CN108715864A

CN108715864A - It is a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application

Info

Publication number: CN108715864A
Application number: CN201810585654.3A
Authority: CN
Inventors: 崔宗斌; 赵熠; 宋桂丽; 李青
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2018-06-06
Filing date: 2018-06-06
Publication date: 2018-10-30

Abstract

The invention discloses a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application.The present invention constructs the endogenous gene mutational vector containing riddled basins and inducible mutational vector carries out the transposase expression vector of genome conformity, utilize electrotransfection method, two kinds of carriers are imported in mouse embryo stem cell jointly, it is analyzed again by inducing transposition expression of enzymes, cell resistance screening, cell clone identification and integration site, effective mutation method of mouse embryo stem cell endogenous gene is built, the cell clone for carrying single mutator and having totipotential differentiation ability is obtained.Mutational vector of the present invention can interfere the transcriptional expression of the endogenous gene in the genome of radom insertion mouse embryo stem cell.Mutation method of the present invention can carry out extensive insertion mutation to mouse embryo stem cell genome, obtain the cell clone library for carrying single mutator and having totipotential differentiation ability, the development for mouse mutant and gene functional research.

Description

A kind of gene insertion mutation system and its mutation method for mouse embryo stem cell And application

Technical field

The present invention relates to the gene insertion mutation of mouse embryo stem cell clone and application technologies, more specifically, this hair It is bright be related to it is a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application.

Background technology

One important means of gene functional research be using genetic manipulation technology to model animal for example mouse, zebra fish, Drosophila etc. carries out gene overexpression or knockout/drop, by observing the exception table in the life processes such as embryonic development, orga- nogenesis Type, molecular mechanism of the parsing gene in embryonic development, orga- nogenesis, disease generating process.Mouse is as mammal pattern Biology has incomparable superiority in terms of studying human gene function, but mouse living gene Knockout technology is time-consuming Arduously, gene functional research process has been seriously affected.Mouse embryo stem cell has self-renewal capacity and can be divided into Three germinal layers, are favored by researcher.Since past more than 20 years, it is thin that international mouse research association is dedicated to mouse embryonic stem Born of the same parents' gene trap and gene-targeted mutant screening, by the end of in January, 2018, international gene trap alliance uses gene trap method 121703 mouse embryo stem cell clones are obtained, 12393 genes are captured.Realize that the mutation of mouse full genome level is ground Study carefully, needs that different gene trap and gene targeting, the gene capturing carrier reported at present is used to capture in murine genes And played important function in terms of gene functional research, but there is also some shortcomings, as gene insertion site Preference, The validity etc. of gene trap.According to the demand of gene functional research, in conjunction with the swivel base advantage of SB transposons in mammals, We construct the polyA gene capturing carriers based on sleeping beauty (Sleeping Beauty, SB) transposons, to excavate more More functional genes.

Invention content

The object of the present invention is to provide a kind of gene insertion mutation system of mouse embryo stem cell and its mutation sides Method and application.The gene insertion mutation system of the present invention can be in the genome of radom insertion mouse embryo stem cell, to Development of Mouse Embryos Tire stem cell gene carries out insertion mutation, and researcher is contributed to carry out functional study to murine genes.

The first aspect of the invention provides a kind of polyA gene trap load transposon-mediated based on sleeping beauty SB Body, the gene capturing carrier include：SA-EXON-SV40polyA-TT gene mutation elements, SV40 promoters, IRES-SD- ARE sequences, riddled basins Neo, FRT sequence, gene press SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT- The sequential series of IRES-SD-ARE.

The construction method of gene capturing carrier described in above-mentioned technical proposal is as follows：By pT2/polyA-trap carriers into Row AatII and SacII double digestion, by SA-EXON-SV40polyA-TT gene mutations element, SV40 promoters, selection markers base It because of Neo, IRES-SD-ARE sequence, is attached, obtains pT2/SA-EXON-SV40PolyA-TT-SV40-Neo-IRES-SD- FRT sequences are then cloned into the both ends SV40-Neo by ARE carriers by PCR method, obtain the gene capturing carrier pT2/ SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE, the nucleic acid sequence of the gene capturing carrier As shown in SEQ ID No.1.

