CN109504708A

CN109504708A - A kind of gene targeting carrier and method of self deletion of selection markers

Info

Publication number: CN109504708A
Application number: CN201811472019.0A
Authority: CN
Inventors: 琚存祥; 赵静; 张明坤; 陶裴裴; 李松; 侯欢欢; 高翔
Original assignee: Jiangsu Collection Pharmacy Biotechnology Co Ltd
Current assignee: Jiangsu Collection Pharmacy Biotechnology Co Ltd
Priority date: 2018-12-03
Filing date: 2018-12-03
Publication date: 2019-03-22

Abstract

The gene targeting carrier and method self deleted the invention discloses a kind of selection markers, it is related to gene modification technical field, which includes following element: recombination site 1, reproduction specificity promoter, recombination enzyme gene, selection markers promoter, riddled basins and recombination site 2；Wherein, reproduction specificity promoter is selected from Prm1 gene promoter, Ddx4 gene promoter, Stra8 gene promoter, AMHR gene promoter or Zp3 gene promoter.Target gene is modified using the gene targeting carrier, during preparing animal model, selection markers self can be deleted in F1 generation, obtain the F1 generation without selection markers, the step of can reducing F1 generation again with the animal mating of expression recombinase to delete selection markers, saves the time of gene targeting removal selection markers.

Description

A kind of gene targeting carrier and method of self deletion of selection markers

Technical field

The present invention relates to gene modification technical field, the gene self deleted in particular to a kind of selection markers is beaten Targeting vector and method.

Background technique

It is to carry out that traditional gene targeting, which is in embryonic stem cell (ES Cell), and targeting vector and target gene pass through together Source recombination occurs segment with target gene and exchanges, and realizes target gene modification.This middle target is the event of extremely low probability, in order to tens of Middle target embryonic stem cell is enriched with and filtered out in ten thousand or even million cell, is needed on targeting vector using Neo, the medicines such as Puro Object resistance screening label, by drug screening, the cell that will can not integrate this kind of drug resistance selection markers is killed, and allows carrying The cell survival of label gets off.These drug selectable markers can enter the genome of Chi-meric mice with embryonic stem cell, and be hereditary to Offspring.Have experiment show the presence of these labels will affect targeted mice phenotype (Colledge et al., 1995； Lantinga-van Leeuwen et al.,2004；Meyers et al.,1998；Olson et al.,1996；van der Hoeven et al., 1996), will affect research, or even draw the wrong conclusion.So being carried out in application gene targeting model Before scientific research, need to remove drug resistance selection markers.

Currently, there are two types of common method drug selectable marker can be removed: 1) being expressed inside middle target embryonic stem cell Recombinase deletes label by the recombining reaction between recombination enzymatic recombination site.On the one hand, it needs to increase primary electricity to turn, The plasmid of expression recombinase is transferred in embryonic stem cell, this transfection procedure can generate shadow to the versatility of embryonic stem cell Ring, so influence the chimeric ability after the cell infusion and later period germline (Germline Transmission, GLT) efficiency；On the other hand, time and human cost also can directly be increased.2) it succeeds after the mouse of middle target, by Chi-meric mice Or the animal mating of F1 generation heterozygote and whole body or reproduction cell specifically expressing recombinase, screening is marked without screening in offspring The offspring of note, which builds, is, but the method can introduce recombinase expressing gene simultaneously, may affect to research, usually sieved Choosing label is deleted after successful offspring, it is also necessary to be bred a generation again, be isolated and do not carry the offspring of recombinase just to build be use In experiment.Therefore, time and human cost can also be greatly increased.

In consideration of it, the present invention is specifically proposed.

Summary of the invention

The purpose of the present invention is to provide the gene targeting carriers that a kind of selection markers self are deleted, and use the gene targeting Carrier modification target gene, during preparing animal model, selection markers self can be deleted in F1 generation, be obtained without screening The F1 generation of label saves base the step of can reduction F1 generation again with the animal mating of expression recombinase to delete selection markers Because of the time for removal selection markers of practicing shooting.

The purpose of the present invention is to provide a kind of methods for constructing genetically modified animal model, construct gene using this method Animal model is modified, during preparing animal model, selection markers self can be deleted in F1 generation, be obtained without selection markers F1 generation, can reduce by F1 generation again with expression recombinase animal mating to delete selection markers the step of, save gene and beat The time of target removal selection markers.

The present invention is implemented as follows:

A kind of gene targeting carrier that selection markers self are deleted comprising following element: recombination site 1, reproduction are special Property promoter, recombination enzyme gene, selection markers promoter, riddled basins and recombination site 2；

Wherein, reproduction specificity promoter is selected from Prm1 gene promoter, Ddx4 gene promoter, Stra8 gene promoter Son, AMHR gene promoter or Zp3 gene promoter.

The promoter that the present invention uses contains more complete transcription factor recognition site and section upstream sequence, than Truncated promoter is compared, and can more effectively realize the transcription of reproduction cell specificity.

Further, in some embodiments of the present invention, the sequence of Prm1 gene promoter such as SEQ ID NO.9 institute Show.

Gene targeting carrier of the invention can specific recognition by that will recombinate enzyme gene, riddled basins and recombinase Recombination site 1 and recombination site 2 jointly building in identical carrier, and using reproduction specificity promoter in reproduction cell Expression characteristic is driven, recombination enzyme gene expression is driven with reproduction specificity promoter, is obtained in this way using the gene targeting carrier Chimeric animal reproduction cell in can express recombination enzyme gene, recombinase can will be located at recombination site 1 and recombination site 2 Between riddled basins delete, i.e., self delete.After chimeric animal hybridizes with wild animal, that is, directly obtain screening Mark the F1 generation deleted.Compared to using existing targeting vector, using carrier of the invention reduce by F1 generation again with The step of animal mating of recombinase is to delete selection markers is expressed, the time of gene targeting removal selection markers is saved.

Further, in some embodiments of the present invention, selection markers promoter is selected from PGK promoter, EM7 starts Son, or both combination；

Preferably, selection markers promoter is the combination of PGK promoter and EM7 promoter.

PGK promoter is the promoter of the wide expression in mammal systemic cell, selection markers can be driven all It is expressed in more mammal cell lines.PGK promoter no activity in the prokaryotes such as bacterium, therefore cannot be driven in bacterium The expression of dynamic resistant gene is unfavorable for targeting vector building.Because building process is also required to resistance screening label, to screen It is enriched with correct carrier.

