CN105440111B - Pair of transcription activator-like effector nucleases (CTFs), coding sequences and application thereof - Google Patents

Abstract

The invention discloses a transcription activator-like effector nuclease for mediating targeted knockout of an enteritis related gene Ace2, a coding sequence thereof and application thereof. The transcription activator-like effector nuclease contains a pair of transcription activator-like effector proteins and monomers or catalytic subunits of DNA endonuclease fused with the transcription activator-like effector proteins respectively, and has the functions of identifying and cutting two adjacent sites on mouse Ace2 gene respectively, so that Ace2 gene is knocked out efficiently and specifically in a targeted manner, a mouse colitis model is obtained quickly, and good genetic resources are provided for colitis research.

Description

A pair of of transcriptional activation increment effector nuclease and its coded sequence and application

Technical field

The present invention relates to gene engineering technology field, more particularly to a pair of of transcriptional activation increment effector nuclease and its Coded sequence and application.

Background technique

With the arrival of genome times afterwards comprehensively, reverse genetics become the important means that people study specific gene function. The foundation of animal disease model is that people comment for studying pathogenesis, drug target screening and the medicine property of medicine of specified disease The essential tool of valence.Wherein, mouse model have small, feeding management is convenient, easily controllable, Reproduction fastly, The advantages such as use cost is low, are constantly subjected to the favor of researcher.Currently, mouse is cultivated by long-term artificial feeding and selection, It has been bred as more than 1000 kinds of inbred strais and outbreeding system.In so numerous mouse species, inbred mouse due to it genetically High consistency, often good comparability, precision are high, data are uniform, inheritance stability for the data obtained using inbred mouse, therefore It is widely used in medical research.Relatively, outbred mice is because its reproductive capacity is strong, weight in high survival rate and biological study The experimental material wanted.

In recent years, gene targeting is increasingly mature, is the important tool of researcher's directed modification genome password, significantly Simplify the gene functional research of cellular level, bion level.However, in traditional gene targeting dependent cells Homologous recombination process, abiogenous probability are hundred a ten thousandths, lead to the inefficiency of gene targeting；Moreover, beating Targeting vector generally requires to design longer homology arm, increases the difficulty of experiment；Furthermore this technology is built upon stem cell skill It is more demanding to stem cells technology on art.So there are low efficiencys, time-consuming and laborious, technology for traditional gene targeting It is required that the deficiencies of high, application receives great limitation.

In recent years, new gene targeting --- Zinc finger nuclease (ZFNs), transcriptional activation increment effector nucleic acid The short palindrome of enzyme (TALENs) and regular cluster interval repeats (CRISPR/Cas9) and comes out one after another, and greatly improves gene editing Efficiency.These three different technologies have the similar mode of action, by programmable albumen (ZFNs and TALENs) or RNA (CRISPR/Cas9) it specifically identifies target site, corresponding nuclease is guided to shear in target site to DNA, generate DNA double Chain is broken (DBS).Normally, the DNA of fracture can repair DNA by the way of non-homologous end joining (NHEJ), and It is this to repair the missing or insertion that often will cause small fragment, thus lead to the inactivation of target gene；It is alternatively possible to be, having In the presence of recovery template, the reparation of DNA can be carried out according to template DNA.Therefore, these can be advantageously applied to gene The fields such as inactivation, gene knock-in, apparent modification.Wherein, since the modularity of ZFNs is not strong, shear active height, a spy are designed Anisotropic good ZFNs is not easy to, and many gene locis are difficult to find that suitable ZFN target spot, therefore greatly limit ZFNs's Using.Although the design of CRISPR/Cas9 technology is simple, there is higher a possibility that missing the target.In comparison, TALENs has effect The advantages such as rate height, high specificity, cytotoxicity be low, are the ideal methods for establishing gene knockout model.TALENs was from 2011 It since starting rise, has been successfully applied on silkworm, zebra fish, mouse, domestic animal and human cell, these successes Case illustrate TALENs have good several species applicability.

Colitis is a kind of relatively common intestines problem, pathogenic process and heredity, environment and stress etc. because Element is closely bound up, its cause of disease is not fully understood at present.Therefore, colitis animal model is developed, is had a good application prospect.

Gene relevant to colitis is more.Wherein, ACE2 albumen is by adjusting intestinal epithelial cell to color ammonia in food The absorption of acid, and then influence the secretion of antibacterial peptide and the balance of enteric microorganism.Once the function of ACE2 albumen changes, Microbial balance in large intestine can be broken, so as to cause the generation of large intestine verifying.

