebrafish (detailed in Example 2) of sexually matured stable heredity, 2
1 milter of bar raun, puts into spawning box, and middle insertion lamina of septum pellucidum is by female milter separately.Spawning box is placed in 26 DEG C of -29 DEG C of perseverances
In greenhouse, dark is overnight raised, and the photoperiod is daytime 14h, dark 10h.Microinjection.In morning next day, unplug lamina of septum pellucidum, protect
Hold quiet environment, allow female milter voluntarily chase spawning.After spawning, collect germ cell immediately, noted by microinjection instrument
Penetrate.Injection system is：GRNA 30ng/ul, phenol red 0.5ul, sterilized water supplies 10ul.Injection site is the animal pole of germ cell,
Whole operation process completes in 45min, is completed with guaranteeing to inject before the First cleavage of cell.Knock out checking.Injection
Germ cell afterwards is cultivated at 28 DEG C.After 24h, matched group takes 10, and every group of two experimental grouies take 10 zebra fish fertilized eggs, respectively
Extract DNA, gRNA target practice site both sides select 500bp about primers, primer sequence such as SEQ ID NO.5 with
Shown in SEQ ID NO.6,
SEQ ID NO.5：GACCGCTACATCACAATCT；
SEQ ID NO.6：TTGGCTTCTGAAGGCATAT.
Expanded with this primer pair zebrafish dna, 98 DEG C of 5min；98 DEG C of 10s, 52 DEG C of 20s, 72 DEG C of 20s follow for 35 totally
Ring, 72 DEG C of 5min.T7 Cobra venom endonuclease I (T7E1) has and identifies and cut incomplete pairing DNA, heteroduplex DNA characteristic, because
This carries out mutation checking with this enzyme, and reaction system is：PCR primer 8.5ul, buffer 1ul, after mix homogeneously, moves back in PCR instrument
Fire.Cycle of annealing is：95 DEG C of 5min, 94 DEG C of 2sec, -0.1 DEG C/cycle, 200times, 75 DEG C 1sec, -0.1 DEG C/cycle,
600times, 16 DEG C of 2min.After the completion of annealing, add 0.5ulT7E1 enzyme, 37 DEG C of incubation 30min.Pass through 2% after the completion of incubation
Agarose gel electrophoresiies detect whether to knock out successfully, and result is as shown in Figure 5.Subsequently it is subcloned into sample presentation in sequencing vector to be sequenced simultaneously
It is compared checking with wildtype gene sequence, confirm effective knockout of target gene.Cut tail detect successful fish (F0) with wild
Type hybridizes, and obtains F1 generation heterozygote, is compared with wild type by sequencing, respectively obtains the base of 5,12 and 8 bases of disappearance
Because of type, cause the frameshift mutation of gene.Result is as shown in Figure 4.
Cas9 transgenic zebrafish enables the effective knockout to MC4R gene, illustrates that Cas9 transgenic zebrafish is built into
Work(.Compared with modern technologies, the invention has the beneficial effects as follows：(1) method implementation process is simple and easy to control；(2) method can obtain
Cas9 transgenic zebrafish；(3) gained Brachydanio rerio strain can be the knockout provides convenient of gene；(4) achieve to MC4R gene
Effective knockout.
The above, only presently preferred embodiments of the present invention, not any to the present invention formal and substantial restriction,
It should be pointed out that for those skilled in the art, on the premise of without departing from the inventive method, also can make
Some improvement and supplement, these improve and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when available disclosed above technology contents make a little more
Equivalent variations that are dynamic, modifying and develop, are the Equivalent embodiments of the present invention；Meanwhile, all substantial technological pair according to the present invention
The change of any equivalent variations, modification and differentiation that above-described embodiment is made, all still fall within the scope of technical scheme
Interior.
Claims (10)
1. a kind of Cas9 expression of enzymes carrier, including：Cas9 expression of enzymes element and be located at described Cas9 expression of enzymes element two ends
Transposon Tol2, described Cas9 expression of enzymes element includes heat-shock inducible promoter HSP70 and Cas9 enzyme gene coded sequence.
2. Cas9 expression of enzymes carrier according to claim 1 is it is characterised in that be located at described Cas9 expression of enzymes element two ends
Transposon Tol2 include the transposon Tol2 that holds positioned at described Cas9 expression of enzymes element 5 ' and be located at described Cas9 expression of enzymes unit
The transposon Tol2 that part 3 ' is held.
3. Cas9 expression of enzymes carrier according to claim 1 it is characterised in that described Cas9 expression of enzymes element also include glimmering
Signal protein sequence, described fluorescent marker protein sequence is connected with Cas9 enzyme gene coded sequence by 2A sequence.
4. Cas9 expression of enzymes carrier according to claim 1 it is characterised in that described Cas9 expression of enzymes carrier contain as
Sequence shown in SEQ ID NO.12.
5. as described in Claims 1 to 4 any claim Cas9 expression of enzymes carrier cultivate Cas9 enzyme transgenic animal in
Purposes.
6. a kind of Cas9 enzyme transgenic zebrafish cultural method, including：Will be as described in Claims 1 to 4 any claim
Cas9 expression of enzymes carrier and Tol2mRNA co-injection enter the unicellular germ cell of wild-type zebrafish, and selection-breeding obtains Cas9 enzyme and turns base
Because of Brachydanio rerio.
7. method according to claim 6 is it is characterised in that described selection includes：Germ cell after injection is entered
Row heat-inducible, specifically includes：Germ cell after injection cultivates 24h under the conditions of 28 DEG C, is then incubated 1h under the conditions of 37 DEG C,
Screening is with fluorescently-labeled fish roe incubation culture to adult fish.
8. method according to claim 6 is it is characterised in that described selection also includes being sheerly Cas9 enzyme transgenic speckle
Express the chimera P0 of fluorescence in the screening of horse fish, first selective fertilization ovum and wild-type zebrafish carries out hybridization and produces heterozygote
F1 generation, fluoresce in screening offspring individuality；By the single individual and wild type fluorescing in described F1 generation carry out test cross obtain miscellaneous
In fit F2 generation, fluoresce in screening offspring individuality；By the raun fluorescing in heterozygote F2 generation and milter selfing, obtain F3 generation；
Gained F3 with wild type test cross and is counted the situation that fluoresces after the incubation of F4 fish roe for single individual continuation, if F4 fish roe is all
Fluoresce it is determined that its parent F3 is pure lines Cas9 enzyme transgenic zebrafish.
9. fish gene is used for by the Cas9 enzyme transgenic zebrafish that claim 6～8 any claim methods described obtains
Purposes in the research of function.
10. a kind of method of researching fish gene function, including：SgRNA for fish gene is expelled to by claim 6
In the unicellular germ cell of Cas9 enzyme transgenic zebrafish that～8 any claim methods describeds obtain, to knock out Brachydanio rerio
Corresponding gene.
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