EM7 promoter is the widely used promoter in prokaryotes, and selection markers can be driven in protokaryons such as bacteriums It is expressed in biology.But EM7 promoter, without activity, cannot drive in eucaryote in the eukaryotic cells such as mammal The expression of resistant gene, therefore cannot be directly used to building drug resistance selection markers, the enrichment and screening of target cell in realization.

PGK/EM7 double-promoter can be used for driving expression of the same gene in protokaryon and eukaryotic system.By PGK and Two promoters of EM7 connect to form double-promoter, can realize same selection markers both in the case where not re-replacing promoter It can express, and can be expressed in eucaryote in prokaryotes, improve the convenience for using the carrier, and do not influence gene Normal expression.

Further, in some embodiments of the present invention, the base sequence of PGK promoter such as SEQ ID NO.1 institute Show.

Further, in some embodiments of the present invention, the base sequence of EM7 promoter such as SEQ ID NO.2 institute Show.

Further, in some embodiments of the present invention, the riddled basins are positive riddled basins；

Preferably, the positive riddled basins are selected from neomycin phosphotransferase gene (neo), hygromycin B phosphoric acid turns Move enzyme gene (hph), xanthine/guanine monophosphate transferase gene (gpt), hypoxanthine phosphoric acid transferase gene (Hprt), Thymidine kinase gene (tk) and puromycin acetyltransferase gene (puro).

Further, in some embodiments of the present invention, the recombinase is selected from iCre recombinase, FLPo recombinase With Dre recombinase；

Wherein, when the recombinase is iCre recombinase, recombination site 1 and recombination site 2 are the site loxP；Cre weight Group enzyme is separated from bacteriophage, and the codon used is still prokaryotes codon.Inventor uses The Cre nucleic acid sequence encoded using mammalian codons, referred to as iCre, the Cre after codon optimization are more properly being fed It is expressed in newborn zooblast, the more original Cre of activity can be improved 2-2.5 times.

When the recombinase is FLPo recombinase, recombination site 1 and recombination site 2 are the site FRT；FLP recombinase is most It is just to be separated from saccharomyces cerevisiae, the temperature of most highly active is 42 DEG C, and the body temperature of mammal is 37 DEG C or so, because This, the recombination activity of FLP is lower.Sequence FLPo after being mutated used in the present invention, is more suitable for recombinating in 37 DEG C of environment, And codon is optimized, it is encoded using the codon of mammal, improves its recombination activity.

When the recombinase is Dre recombinase, recombination site 1 and recombination site 2 are the site rox.

Further, in some embodiments of the present invention, iCre recombinates the base sequence such as SEQ ID of enzyme gene Shown in NO.3.

Further, in some embodiments of the present invention, the amino acid sequence of iCre recombinase such as SEQ ID NO.4 It is shown.

Further, in some embodiments of the present invention, FLPo recombinates the base sequence such as SEQ ID of enzyme gene Shown in NO.5.

Further, in some embodiments of the present invention, the amino acid sequence of FLPo recombinase such as SEQ ID NO.6 It is shown.

Further, in some embodiments of the present invention, Dre recombinates the base sequence such as SEQ ID of enzyme gene Shown in NO.7.

Further, in some embodiments of the present invention, the amino acid sequence of Dre recombinase such as SEQ ID NO.8 It is shown.

Recombinase with above-mentioned sequence is the recombinase after codon optimization, is had in mammalian cells higher Activity.

It should be noted that recombinase used in the present invention can be recombinated with the mutant of above-mentioned iCre recombinase, FLPo The mutant of enzyme or the mutant of Dre recombinase, as long as it has the activity of sequence between deletion recombination site.

Further, in some embodiments of the present invention, recombination site 1 is identical with 2 direction of recombination site.

Further, in some embodiments of the present invention, the gene targeting carrier is in 2 downstream of recombination site It further include having the recombination site 3 not identified by the recombinase, the recombination site 3 is selected from the site loxP, the site FRT and rox Site.

By introducing recombination site 3, in this way, after selection markers, that is, above-mentioned positive selection markers are deleted, in cytogene Recombination site 3 is also remained in group.The identical recombination site 3 in the direction integrated in advance on the site and its upstream and downstream other positions Combination, the inducibility that target fragment may be implemented are deleted, and the animal model obtained in this way can not only be used for conditionity and knock out mould Type, after its animals with the specific expressed recombinase that can identify recombination site 3, both can be obtained in specific cells or The animal model of the regional sequence is knocked out in tissue.It similarly, can be with if the recombination site 3 integrated in advance is opposite direction Realize that the segment of inducibility is inverted, the conditionity for gene is expressed.Such as fluorescent reporter gene or mutated gene.

It should be noted that the classification of recombination site 3 is different from recombination site 2, the recombination of identified recombination site 2 is avoided Enzyme is deleted.For example, when recombinase is FLPo recombinase, recombination site FRT, then recombination site 3 is the site rox or loxP Site.

Further, in some embodiments of the present invention, the sequence of loxP recombination site such as SEQ ID NO.10 institute Show.

Further, in some embodiments of the present invention, the sequence of FRT recombination site such as SEQ ID NO.11 institute Show.

Further, in some embodiments of the present invention, the sequence of rox recombination site such as SEQ ID NO.12 institute Show.

Further, in some embodiments of the present invention, the structure of the gene targeting carrier be linear structure or Ring structure.

Further, in some embodiments of the present invention, the upstream of recombination site 1 be also connected with 5 ' end it is homologous Arm, the downstream of recombination site 2 are also connected with 3 ' end homology arms.

It should be noted that the sequence of 5 ' end homology arms and 3 ' end homology arms can be according to the target gene wait knock out or knock in The upstream and downstream sequence design in site.

Further, in some embodiments of the present invention, the downstream connection of 3 ' end homology arms has negative selection to mark base Cause.

Further, in some embodiments of the present invention, negative selection marker gene is selected from DTA or HSV-TK.

On the other hand, the present invention provided a kind of method for constructing genetically modified animal model comprising following steps:

The gene targeting carrier that above-mentioned selection markers self are deleted imports animal embryonic stem cell；

It after identified correct embryonic stem cells to blastaea, is transplanted in the female uterus of false pregnancy, obtains Chimera；

Chimera is mated with wild animal, obtains the F1 generation animal model of selection markers deletion.

Genetically modified animal model is constructed using this method, during preparing animal model, selection markers can be in F1 For self delete, obtain without selection markers F1 generation, can reduce in this way by F1 generation again with expression recombinase animal mating with The step of deleting selection markers saves the time of gene targeting removal selection markers, improves efficiency.

Further, in some embodiments of the present invention, the animal is non-human mammal.