Currently, researcher has used traditional gene targeting method in inbred mouse, several strains are constructed Colitis mouse model, but its inefficiency are difficult to be quickly obtained a large amount of model mice, to meet subsequent disease machine The research such as reason, drug toxicology, evaluating drug effect.Also, traditional shooting method can introduce marker gene.Some researches show that labels Gene may affect to experimental result, although this saying there is no final conclusion, when constructing model, answer The introducing of marker gene is avoided as far as possible.

Therefore, a kind of efficient, unmarked model building method needs are developed.Meanwhile large intestine all at present Inflammation research all concentrates on inbred mouse, and the building of outbred mice model has not been reported.

Summary of the invention

It is an object of the invention to propose to mediate a pair of of transcription activator of the targeting knockout of colitis related gene Ace2 Sample effector nuclease, coded sequence and its application.The transcriptional activation increment effector nuclease of the application can efficiently, Specifically targeting knockout Ace2 gene.

To achieve this purpose, the present invention adopts the following technical scheme:

In a first aspect, the present invention provides a pair of of polypeptide, wherein a polypeptide has the amino acid sequence as shown in SEQ ID NO:1 It arranges or with amino acid sequence shown in SEQ ID NO:1 with >=90%, preferably >=95%, more preferably >=98%, most preferably >= The amino acid sequence of 99% homology, another polypeptide have the amino acid sequence as shown in SEQ ID NO:2 or with SEQ ID Amino acid sequence shown in NO:1 has >=90%, preferably >=95%, more preferably >=98%, most preferably >=99% amino of homology Acid sequence.

Preferably, the pair of polypeptide respectively has the function of the nucleotide sequence of specific recognition T (N) nA structure, Middle N is any one base in A, G, C or T, and n is any number between 30 to 70；

It is further preferred that the wherein polypeptid specificity in the pair of polypeptide is identified as described in SEQ ID NO:3 Nucleotide, or nucleotide as described in SEQ ID NO:3 through one or more, preferably 2 nucleotide replace derived from core Thuja acid, another polypeptid specificity identify the nucleotide as described in SEQ ID NO:4, or the nucleosides as described in SEQ ID NO:4 Acid replaces derivative nucleotide through one or more, preferably 2 nucleotide.

Second aspect, the present invention provides a pair of of polynucleotides 1, selected from the following group:

(1) a pair of of polynucleotides of a pair of of polypeptide as described in relation to the first aspect are encoded；

(2) it is respectively provided with the nucleotide sequence as shown in SEQ ID NO:5 or with it with >=90%, preferably >=95%, more It is preferred that >=98%, most preferably >=99% nucleotide sequence of homology, and the nucleotide sequence as shown in SEQ ID NO:6 or with It has >=90%, preferably >=95%, more preferably >=98%, most preferably >=99% nucleotide sequence of homology.

The third aspect, the present invention provide a pair of of fusion protein, and a pair of of polypeptide as described in first aspect is cut with DNA respectively Two monomers or two subunits for cutting element merge, and the DNA cutting element needs the poly- of two monomers or two subunits Collection is to exercise DNA cutting function.The pair of fusion protein is transcriptional activation increment effector nucleic acid of the present invention Enzyme (TALEN), hereinafter referred to as TALEN-L and TALEN-R.

Preferably, the pair of fusion protein is respectively provided with the amino acid sequence as shown in SEQ ID NO:7 or and SEQ Amino acid sequence shown in ID NO:7 has >=90%, preferably >=95%, more preferably >=98%, most preferably >=99% homology Amino acid sequence, and the amino acid sequence as shown in SEQ ID NO:8 or with amino acid sequence shown in SEQ ID NO:8 have >= 90%, preferably >=95%, more preferably >=98%, the amino acid sequence of most preferably >=99% homology.

Fourth aspect, the present invention provides a pair of of polynucleotides 2, selected from the following group:

(1) a pair of of polynucleotides such as the pair of fusion protein of the third aspect are encoded；

(2) it is respectively provided with the nucleotide sequence as shown in SEQ ID NO:9 or with it with >=90%, preferably >=95%, more It is preferred that >=98%, most preferably >=99% nucleotide sequence of homology, and the nucleotide sequence as shown in SEQ ID NO:10 or There is >=90%, preferably >=95%, more preferably >=98%, most preferably >=99% nucleotide sequence of homology with it.