The second aspect of the invention, provides a kind of derivable transposase expression vector, and the swivel base expression of enzymes carries Body includes CMV promoter, Tet-on systems (rtTA and TRE elements), SB11 transposases, β actin polyA sequences, and gene is pressed The sequential series of pCMV-rtTA-TRE-SB11- β actin polyA.

The construction method of transposase expression vector described in above-mentioned technical proposal is as follows：

TRE-miniCMV segments are cloned into carrier PCMV-rtTA- by digestion AgeI/BsiWI double enzyme sites On SB11- β actin polyA, the transposase expression vector PCMV-rtTA-TRE-SB11- β actin polyA are obtained. The nucleic acid sequence of the carrier is as shown in SEQ ID No.2.

Present invention transposase expression vector only induced expression SB11 swivel bases in the presence of DOX described above Enzyme, SB11 transposases are attached on SB transposon vectors, and capturing carrier is inserted into genome by transposition mechanism.

The third aspect of the present invention provides a kind of gene insertion mutation system for mouse embryo stem cell, described Gene insertion mutation system include：

(1) the polyA gene capturing carrier transposon-mediated based on sleeping beauty SB described in；With

(2) the derivable transposase expression vector described in.

The fourth aspect of the present invention provides the gene insertion mutation method of mouse embryo stem cell described above, institute The method of stating includes the following steps：

(a) structure of insertion mutation system, including the polyA gene trap transposon-mediated based on sleeping beauty SB The structure for the transposase expression vector that the structure and control gene capturing carrier of carrier play a role；

(b) mouse embryo stem cell genetic transformation and cystic cancer cell line；

(c) embryonic stem cell insertion mutation Clonal integration Locus Analysis in Shoots；

(d) expression analysis of mutator.

Further, the genetic transformation of mutational vector importing embryonic stem cell is specially in above-mentioned technical proposal step (b) Electrotransformation, specific electrotransformation process are as follows：ES cells, quantity 1x10 are collected in digestion⁷A cell, mutational vector DNA are used Amount is 25 μ g, and carrying out electricity using Bio-rad electroporations turns, and parameter is：Voltage 250, capacitance 500, resistance ∞, electric revolving cup diameter 4mm, wherein：The mutational vector includes gene capturing carrier and transposase expression vector, the two mass ratio 1：1.

Further, the insertion mutation colony screening in above-mentioned technical proposal is analyzed with insertion point, and detailed process is as follows： 1 μ g/ml Dox are added after electricity turns in cell in culture solution, induce the expression of SB11 transposases, 200 μ g/ are utilized after 24 hours Ml G418 screen transfectional cell, replace a subculture daily, and screening is for 1 week, expand culture to the mES monoclonals of survival Afterwards, different cell clones are chosen and carry out positive colony screening and insertion point analysis, the segment of acquisition are sequenced, database It compares analysis and obtains capture gene loci.

Preferably, the positive-selecting primer sequence described in above-mentioned technical proposal includes：

Primers F：5'-GACCACCAAGCGAAACATCG-3'(SEQ ID No.3)；

Primer R：5'-ATATCACGGGTAGCCAACGC-3'(SEQ ID No.4).

Preferably, the insertion point analysis described in above-mentioned technical proposal is specifically to be carried out using Splinker PCR methods Insertion point is analyzed.

Further, the expression analysis of the mutator described in above-mentioned technical proposal step (d), including fusion transcript point Analysis, quantitative fluorescent PCR and Southern blot analyses.

The fifth aspect of the present invention provides the application of gene insertion mutation method described above, is mainly used for small Mouse embryonic stem cell genome carries out extensive gene insertion mutation, and batch screens the single cell clone of different genes insertion mutation Library obtains the cell clone library for carrying single mutator and having totipotential differentiation ability, and then grinding for mouse mutant System and gene functional research.