Further, in some embodiments of the present invention, the non-human mammal be selected from mouse, rat, horse, Ox, sheep, pig, monkey and ape.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

The structural schematic diagram for the gene targeting carrier that the selection markers that Fig. 1 is 1-4 of the embodiment of the present invention self are deleted.

Fig. 2 is that the structural schematic diagram of the rosa26-DDSDC1 targeting vector constructed in experimental example of the present invention and its digestion are tested Demonstrate,prove result.

Fig. 3 is that the structural schematic diagram of the rosa26-DDSDC2 targeting vector constructed in experimental example of the present invention and its digestion are tested Demonstrate,prove result.

Fig. 4 is that the structural schematic diagram of the rosa26-DDSDC3 targeting vector constructed in experimental example of the present invention and its digestion are tested Demonstrate,prove result.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

The structure for the gene targeting carrier that selection markers provided in this embodiment self are deleted is as shown in figure 1 shown in A, the gene Targeting vector has following sequentially connected Expression element:

Recombination site 1, reproduction specificity promoter, recombination enzyme gene, selection markers promoter, riddled basins and again Group site 2；

Wherein, recombination site 1 and recombination site 2 are loxP recombination site (SEQ ID NO.10), two recombination sites Direction is identical, and reproduction specificity promoter is Prm1 gene promoter (SEQ ID NO.9), which has Round spermatid rank Section specifically expressing characteristic, recombination enzyme gene are that the Cre through codon optimization recombinates enzyme gene, that is, iCre (SEQ ID NO.3), sieve Choosing label promoter is the combination of PGK promoter (SEQ ID NO.1) and EM7 promoter (SEQ ID NO.2), PGK promoter Positioned at EM7 promoter upstream, riddled basins Neo has ployA downstream.

The gene targeting carrier of the present embodiment is named as DDSDC1.

The gene targeting carrier can synthesize to obtain by way of gene chemical synthesis.

When carrying out gene modification using the gene targeting carrier, it can be held according to target gene site 5 ' and 3 ' terminal sequences, it can be 1 upstream of recombination site of the gene targeting carrier connects 5 ' homology arms, 2 downstream connection of recombination site, 3 ' homology arm.

Embodiment 2

The structure for the gene targeting carrier that selection markers provided in this embodiment self are deleted is as shown in figure 1 shown in B, the gene Targeting vector, which has, is sequentially connected Expression element as follows:

Recombination site 1, reproduction specificity promoter, recombination enzyme gene, selection markers promoter, riddled basins, again Group site 2 and recombination site 3；

Wherein, recombination site 1 and recombination site 2 are FRT recombination site (SEQ ID NO.11), the side of two recombination sites To identical, reproduction specificity promoter is Prm1 gene promoter (SEQ ID NO.9), and recombination enzyme gene is through codon optimization FLP recombinate enzyme gene, that is, FLPo (SEQ ID NO.5), selection markers promoter be PGK promoter (SEQ ID NO.1) and The combination of EM7 promoter (SEQ ID NO.2), PGK promoter are located at EM7 promoter upstream, and riddled basins are Neo under it Downstream belt has ployA, and recombination site 3 is loxP recombination site.

The gene targeting carrier of the present embodiment is named as DDSDC2.

Embodiment 3

The structure for the gene targeting carrier that selection markers provided in this embodiment self are deleted is as shown in figure 1 shown in C, the gene Targeting vector, which has, is sequentially connected Expression element as follows:

Wherein, recombination site 1 and recombination site 2 are rox recombination site (SEQ ID NO.12), the side of two recombination sites To identical, reproduction specificity promoter is Prm1 gene promoter (SEQ ID NO.9), and recombination enzyme gene is Dre recombinase base Because of (SEQ ID NO.7), selection markers promoter is PGK promoter (SEQ ID NO.1) and EM7 promoter (SEQ ID NO.2 combination), PGK promoter are located at EM7 promoter upstream, and riddled basins Neo has ployA, recombination downstream Site 3 is loxP recombination site.

The gene targeting carrier of the present embodiment is named as DDSDC3.

Embodiment 4

The structure for the gene targeting carrier that selection markers provided in this embodiment self are deleted is as shown in figure 1 shown in D, the gene Targeting vector has following sequentially connected Expression element:

Wherein, recombination site 1 and recombination site 2 are rox recombination site, and the direction of two recombination sites is identical, and reproduction is special Specific Promoters are Prm1 gene promoter (SEQ ID NO.9), are recombinated enzyme gene Dre recombinase (SEQ ID NO.7), screening Label promoter is the combination of PGK promoter (SEQ ID NO.1) and EM7 promoter (SEQ ID NO.2), PGK promoter position In EM7 promoter upstream, riddled basins Neo has ployA downstream.

The gene targeting carrier of the present embodiment is named as DDSDC3.1.

Experimental example 1

Test self deletion efficiency of the gene targeting carrier of above-described embodiment 1-3

(1) it is constructed using conventional molecule clone technology and RED/ET methods of homologous recombination (Liu et al., 2003) The targeting vector in the site mouse Rosa26.

In addition, being easy when in building using PCR amplification preparation long segment because DDSDC marks bigger, about 5400bp Mutation is generated, to avoid generating mutation in amplification, after the completion of building, needs that segment is sequenced.On the other hand, it is also necessary to DDSDC marks suitable position on two sides and targeting vector to place specific restriction enzyme site, to be cloned on targeting vector.It is logical Often, gene targeting carrier length is above 10000bp, belongs to bigger segment, and the digestion joint efficiency between large fragment is logical Chang Bugao, and influenced by restriction enzyme site, the end that different digestions generates is different, toughness end peace end, connection Complexity is also different.For convenience of building targeting vector, avoids PCR amplification from introducing mutation, beaten in the gene of embodiment 1-3 In the structure basis of targeting vector, attL1, attL2 recombination site, by attL1-DDSDC- are added on the outside of DDSDC label This structure of attL2 is inserted into a carrier, as fixed tool carrier.When needing to be inserted into DDSDC to targeting vector, only It needs first to recombinate a small resistance marker in targeting vector, introduces the two sites attR1-attR2, then will practice shooting and carry The tool carrier of body and above-mentioned carrying attL1-DDSDC-attL2 is put together, and LR is passed throughRecombinase DDSDC mark group, can be efficiently attached to the target practice for carrying attR1 and attR2 by (Invitrogen Products) catalysis On carrier, loaded down with trivial details PCR amplification and digestion connection are eliminated.