5th aspect, the present invention provide a kind of comprising in the pair of polynucleotides 2 of such as fourth aspect more than any one The carrier of nucleotide；Preferably, wherein further including the nucleotide sequence for encoding nuclear localization signal in the upstream of the polynucleotides.

6th aspect, the present invention provide a kind of host cell with the carrier conversion as described in terms of the 5th.

7th aspect, the present invention provide a kind of such as the pair of fusion protein of the third aspect or as fourth aspect is the pair of Application of the polynucleotides 2 in the Ace2 gene target to mouse knocks out.

Eighth aspect, the present invention provide a kind of construction method of mouse colitis model comprising: it will be such as third aspect institute State a pair of of fusion protein or such as the pair of polynucleotides 2 of fourth aspect or respectively containing such as the pair of multicore glycosides of fourth aspect The carrier of acid 2 is transferred to the fertilized eggs of mouse；

Preferably, it will be transcribed in vitro respectively such as the pair of polynucleotides 2 of fourth aspect at corresponding mRNA, later by institute State the fertilized eggs of mRNA injection mouse；It is further preferred that wherein, a pair of of polynucleotides 2 as described in fourth aspect it is respective Upstream further include encode nuclear localization signal nucleotide sequence, by nuclear localization signal is introduced into transcribe after mRNA in.

In a preferred embodiment, the present invention respectively will be as shown in SEQ ID NO:9 and SEQ ID NO:10 Nucleotide sequence is transcribed into corresponding mRNA, and introduces nuclear localization signal NLS during transcribing in vitro, later infuses mRNA The fertilized eggs for penetrating mouse, the integration that this avoids DNA sequence dnas in mouse genome；After mRNA translates into albumen in the cell, Enter nucleus by nuclear localization signal NLS, then identify special aim sequence, and then nuclease FokI is sheared.

9th aspect, the present invention provides a kind of kits comprising a pair of of polypeptide as described in relation to the first aspect, or such as the A pair of of polynucleotides 1 described in two aspects, or a pair of of fusion protein as described in the third aspect, or one as described in fourth aspect To polynucleotides 2, or the carrier as described in terms of the 5th, or the host cell of the conversion as described in terms of the 6th.Of the invention Transcriptional activation increment effector nuclease (TALEN) can efficiently cause target gene --- colitis related gene Ace2 Inactivation, to be quickly obtained mouse colitis model, and pass on and stablize, good heredity is provided provides for the research of colitis Source.

Detailed description of the invention

Fig. 1 be mouse Ace2 gene structure chart and transcriptional activation increment effector nuclease (TALEN) of the present invention Binding site.Grey square frame is the noncoding region of gene, and black box is the code area of gene.

Fig. 2 is the RFLP-DraI qualification result that the mouse obtained after the TALEN mRNA is injected in the embodiment of the present invention.

Fig. 3 is the change that the genome sequence of the mouse obtained after the TALEN mRNA is injected in the embodiment of the present invention. Wherein, red font part is binding site of the TALEN-L and TALEN-R on Ace2 gene, " ♂ " and " ♀ " difference table Show public mouse and female rat, " △ " and "+" respectively indicate the missing and insertion of base, digital representation missing, the base of insertion thereafter Number, " frameshit " indicate frameshift mutation.

Fig. 4 shows the colitis model mice of preparation of the embodiment of the present invention because of weight loss caused by diarrhea；Wherein, " wild Raw type, control " indicates that wild-type mice feeds common drinking water, and " Ace2 is knocked out, control " indicates that Ace2 knock out mice is raised Common drinking water is fed, " wild type, DSS " indicate the drinking water of wild-type mice feeding addition DSS, and " Ace2 is knocked out, and DSS " is indicated The drinking water of Ace2 knock out mice feeding addition DSS." * " indicates P < 0.05, and " * * " indicates P < 0.01.

Fig. 5 is the colon lengths of the colitis model mice of preparation of the embodiment of the present invention；Wherein, " wild type, control " table Show that wild-type mice feeds common drinking water, " Ace2 is knocked out, control " indicates that Ace2 knock out mice feeds common drinking water, " wild type, DSS " indicate the drinking water of wild-type mice feeding addition DSS, and " Ace2 is knocked out, and DSS " indicates Ace2 gene knockout The drinking water of mouse feeding addition DSS." * " indicates P < 0.05, and " * * " indicates P < 0.01.