Further, the present invention carries out totipotential differentiation capability analysis and foundation to the cell clone for carrying term single gene mutation Single cell clone library, including the screening of the batch of the detection of expression of cell clone embryonic stem cell marker gene and single cell clone. Using the antibody of molecular marker gene SSEA-1, OCT-4, immunofluorescence dyeing is carried out, meanwhile, fluorescence quantitative PCR detection molecule The expression of marker gene SSEA-1, OCT-4, REX-1, NANOG, and carry out alkaline phosphatase staining and karyotype point Analysis determines that the cell clone that the mutation method obtains has embryonic stem cell totipotency.Through Markers for Detection, karyotyping table The bright ES cells clone that we obtain gene mutation has higher reproduction chimaeric adenovirus ability, further to utilize its preparation The mouse of genetic modification is laid a good foundation.

Mouse mutant development is carried out using the cell clone for carrying single mutator described in above-mentioned technical proposal, Detailed process is as follows, and by the method for blastaea microinjection, the normal embryonic stem cell line of caryogram is injected C57BL/6J mouse Blastaea in, obtain allophenic mice, then the mouse of the high chimeric degree of selection and C57BL/6J mouse hybrids, obtain and carry mutation The hybrid mice of gene, then carry out selfing and obtain homozygous mutation body mouse.

Compared with prior art, of the invention for the gene insertion mutation system of mouse embryo stem cell and its mutation side Method and application have the advantages that：

(1) gene insertion mutation system of the invention include based on polyA gene capturing carriers transposon-mediated SB and Derivable transposase expression vector, the present invention utilize sleeping beauty transposon stand System-mediated gene mutation element and gene trap member Part carries out the mutation of mouse embryo stem cell genome conformity, it can be achieved that the gene to mES is quickly and effectively mutated, and helps to inquire into Function of the gene in embryonic development and disease process.

(2) the gene mutation method of mouse embryo stem cell of the invention includes the insertion mutation based on sleeping beauty transposon stand Structure, mouse embryo stem cell genetic transformation and the screening of system, embryonic stem cell insertion mutation Clonal integration Locus Analysis in Shoots, base Because of expression analysis etc..The gene mutation method aims to overcome that gene capturing carrier is not in existing mouse embryo stem cell Foot, provides effective gene mutation method.

Description of the drawings

Fig. 1 is the structure of the polyA gene capturing carrier transposon-mediated based on sleeping beauty SB described in the embodiment of the present invention 1 Build schematic diagram；

Fig. 2 is the structure schematic diagram of the derivable transposase expression vector described in the embodiment of the present invention 2；

Fig. 3 is the embodiment of the present invention 3, the electrotransformation described in embodiment 4 and insertion mutation colony screening process schematic；

Fig. 4 is the capture gene expression analysis figure described in the embodiment of the present invention 5；Wherein：A- gene capturing carriers are inserted into base Because of a group site schematic diagram, B- gene capturing carriers analyze the primer position view, C- genes with endogenous gene amalgamation and expression Capturing carrier and endogenous gene amalgamation and expression analytical electrophoresis figure, D- capture gene expression analysis, and E-Southern blot analyze base Because of capturing carrier genome copy numbers；

Fig. 5 is the cellular omnipotency analysis chart described in the embodiment of the present invention 6, wherein：A- immunofluorescence dyeings detect ES marks Remember that gene expression, the expression of B- fluorescence quantitative PCR detection ES marker gene, C-ES cell clone alkaline phosphatase activities are analyzed, D-ES cell clone chromosome karyotype analysis.

Specific implementation mode

Technical scheme of the present invention is described in detail below by specific embodiment and attached drawing.Following reality It is preferred embodiments of the present invention to apply example only, is not the restriction that other forms are done to the present invention, any skill for being familiar with this profession The equivalent embodiment that art personnel are changed to change on an equal basis possibly also with the technology contents of the disclosure above.It is every without departing from this hair Bright plan content, any simple modification made according to the technical essence of the invention to following embodiment or equivalent variations, fall Within the scope of the present invention.

Experimental method in various embodiments of the present invention, routinely condition carries out, and with specific reference to such as Pehanorm Brooker etc., divides Experimental method described in sub- cloning experimentation guide (second edition, 1992, Science Press).