It will be inserted into PL253 carrier framework comprising Rosa26 gene loci and its upstream and downstream homology arm segment, and recombinate carrying After the resistance marker of attR1-attR2, it can react to obtain the targeting vector in the site Rosa26 by through above-mentioned Gateway, and I-CeuI single endonuclease digestion site is added in 5 ' homology arm upstreams, in 3 ' homology arm downstreams addition MC1-DTA negative selection label, targeting vector It is respectively designated as Rosa26-DDSDC1, Rosa26-DDSDC2 and Rosa26-DDSDC3.

The structure and digestion verification screenshot of obtained Rosa26 targeting vector are as in Figure 2-4,

In Fig. 2, restriction enzyme title and stripe size are as follows:

ApaLI:7883,7132,3497,1246,298；

BglII:9318,4719,3135,1367,769,748；

BamHI:7027,5894,3052,1872,1832,379。

In Fig. 3, restriction enzyme title and stripe size are as follows:

BglII:9318,4753,3135,2379,748；

EcoRV:10244,5211,2309,1559,574,436；

SpeI:11290,5370,2560,572,480,61。

In Fig. 4, restriction enzyme title and stripe size:

BglII:9318,4753,3135,1503,748,570,76；

EcoRV:10244,4979,2309,1559,574,436；

SpeI:11288,5368,2332,572,480,61。

The results show that Rosa26-DDSDC1, Rosa26-DDSDC2 and Rosa26-DDSDC3 targeting vector construct successfully, Sequence is identical as design.

(2) ES cell screening

Using I-Ceu I (Cat#R0699L, NEB) linearization for enzyme restriction targeting vector, through phenol: chloroform purifying, ethyl alcohol DdH is dissolved in after precipitating₂In O, electricity is gone to practices shooting from the W4 ES Cell line of 129S6/SvEvTac strain, passes through After G418 screening, the ES of 96 G418 resistances of each picking is cloned, and is extracted DNA and is carried out LR-PCR identification.Select LR-PCR identification just True clone carries out Q-PCR and Southern blot identification.

It the results are shown in Table 1.

1 DDSDC of table marks target practice result statistics

A.LR-PCR detects positive colony number/LR-PCR detection clone's sum；

B.Q-PCR detects positive colony number/Q-PCR detection clone's sum；

C.Southern detects positive colony number/Southern detection clone's sum.

96 resistance clones of each picking extract genomic DNA, and using the primer across homology arm, (i.e. a primer is placed on On genome on the outside of homology arm, another primer is placed on the exogenous sequences in insertion genome, i.e., on DDSDC label) it carries out Whether long range PCR (LR-PCR) identifies both ends by being pre-designed generation homologous recombination, rosa26-DDSDC1 positive rate respectively For 23.9% (23/96), rosa26-DDSDC2 positive rate is 19.8% (19/96), and rosa26-DDSDC3 positive rate is 7.3% (7/96)。

It selects 6 LR-PCR and identifies correct ES clonal expansion, extract genomic DNA and (fluorescence is fixed in real time using Q-PCR Measure PCR) analysis chromosome G banding, identify whether the caryogram (chromosome number) of ES cell is correct, and the ES cell of chromosome abnormalities is not Gene modification offspring that can be hereditary can be generated.Rosa26-DDSDC1, rosa26-DDSDC2 and rosa26- as the result is shown The correct clone's number of Q-PCR identification of DDSDC3 is respectively 4 (4/6), and 5 (5/6) and 6 (6/6).

Long range PCR (LR-PCR) is that the method for carrying out PCR amplification using across homology arm primer determines whether to occur correctly Recombination, the clone to ensure to filter out occur in the genome correctly recombinate, inventor select second method Southern blot is identified again.Southern blot is nucleic acid hybridization, and detailed process is, first using being located at Restriction enzyme site on the outside of homology arm and above insertion exogenous sequences cuts genomic DNA, by digestion products through Ago-Gel electricity Swimming is separated, and is transferred on nylon membrane, and one section of specific sequence being located on the outside of homology arm on genome is selected to be used as spy Needle hybridizes after digoxin with the digestion DNA product on nylon membrane on label, and the chemiluminescence finally by detection Gaoxin label is believed Number, specific electrophoretic band position is obtained, the clone correctly recombinated is judged whether it is.Inventor has selected three above project Q- Correctly clone carries out Southern blot identification, rosa26-DDSDC1, rosa26-DDSDC2 and rosa26- for PCR identification It is respectively 3 (3/6), 4 (4/6) and 6 (6/6) that the identification of DDSDC3, which is correctly cloned,.

It chooses target clone in 2 identifications correctly and is injected into C57BL/6J-Tyrc-2J blastaea, the ICR for being transplanted to false pregnancy is female Mouse intrauterine, to obtain Chi-meric mice.

(3) it injects and breeds

The C57BL/6J female mice of 4 week old is selected to carry out super row, every intraperitoneal injection 5IU PMSG is injected intraperitoneally after 48 hours 5IU HCG is simultaneously mated with the 12 week old hero mouse singly let off 3 days, examines bolt after 72 hours, will be seen that bolt female mice is taken out, is denoted as E0.5 days. See that bolt takes E2.5 days embryos two days later, in vitro culture is overnight to after E3.5 days embryo (blastaea), by ready targeted ES clones Be injected in the blastocoele of embryo, the blastaea after injection is placed on restored in M16 culture solution after, can migrate to and see bolt 2.5 It ICR strain false pregnancy female mice intrauterine.After 18-20 days, Chi-meric mice birth selects the positive Male chimeras mouse of identified for genes It is returned with C57BL/6J female mice, obtains F1 generation heterozygote.

When using traditional selection markers (adding loxP, the site FRT or rox in both ends), need by F0 generation or F1 generation it is small Mouse mates with the tool mouse for expressing specific recombinase, to obtain the offspring that label is deleted, according to having there is experience sum number evidence, screens mark Remember that the tool mouse and recombination system of deletion efficiency and selection are related, the recombination expression of enzymes region of different promoter drivings and table Up to intensity difference, the ability of difference recombination enzymatic recombination is also different.It using from the purpose for deleting label is obtained in F1 generation Selection markers and the successful mouse of gene modification are not carried, are the different DDSDC label of determination from deletion efficiency, inventor detects Residual/deletion situation of selection markers, the results are shown in Table 2, DDSDC1, DDSDC2 and DDSDC3 and all detects completely in F1 generation Delete the F1 generation of selection markers, it was demonstrated that this 3 kinds labels of DDSDC1, DDSDC2 and DDSDC3 that we develop can be in Chi-meric mice This self deletion in the process of F1 generation is bred, but deletion efficiency is different.From the point of view of the data obtained, the deletion efficiency of DDSDC1 Highest detects that 22 have been deleted label (the remaining offspring of label is not detected) completely in 63 F1 generations；DDSDC3 is 99 Detect that 35 are deleted label completely in F1 generation, 3 labels remain, and deletion efficiency is suitable with DDSDC1；DDSDC2 is at 90 Detect that 8 are deleted label completely in F1 generation, 29 still with remaining selection markers.In terms of result, the deletion of FLP/FRT Efficiency is lower, and that deletes completely in GLT offspring only accounts for 21.6% (8/37), Cre/loxP and Dre/rox system-kill efficiency It is all very high, respectively reach 100% (22/22) and 92.1% (35/38).