Fig. 6-1,6-2,6-3,6-4 are respectively " wild type, control " group in the embodiment of the present invention, " Ace2 is knocked out, right According to " group, " wild type, DSS " group and " Ace2 knockout, the colon pathological section of DSS " group mouse；Wherein, the band position " * " indicates scorching The infiltration of property cell；" WT, control " indicate that wild-type mice feeds common drinking water, and " Ace2 is knocked out, control " indicates Ace2 Knock out mice feeds common drinking water, " wild type, DSS " indicate the drinking water of wild-type mice feeding addition DSS, " Ace2 is knocked out, and DSS " indicates the drinking water of Ace2 knock out mice feeding addition DSS.

Specific embodiment

To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing to the present invention make into One step elaborates.Test method without specific conditions in embodiment carries out usually according to normal condition.

Kunming mice used in embodiment is purchased from Guangdong Medical Lab Animal Center.

Embodiment 1, the analysis of the sequence of mouse Ace2 gene and the design in TALEN target practice site

Ncbi database shows that (gene I/D: 70008) overall length 49,077bp, coding are aobvious outside by 19 for mouse Ace2 gene Molecular mRNA, further encodes the protein being made of 805 amino acid residues, and gene structure is as shown in Figure 1.

According to the design feature of mouse Ace2 gene, with Second Exon for TALEN target practice site in the present embodiment, and select Selecting recognition site of the TALEN on mouse Ace2 gene is 5 '-TCACCGAGGAAAATGCCAAGACATTTTTAAACAACTTT AATCAGGAAGCTGA- 3 ', wherein underscore part is respectively TALEN-L and TALEN-R recognition site, i.e. SEQ ID NO.3 With SEQ ID NO.4.

According to variable module NI identification A base, NG identification T base, NN identification G base, the principle of HD identification C base, press According to the method for Golden Gate, the polypeptide in the above-mentioned two site of specific recognition is designed (respectively such as SEQ ID NO:1 and SEQ Shown in ID NO:2) and encoding such polypeptides nucleotide sequence (respectively such as SEQ ID NO:5 and SEQ ID NO:6 institute Show), and in the nucleotide sequence of the sequence of their upstream setting T3 promoter and coding nuclear localization signal NLS, downstream The nucleotide sequence of code nucleic acid enzyme FokI is set.

Being from upstream to downstream successively includes T3 promoter, nucleotide sequence, such as SEQ ID NO:5 or SEQ for encoding NLS Nucleotide sequence shown in ID NO:6 and coding FokI nucleotide sequence total nucleotide sequence such as SEQ ID NO:11 and Shown in SEQ ID NO:12, they correspond respectively to the position the 71-90 in sequence nucleotide sequence, 118-138 nucleotide sequences, 139-2319 nucleotide sequences and 2320-3084 nucleotide sequences；As shown in SEQ ID NO:11 and SEQ ID NO:12 Nucleotide sequence is separately encoded the nucleotide sequence of TALEN-L and TALEN-R with nuclear localization signal NLS.

It is respectively that nucleotide sequence shown in SEQ ID NO:11 and SEQ ID NO:12 is same using AflII and XhoI restriction endonuclease Shi Jinhang digestion, recycling endonuclease bamhi are used as the template being transcribed in vitro.It is transcribed in vitro and is tried using mMESSAGE mMACHINE T3 Agent box (Life technologies, the U.S.) carries out, the condition of in-vitro transcription are as follows: 100ng template is in 37 DEG C, 1u transcriptase Under the conditions of be incubated for 2 hours.Then, reaction solution polyA tailing kit (Epicentre, the U.S.) is carried out plus A is anti- It answers, reaction condition are as follows: 37 DEG C are incubated for 1 hour.Subsequently, the DnaseI of 1u, 37 DEG C of incubations, 15 minutes use are added into reaction solution To digest template DNA.Be stripped finally, transcribing mRNA obtained using the method for phenol chloroform, mRNA be placed in -80 DEG C it is standby With.

The preparation of embodiment 2, Ace2 knock out mice

MRNA prepared by embodiment 1 is admixed together according to the final concentration of each 50ng/uL of TALEN-L and TALEN-R, will Mixed liquor is injected into the cytoplasm of kunming mice fertilized eggs, and injection volume is 1pL to 50pL.Later, the fertilized eggs of survival are moved In the uterus for planting the pregnant mouse of back substitution.By about 21 days period of pregnancys, be born 49 mouse altogether.The tail point for taking mouse, using phenol- The method of chloroform extracts DNA.Whether mouse is practiced shooting and is successfully determined using RFLP-DraI, specifically:

Using following primer, sequence are as follows:

ACE2-F:5’-CTTCTCAGTGCCCAACCCA-3’

ACE2-R:5’-GGATCAGAGCTACAGAGGCAGT-3’

Using mouse DNA as template, carry out PCR amplification, obtain the amplified production of 432bp, the product include TALEN-L and The sequence that TALEN-R is identified, and in DraI endonuclease recognized site between the two.