2 DDSDC of table label deletes Efficiency Statistics certainly

A. it mixes: the mixed type that " label residual " and " label is deleted " two kinds of genotype coexist.

When making genetically modified animal model with traditional ES shooting method, homologous recombination is extremely low probability event, in order to Middle target clone is screened, needs to add medicine sieve label (such as: Neo, Puro etc.) on targeting vector, passes through antibiotic-screening richness Collection carries the ES cell of targeting vector, so that middle target clone be made to be easier to be detected.However, more and more researchers It was found that stay in target gene medicine sieve label target gene function can be had an impact and generate phenotype (Colledge et al., 1995；Lantinga-van Leeuwen et al.,2004；Meyers et al.,1998；Olson et al.,1996； Van der Hoeven et al., 1996), cause experimental result increasingly complex, or even draw the wrong conclusion.Therefore, at present Countermeasure be medicine sieve label two sides addition specific recombination site (such as loxP or FRT), obtain targeted ES clones or After mouse, by transfection recombinase expression plasmid or with expression recombinase the methods of tool mouse breeding line mating, through Cre/loxP Or medicine sieve label is deleted in FLP/FRT recombination.However, the plasmid for expressing recombinase need to be turned if selection removes label in the ES stage Dye is into targeted ES cells, this operation needs to spend time, manpower and cost, it is even more important that transfection procedure is to ES cell Totipotency have a negative impact, reduce its each system's heredity GLT (Germline Transmission) potentiality.Another option is that The mouse for carrying medicine sieve label is first obtained, then the tool mouse for expressing recombinase in tape label mouse and whole body or reproduction cell is handed over Match, obtain the offspring of genetic modification Yu the recombinase Gene Double positive, realizes that label is deleted in this double positive offsprings.It is aobvious and easy See, this method needs to increase 1-2 for mouse breeding cycle, and the expression region of recombinase and efficiency is simultaneously in many tool mouse Undesirable, post-coitum would generally be marked the offspring of " part removes ", i.e., medicine selection markers are only deleted in the cell of part, because This, shown in identified for genes drug sieve label delete and residual genotype admixture, need to by this mouse genotypes again with Tool mouse or wild type mate a generation, could obtain complete deletion drug labelling offspring (Buchholz et al., 1998； Meyers et al.,1998)。

And the gene targeting carrier for using selection markers provided in an embodiment of the present invention self to delete, germline mistake Specifically expressed promoter expresses recombinase in journey, can be returned in succeeding generations in Chi-meric mice and realize self deleting for selection markers It removes, is bred without additional cell transfecting and mouse, can be obtained the F1 generation heterozygote without medicine sieve label.For convenience of structure It builds, we are added to the site attL1 and attL2 at targeting vector both ends, can pass through a step LRClone beats gene Targeting vector is added to skeleton carrier.It is provided in an embodiment of the present invention relative to the gene targeting method for using common selection markers The gene targeting carrier that selection markers self are deleted can will obtain the time advance 1-2 generation (3-6 of removal medicine sieve label heterozygote Month), without changing production procedure, extra cost is not generated, deletion efficiency is high.Although some researches show that tetraploid fusion (Wen Et al., 2014), the technologies such as 8-cell injection (Dechiara et al., 2009) can also be accelerated to obtain the process of heterozygote, But the These characteristics for the gene targeting carrier that selection markers provided in an embodiment of the present invention self are deleted make it have highly significant Advantage, if being combined with tetraploid fusion technology and ROSI (injection of circle essence) (Ogonuki et al., 2009) technology, it will into One step shortens the time for obtaining heterozygote.

In addition, in testing it was found that the deletion efficiency of different recombination system has apparent difference, Cre/loxP and The deletion efficiency of Dre/rox system is significantly higher than FLP/FRT system, according to experimental result, it is proposed that preferential selection uses The DDSDC1 and DDSDC3 of Cre/loxP and Dre/rox system repair the extremely complicated gene that two kinds of recombination systems cannot achieve The DDSDC2 based on FLP/FRT system may be selected in decorations.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

SEQUENCE LISTING

<110>Jiangsu treasury Yao Kang Biotechnology Co., Ltd

<120>gene targeting carrier and method that a kind of selection markers self are deleted

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 508

<212> DNA

<213>artificial sequence

<400> 1

ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 60

gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 120

taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 180

agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 240

agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 300

cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 360

aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 420

gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 480

ctcttcctca tctccgggcc tttcgacc 508

<210> 2

<211> 66

<212> DNA

<213>artificial sequence

<400> 2

gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac 60

taaacc 66

<210> 3

<211> 1056

<212> DNA

<213>artificial sequence

<400> 3

atggtgccca agaagaagag gaaagtctcc aacctgctga ctgtgcacca aaacctgcct 60

gccctccctg tggatgccac ctctgatgaa gtcaggaaga acctgatgga catgttcagg 120

gacaggcagg ccttctctga acacacctgg aagatgctcc tgtctgtgtg cagatcctgg 180

gctgcctggt gcaagctgaa caacaggaaa tggttccctg ctgaacctga ggatgtgagg 240

gactacctcc tgtacctgca agccagaggc ctggctgtga agaccatcca acagcacctg 300

ggccagctca acatgctgca caggagatct ggcctgcctc gcccttctga ctccaatgct 360

gtgtccctgg tgatgaggag aatcagaaag gagaatgtgg atgctgggga gagagccaag 420

caggccctgg cctttgaacg cactgacttt gaccaagtca gatccctgat ggagaactct 480

gacagatgcc aggacatcag gaacctggcc ttcctgggca ttgcctacaa caccctgctg 540

cgcattgccg aaattgccag aatcagagtg aaggacatct cccgcaccga tggtgggaga 600

atgctgatcc acattggcag gaccaagacc ctggtgtcca cagctggtgt ggagaaggcc 660

ctgtccctgg gggttaccaa gctggtggag agatggatct ctgtgtctgg tgtggctgat 720

gaccccaaca actacctgtt ctgccgggtc agaaagaatg gtgtggctgc cccttctgcc 780

acctcccaac tgtccacccg ggccctggaa gggatctttg aggccaccca ccgcctgatc 840

tatggtgcca aggatgactc tgggcagaga tacctggcct ggtctggcca ctctgccaga 900

gtgggtgctg ccagggacat ggccagggct ggtgtgtcca tccctgaaat catgcaggct 960

ggtggctgga ccaatgtgaa catagtgatg aactacatca gaaacctgga ctctgagact 1020

ggggccatgg tgaggctgct cgaggatggg gactga 1056

<210> 4

<211> 351

<212> PRT

<213>artificial sequence

<400> 4

Met Val Pro Lys Lys Lys Arg Lys Val Ser Asn Leu Leu Thr Val His

1 5 10 15

Gln Asn Leu Pro Ala Leu Pro Val Asp Ala Thr Ser Asp Glu Val Arg

20 25 30

Lys Asn Leu Met Asp Met Phe Arg Asp Arg Gln Ala Phe Ser Glu His

35 40 45

Thr Trp Lys Met Leu Leu Ser Val Cys Arg Ser Trp Ala Ala Trp Cys

50 55 60

Lys Leu Asn Asn Arg Lys Trp Phe Pro Ala Glu Pro Glu Asp Val Arg

65 70 75 80

Asp Tyr Leu Leu Tyr Leu Gln Ala Arg Gly Leu Ala Val Lys Thr Ile

85 90 95

Gln Gln His Leu Gly Gln Leu Asn Met Leu His Arg Arg Ser Gly Leu

100 105 110

Pro Arg Pro Ser Asp Ser Asn Ala Val Ser Leu Val Met Arg Arg Ile

115 120 125

Arg Lys Glu Asn Val Asp Ala Gly Glu Arg Ala Lys Gln Ala Leu Ala

130 135 140

Phe Glu Arg Thr Asp Phe Asp Gln Val Arg Ser Leu Met Glu Asn Ser

145 150 155 160

Asp Arg Cys Gln Asp Ile Arg Asn Leu Ala Phe Leu Gly Ile Ala Tyr

165 170 175

Asn Thr Leu Leu Arg Ile Ala Glu Ile Ala Arg Ile Arg Val Lys Asp

180 185 190

Ile Ser Arg Thr Asp Gly Gly Arg Met Leu Ile His Ile Gly Arg Thr

195 200 205

Lys Thr Leu Val Ser Thr Ala Gly Val Glu Lys Ala Leu Ser Leu Gly

210 215 220

Val Thr Lys Leu Val Glu Arg Trp Ile Ser Val Ser Gly Val Ala Asp

225 230 235 240

Asp Pro Asn Asn Tyr Leu Phe Cys Arg Val Arg Lys Asn Gly Val Ala

245 250 255

Ala Pro Ser Ala Thr Ser Gln Leu Ser Thr Arg Ala Leu Glu Gly Ile

260 265 270

Phe Glu Ala Thr His Arg Leu Ile Tyr Gly Ala Lys Asp Asp Ser Gly

275 280 285

Gln Arg Tyr Leu Ala Trp Ser Gly His Ser Ala Arg Val Gly Ala Ala

290 295 300

Arg Asp Met Ala Arg Ala Gly Val Ser Ile Pro Glu Ile Met Gln Ala

305 310 315 320