PCR product is subjected to digestion with DraI restriction endonuclease, if PCR product can be cut completely through, determines that the mouse is Wild-type mice；If PCR product cannot be cut open, illustrate that restriction enzyme site is destroyed by TALEN, determines that the mouse is double equipotentials Knock out mice；If PCR product can be partially cut open, illustrate that in mouse genome a allele is destroyed, Determine the mouse for monoallelic knock-out mice.Through detecting, the digestion result of PCR product is as shown in Fig. 2, from Figure 2 it can be seen that 49 There are 28 in mouse for knock out mice, knocking out efficiency is 57%.

Further, PCR product is connected to pMD18-T carrier, converts bacillus coli DH 5 alpha.Using above-mentioned RFLP- The method of DraI identifies obtained bacterial clone.Then, 6 knockout clones of every mouse picking, carry out Sanger Sequencing.Sequencing result is as shown in figure 3, as seen from Figure 3, sequencing result is consistent with RFLP-DraI result.Particularly, frameshift mutation Individual be considered as final successful knockout mouse.It is important to note that certain individual mices, as 8#, 22#, 23#, 24# and 38# has more genotype, it is considered to be gene chimeric mouse.

The foundation of embodiment 3, colitis mouse model

The public mouse that diallele knocks out is selected from Ace2 knock out mice prepared by embodiment 2, is randomly divided into two A group, every group of 6 mouse: it wherein will feed common drinking water by one group of mouse, and be denoted as " Ace2 is knocked out, control "；Another set is raised The drinking water that 5% dextran sulfate sodium (DSS) is added is fed, " Ace2 knockout, DSS " are denoted as.Meanwhile choosing week identical as mouse is knocked out The Kunming public affairs mouse in age is randomly divided into two groups, every group of 6 mouse: wherein will feed common drinking water by one group of mouse, and be denoted as " wild Raw type, control "；The drinking water of 5% dextran sulfate sodium (DSS) is added in another set feeding, is denoted as " wild type, DSS ".It is raising During feeding, there is the phenomenon that diarrhea in mouse.Also, with the extension of feeding time, diarrhoea degree is also aggravated, or even is occurred The case where hematochezia.

In order to evaluate the diarrhoea degree of mouse, the weight of every mouse is weighed daily, is come with (Wti-Wt0)/Wt0*100% Calculate mouse in the body weight loss of experiment i-th day, as a result as shown in Figure 4.Fig. 4 the result shows that, " Ace2 knock out, DSS " organize mouse Body weight loss more than other groups (P < 0.05), show that the diarrhoea degree of this group of mouse is more serious.

After 7 days administering transgenics, mouse is put to death, takes mouse Colon to measure its length, as a result as shown in Figure 5.Equally Ground, Fig. 5 is the results show that " Ace2 is knocked out, and the colon of the more other groups of mouse of colon of DSS " group mouse is obviously shortened (P < 0.01).

On the other hand, it in order to observe the pathological change of mouse Colon, takes colon to be embedded in paraffin, is cut into 5 μ m-thicks Thin slice, paraffin section are dyed through Hematoxylin-eosin, specifically: slice is dewaxed (dimethylbenzene I15 minutes；Dimethylbenzene II10 points Clock), aquation (absolute alcohol I1-2 minutes；Absolute alcohol II1-2 minutes；95% alcohol 1-2 minutes；80% alcohol 1-2 minutes； Tap water rinses a moment), dyeing (distilled water flushing a moment；Haematoxylin dyeing 10-15 minutes；Tap water rinses a moment；1% salt Acid differentiation 0.5-1 minutes；Flowing water rinses；It redyes 2-5 minutes in 0.5% Yihong；Tap water rinses a moment；95% alcohol I1-2 points Clock；95% alcohol II1-2 minutes；Absolute alcohol I1-2 minutes；Absolute alcohol II1-2 minutes), transparent (dimethylbenzene I5-10 points Clock；Dimethylbenzene II5-10 minutes).Lesion situation on microscopically observation slice.