Gly Gly Trp Thr Asn Val Asn Ile Val Met Asn Tyr Ile Arg Asn Leu

325 330 335

Asp Ser Glu Thr Gly Ala Met Val Arg Leu Leu Glu Asp Gly Asp

340 345 350

<210> 5

<211> 1299

<212> DNA

<213>artificial sequence

<400> 5

atggctccta agaagaagag gaaggtgatg agccagttcg acatcctgtg caagaccccc 60

cccaaggtgc tggtgcggca gttcgtggag agattcgaga ggcccagcgg cgagaagatc 120

gccagctgtg ccgccgagct gacctacctg tgctggatga tcacccacaa cggcaccgcc 180

atcaagaggg ccaccttcat gagctacaac accatcatca gcaacagcct gagcttcgac 240

atcgtgaaca agagcctgca gttcaagtac aagacccaga aggccaccat cctggaggcc 300

agcctgaaga agctgatccc cgcctgggag ttcaccatca tcccttacaa cggccagaag 360

caccagagcg acatcaccga catcgtgtcc agcctgcagc tgcagttcga gagcagcgag 420

gaggccgaca agggcaacag ccacagcaag aagatgctga aggccctgct gtccgagggc 480

gagagcatct gggagatcac cgagaagatc ctgaacagct tcgagtacac cagcaggttc 540

accaagacca agaccctgta ccagttcctg ttcctggcca cattcatcaa ctgcggcagg 600

ttcagcgaca tcaagaacgt ggaccccaag agcttcaagc tggtgcagaa caagtacctg 660

ggcgtgatca ttcagtgcct ggtgaccgag accaagacaa gcgtgtccag gcacatctac 720

tttttcagcg ccagaggcag gatcgacccc ctggtgtacc tggacgagtt cctgaggaac 780

agcgagcccg tgctgaagag agtgaacagg accggcaaca gcagcagcaa caagcaggag 840

taccagctgc tgaaggacaa cctggtgcgc agctacaaca aggccctgaa gaagaacgcc 900

ccctacccca tcttcgctat caagaacggc cctaagagcc acatcggcag gcacctgatg 960

accagctttc tgagcatgaa gggcctgacc gagctgacaa acgtggtggg caactggagc 1020

gacaagaggg cctccgccgt ggccaggacc acctacaccc accagatcac cgccatcccc 1080

gaccactact tcgccctggt gtccaggtac tacgcctacg accccatcag caaggagatg 1140

atcgccctga aggacgagac caaccccatc gaggagtggc agcacatcga gcagctgaag 1200

ggcagcgccg agggcagcat cagatacccc gcctggaacg gcatcatcag ccaggaggtg 1260

ctggactacc tgagcagcta catcaacagg cggatctga 1299

<210> 6

<211> 432

<212> PRT

<213>artificial sequence

<400> 6

Met Ala Pro Lys Lys Lys Arg Lys Val Met Ser Gln Phe Asp Ile Leu

1 5 10 15

Cys Lys Thr Pro Pro Lys Val Leu Val Arg Gln Phe Val Glu Arg Phe

20 25 30

Glu Arg Pro Ser Gly Glu Lys Ile Ala Ser Cys Ala Ala Glu Leu Thr

35 40 45

Tyr Leu Cys Trp Met Ile Thr His Asn Gly Thr Ala Ile Lys Arg Ala

50 55 60

Thr Phe Met Ser Tyr Asn Thr Ile Ile Ser Asn Ser Leu Ser Phe Asp

65 70 75 80

Ile Val Asn Lys Ser Leu Gln Phe Lys Tyr Lys Thr Gln Lys Ala Thr

85 90 95

Ile Leu Glu Ala Ser Leu Lys Lys Leu Ile Pro Ala Trp Glu Phe Thr

100 105 110

Ile Ile Pro Tyr Asn Gly Gln Lys His Gln Ser Asp Ile Thr Asp Ile

115 120 125

Val Ser Ser Leu Gln Leu Gln Phe Glu Ser Ser Glu Glu Ala Asp Lys

130 135 140

Gly Asn Ser His Ser Lys Lys Met Leu Lys Ala Leu Leu Ser Glu Gly

145 150 155 160

Glu Ser Ile Trp Glu Ile Thr Glu Lys Ile Leu Asn Ser Phe Glu Tyr

165 170 175

Thr Ser Arg Phe Thr Lys Thr Lys Thr Leu Tyr Gln Phe Leu Phe Leu

180 185 190

Ala Thr Phe Ile Asn Cys Gly Arg Phe Ser Asp Ile Lys Asn Val Asp

195 200 205

Pro Lys Ser Phe Lys Leu Val Gln Asn Lys Tyr Leu Gly Val Ile Ile

210 215 220

Gln Cys Leu Val Thr Glu Thr Lys Thr Ser Val Ser Arg His Ile Tyr

225 230 235 240

Phe Phe Ser Ala Arg Gly Arg Ile Asp Pro Leu Val Tyr Leu Asp Glu

245 250 255

Phe Leu Arg Asn Ser Glu Pro Val Leu Lys Arg Val Asn Arg Thr Gly

260 265 270

Asn Ser Ser Ser Asn Lys Gln Glu Tyr Gln Leu Leu Lys Asp Asn Leu

275 280 285

Val Arg Ser Tyr Asn Lys Ala Leu Lys Lys Asn Ala Pro Tyr Pro Ile

290 295 300

Phe Ala Ile Lys Asn Gly Pro Lys Ser His Ile Gly Arg His Leu Met

305 310 315 320

Thr Ser Phe Leu Ser Met Lys Gly Leu Thr Glu Leu Thr Asn Val Val

325 330 335

Gly Asn Trp Ser Asp Lys Arg Ala Ser Ala Val Ala Arg Thr Thr Tyr

340 345 350

Thr His Gln Ile Thr Ala Ile Pro Asp His Tyr Phe Ala Leu Val Ser

355 360 365

Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser Lys Glu Met Ile Ala Leu Lys

370 375 380

Asp Glu Thr Asn Pro Ile Glu Glu Trp Gln His Ile Glu Gln Leu Lys

385 390 395 400

Gly Ser Ala Glu Gly Ser Ile Arg Tyr Pro Ala Trp Asn Gly Ile Ile

405 410 415

Ser Gln Glu Val Leu Asp Tyr Leu Ser Ser Tyr Ile Asn Arg Arg Ile

420 425 430

<210> 7

<211> 1044

<212> DNA

<213>artificial sequence

<400> 7

atgggtgcta gcgagctgat catctctggc tcctctggag gattcctgag gaacatcggc 60

aaggagtacc aggaggctgc tgagaacttc atgagattca tgaatgacca gggagcctac 120

gcccctaaca ccctgagaga cctgaggctg gtgttccact cctgggctag atggtgccac 180

gctagacagc tggcctggtt ccctatctct cctgagatgg ctagggagta cttccttcag 240

ctgcacgatg ctgacctggc ctctaccacc atcgacaagc actacgccat gctgaacatg 300

ctgctgtccc actgtggcct gcctcctctg tctgatgaca agtctgtgag cctggccatg 360

aggagaatcc ggagagaggc tgccaccgag aagggagaga gaaccggcca ggccatccct 420

ctgagatggg atgacctgaa gctgctggat gtgctgctgt ctagatctga gagactggtg 480

gacctgagga atagggcctt cctgtttgtg gcctacaaca ccctgatgag gatgtctgag 540

atctctagga tcagagtggg agacctggac cagaccggag acaccgtgac cctgcacatc 600

tcccacacca agaccatcac caccgctgct ggcctggaca aagtgctgtc taggaggacc 660

accgctgtgc tgaatgactg gctggatgtg tctggcctga gagagcaccc tgacgctgtg 720

ctgttccctc ctatccaccg gagcaacaag gctaggatca ccaccacccc tctgaccgcc 780

cctgccatgg agaagatttt tagcgatgcc tgggtgctgc tgaacaagag ggatgccacc 840

cctaacaagg gccgctaccg gacctggacc ggccactctg ctagagtggg agctgccatc 900

gacatggctg agaagcaagt gtccatggtg gagatcatgc aggagggcac ctggaaaaag 960

cctgagacac tgatgagata cctgaggagg ggaggagtgt ctgtgggagc caactctagg 1020

ctgatggact ccgctagcgg cgcc 1044

<210> 8

<211> 348

<212> PRT

<213>artificial sequence

<400> 8

Met Gly Ala Ser Glu Leu Ile Ile Ser Gly Ser Ser Gly Gly Phe Leu

1 5 10 15

Arg Asn Ile Gly Lys Glu Tyr Gln Glu Ala Ala Glu Asn Phe Met Arg

20 25 30

Phe Met Asn Asp Gln Gly Ala Tyr Ala Pro Asn Thr Leu Arg Asp Leu

35 40 45

Arg Leu Val Phe His Ser Trp Ala Arg Trp Cys His Ala Arg Gln Leu

50 55 60

Ala Trp Phe Pro Ile Ser Pro Glu Met Ala Arg Glu Tyr Phe Leu Gln

65 70 75 80

Leu His Asp Ala Asp Leu Ala Ser Thr Thr Ile Asp Lys His Tyr Ala

85 90 95

Met Leu Asn Met Leu Leu Ser His Cys Gly Leu Pro Pro Leu Ser Asp

100 105 110

Asp Lys Ser Val Ser Leu Ala Met Arg Arg Ile Arg Arg Glu Ala Ala

115 120 125

Thr Glu Lys Gly Glu Arg Thr Gly Gln Ala Ile Pro Leu Arg Trp Asp

130 135 140

Asp Leu Lys Leu Leu Asp Val Leu Leu Ser Arg Ser Glu Arg Leu Val

145 150 155 160

Asp Leu Arg Asn Arg Ala Phe Leu Phe Val Ala Tyr Asn Thr Leu Met

165 170 175

Arg Met Ser Glu Ile Ser Arg Ile Arg Val Gly Asp Leu Asp Gln Thr

180 185 190

Gly Asp Thr Val Thr Leu His Ile Ser His Thr Lys Thr Ile Thr Thr

195 200 205

Ala Ala Gly Leu Asp Lys Val Leu Ser Arg Arg Thr Thr Ala Val Leu

210 215 220

Asn Asp Trp Leu Asp Val Ser Gly Leu Arg Glu His Pro Asp Ala Val

225 230 235 240

Leu Phe Pro Pro Ile His Arg Ser Asn Lys Ala Arg Ile Thr Thr Thr

245 250 255

Pro Leu Thr Ala Pro Ala Met Glu Lys Ile Phe Ser Asp Ala Trp Val

260 265 270

Leu Leu Asn Lys Arg Asp Ala Thr Pro Asn Lys Gly Arg Tyr Arg Thr

275 280 285

Trp Thr Gly His Ser Ala Arg Val Gly Ala Ala Ile Asp Met Ala Glu

290 295 300

Lys Gln Val Ser Met Val Glu Ile Met Gln Glu Gly Thr Trp Lys Lys

305 310 315 320

Pro Glu Thr Leu Met Arg Tyr Leu Arg Arg Gly Gly Val Ser Val Gly

325 330 335

Ala Asn Ser Arg Leu Met Asp Ser Ala Ser Gly Ala

340 345

<210> 9

<211> 986

<212> DNA

<213>artificial sequence

<400> 9

cccccccaaa aaaaaggtgc ctatcaccct tctgcctacc tgtgtatcct caggacatgg 60

tgggcctctc tggttggcag aaagcacaac aaagcctctt catcctatac tacttctctt 120

gaccagatgc caagtctaat atgaactgca atcctctata caccaaaagt tcatgggggc 180

accgtgaggt cccactccac ctcagccaat tccttgttgc tgccccactc acctcctgag 240

cctccctctg tttcctgtcc acctttcagc ttccctctca ggctgggagc aggggccagt 300

agcagcaccc acgtccacct tctgtctagt aatgtccaac acctccctca gtccaaacac 360

tgctctgcat ccatgtggct cccatttata cctgaagcac ttgatggggc ctcaatgttt 420

tactagagcc cacccccctg caactctgag accctctgga tttgtctgtc agtgcctcac 480

tggggcgttg gataatttct taaaaggtca agttccctca gcagcattct ctgagcagtc 540

tgaagatgtg tgcttttcac agttcaaatc catgtggctg tttcacccac ctgcctggcc 600

ttgggttatc tatcaggacc tagcctagaa gcaggtgtgt ggcacttaac acctaagctg 660

agtgactaac tgaacactca agtggatgcc atctttgtca cttcttgact gtgacacaag 720

caactcctga tgccaaagcc ctgcccaccc ctctcatgcc catatttgga catggtacag 780

gtcctcactg gccatggtct gtgaggtcct ggtcctcttt gacttcataa ttcctagggg 840

ccactagtat ctataagagg aagagggtgc tggctcccag gccacagccc acaaaattcc 900

acctgctcac aggttggctg gctcgaccca ggtggtgtcc cctgctctga gccagctccc 960

ggccaagcca gcggcgcgcc gccacc 986

<210> 10

<211> 34

<212> DNA

<213>artificial sequence

<400> 10

ataacttcgt ataatgtatg ctatacgaag ttat 34

<210> 11

<211> 34

<212> DNA

<213>artificial sequence

<400> 11

gaagttccta ttctctagaa agtataggaa cttc 34

<210> 12

<211> 32

<212> DNA

<213>artificial sequence

<400> 12

taactttaaa taattggcat tatttaaagt ta 32

Claims

1. the gene targeting carrier that a kind of selection markers self are deleted, which is characterized in that it includes following element: recombination site 1, Reproduction specificity promoter, recombination enzyme gene, selection markers promoter, riddled basins and recombination site 2；

Wherein, reproduction specificity promoter be selected from Prm1 gene promoter, Ddx4 gene promoter, Stra8 gene promoter, AMHR gene promoter or Zp3 gene promoter.

2. the gene targeting carrier that selection markers according to claim 1 self are deleted, which is characterized in that selection markers open Mover be selected from PGK promoter, EM7 promoter, or both combination；

3. the gene targeting carrier that selection markers according to claim 1 or 2 self are deleted, which is characterized in that the sieve Marker gene is selected to be positive riddled basins；

Preferably, the positive riddled basins are selected from neomycin phosphotransferase gene (neo), hygromycin B phosphotransferase Gene (hph), xanthine/guanine monophosphate transferase gene (gpt), hypoxanthine phosphoric acid transferase gene (Hprt), thymus gland Pyrimidine kinase gene (tk) and puromycin acetyltransferase gene (puro).

4. the gene targeting carrier that selection markers according to claim 1 or 2 self are deleted, which is characterized in that described heavy Group enzyme is selected from iCre recombinase, FLPo recombinase and Dre recombinase；

Wherein, when the recombinase is iCre recombinase, recombination site 1 and recombination site 2 are the site loxP；

When the recombinase is FLPo recombinase, recombination site 1 and recombination site 2 are the site FRT；

5. the gene targeting carrier that selection markers according to claim 4 self are deleted, which is characterized in that recombination site 1 It is identical with 2 direction of recombination site.

6. the gene targeting carrier that selection markers according to claim 5 self are deleted, which is characterized in that the gene is beaten Targeting vector further includes having the recombination site 3 not identified by the recombinase in 2 downstream of recombination site, the recombination site 3 Selected from the site loxP, the site FRT and the site rox.

7. the gene targeting carrier that selection markers according to claim 1 or 2 self are deleted, which is characterized in that the base Because the structure of targeting vector is linear structure or ring structure.

8. a kind of method for constructing genetically modified animal model, which is characterized in that it includes the following steps:

It is dry thin that the gene targeting carrier that the described in any item selection markers of claim 1-7 self are deleted imports animal embryo Born of the same parents；

After identified correct embryonic stem cells to blastaea, it is transplanted in the female uterus of false pregnancy, is fitted into Body；

9. the method for building genetically modified animal model according to claim 8, which is characterized in that the animal is inhuman Mammal.

10. according to the method described in claim 9, it is characterized in that, the non-human mammal be selected from mouse, rat, horse, Ox, sheep, pig, monkey and ape.