As a result as shown in fig. 6, Fig. 6 is shown: " Ace2 is knocked out, and there are a large amount of inflammatory cells in the colon of DSS " group mouse Infiltration (region shown in " ↓ " in figure) and mucosal structure serious destruction.In contrast, " wild type, DSS " organize mouse Pathology characterization want light.

These results indicate that using the Ace2 gene of TALEN knock-out mice of the invention, so that mouse makes DSS induction At intestinal tract injury become sensitive, compared with wild-type mice, show even more serious intestinal inflammation, including serious abdomen It rushes down, colon shortens, a large amount of infiltrations of inflammatory cell and the serious destruction of mucosal structure in colonic tissue.It further illustrates, TALEN of the invention can be used for efficiently constructing mouse colitis model, and success is achieved in kunming mice.

Finally, it should be noted that above embodiments are only limited with technical solution of the present invention is illustrated, although passing through Referring to the embodiment of optimization of the invention, invention has been described, but those skilled in the art should manage Solution, can make various changes, without departing from defined by the appended claims to it in the form and details The spirit and scope of the present invention.

Claims (14)

1. a pair of of polypeptide, which is characterized in that wherein a polypeptide has an amino acid sequence as shown in SEQ ID NO:1, and another Polypeptide has the amino acid sequence as shown in SEQ ID NO:2；

Wherein a polypeptid specificity identifies the nucleotide as described in SEQ ID NO:3, another polypeptid specificity identification such as SEQ Nucleotide described in ID NO:4.

2. a pair of of polynucleotides 1, which is characterized in that selected from the following group:

(1) a pair of of polynucleotides of coding a pair of of polypeptide as described in claim 1；

(2) nucleotide sequence as shown in SEQ ID NO:5, and the nucleotide sequence as shown in SEQ ID NO:6 are respectively provided with.

3. a pair of of fusion protein, which is characterized in that by a pair of of polypeptide described in claim 1 respectively with DNA cutting element two A monomer or two subunits merge, and the DNA cutting element needs the aggregation of two monomers or two subunits to exercise DNA Cutting function.

4. fusion protein according to claim 3, which is characterized in that the DNA cutting element is nuclease FokI.

5. fusion protein according to claim 4, which is characterized in that the pair of fusion protein is respectively provided with such as SEQ ID Amino acid sequence shown in NO:7, and the amino acid sequence as shown in SEQ ID NO:8.

6. a pair of of polynucleotides 2, which is characterized in that selected from the following group:

(1) a pair of of polynucleotides such as any one of claim 3-5 the pair of fusion protein are encoded；

(2) nucleotide sequence as shown in SEQ ID NO:9, and the nucleotide sequence as shown in SEQ ID NO:10 are respectively provided with.

7. a kind of carrier comprising any one polynucleotides in a pair of of polynucleotides 2 as claimed in claim 6.

8. carrier according to claim 7, which is characterized in that the upstream of the polynucleotides further include coding nuclear location The nucleotide sequence of signal.

9. a kind of host cell with the carrier conversion of claim 7 or 8.

10. a kind of the pair of fusion protein of any one of such as claim 3-5 or one-to-many nucleosides as claimed in claim 6 Application of the acid 2 in the Ace2 gene target to mouse knocks out.

11. a kind of construction method of mouse colitis model, comprising: will be such as the pair of fusion of any one of claim 3-5 Albumen, as claimed in claim 6 a pair of of polynucleotides 2 or the carrier for containing a pair of of polynucleotides 2 as claimed in claim 6 respectively It is transferred to the fertilized eggs of mouse.

12. according to the method for claim 11, which is characterized in that respectively will a pair of of polynucleotides 2 as claimed in claim 6 Corresponding mRNA is transcribed in vitro into, later by the fertilized eggs of mRNA injection mouse.

13. method according to claim 11 or 12, which is characterized in that wherein, one-to-many nucleosides as claimed in claim 6 The respective upstream of acid 2 further includes the nucleotide sequence for encoding nuclear localization signal, after nuclear localization signal is introduced transcription In mRNA.

14. a kind of kit comprising a pair of polypeptide as described in claim 1, or a pair of of multicore as claimed in claim 2 Thuja acid 1, or a pair of of fusion protein as described in any one of claim 3-5, or one-to-many nucleosides as claimed in claim 6 Acid 2 or carrier as claimed in claim 7 or 8, or the host cell converted as claimed in claim 9.