The present invention is dashed forward by building gene mutation carrier express containing riddled basins and derivable mediation gene It is thin to be imported mouse embryonic stem using electrotransfection method by the expression vector for becoming carrier progress genome conformity jointly for two kinds of carriers In born of the same parents, by induced expression, cell resistance screening and cell clone identification and analysis, structure mouse embryo stem cell gene is effectively prominent Change method obtains the cell clone for carrying single mutator and having totipotential differentiation ability.

The mutational vector of the present invention can be in the genome of radom insertion mouse embryo stem cell, including exon, introne And noncoding region.Using this method, mutational vector insertion is incorporated into the introne of murine genes X, the endogenous gene is interfered Transcriptional expression.This method can be in the genome of radom insertion mouse embryo stem cell, can be to mouse embryonic stem with this method Cellular genome carries out extensive gene insertion mutation, obtains the cell for carrying single mutator and having totipotential differentiation ability Clone bank, and the development for mouse mutant and gene functional research.

Embodiment 1

A kind of polyA gene capturing carrier transposon-mediated based on sleeping beauty SB of the present embodiment, the gene trap Carrier includes：SA-EXON-SV40polyA-TT gene mutation elements, SV40 promoters, IRES-SD-ARE sequences, selection markers Gene Neo, FRT sequence, gene press the sequence of SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE Series connection.

The construction method of the polyA gene capturing carrier transposon-mediated based on sleeping beauty SB described above is as follows：

As shown in Figure 1, it is bis- that pT2/polyA-trap carriers (Song, Li et al.2012) are carried out AatII and SacII Digestion, by SA-EXON-SV40polyA-TT gene mutations element, SV40 promoters, IRES-SD-ARE sequences, selection markers base Because Neo is attached, pT2/SA-EXON-SV40PolyA-TT-SV40-Neo-IRES-SD-ARE carriers are obtained, then by FRT Sequence is cloned into the both ends SV40-Neo by PCR method, obtains capturing carrier pT2/SA-EXON-SV40PolyA-TT-FRT- SV40-Neo-FRT-IRES-SD-ARE, the nucleic acid sequence of the capturing carrier is as shown in SEQ ID No.1.

Embodiment 2

A kind of derivable transposase expression vector of the present embodiment, the transposase expression vector include CMV promoter, Tet-on systems (rtTA and TRE elements), SB11 transposases, β actin polyA sequences, gene press PCMV-rtTA-TRE- The sequential series of SB11- β actin polyA.

The construction method of transposase expression vector described above is as follows：

As shown in Fig. 2, TRE-miniCMV segments are cloned into carrier by digestion AgeI/BsiWI double enzyme sites On PCMV-rtTA-SB11- β actin polyA, the transposase expression vector pCMV-rtTA-TRE-SB11- β are obtained Actin polyA, the nucleic acid sequence of the transposase expression vector is as shown in SEQ ID No.2.

The present embodiment transposase expression vector described above only induced expression SB11 swivel bases in the presence of DOX Enzyme, SB11 transposases are attached on SB transposon vectors, and capturing carrier is inserted into genome by transposition mechanism.

3 Mouse Embryonic Stem Cell Culture of embodiment and electrotransformation

Mouse embryo stem cell (mES) is that we use R1/E cell lines (ATCC:SCRC-1036^TM), condition of culture It is：37 DEG C, 5% carbon dioxide, ES complete medium components：DMEM (invitrogen 12430), ES grades of FBS (biochrom S4115), Glutamax (invitrogen 35050), NEAA (invitrogen 11140), LIF (Millipore ESG1107), β-Mer (invitrogen 21985).

Feeder cells used in R1/E in the present embodiment：MEF-G418 (Kunming white).

The present embodiment mutational vector (including gene capturing carrier and transposase expression vector, the two mass ratio are 1：1) it leads Enter the electrotransformation of mouse embryo stem cell, detailed process is as shown in Figure 3：ES cells, quantity 1x10 are collected in digestion⁷It is a thin Born of the same parents, mutational vector DNA (including gene capturing carrier and transposase expression vector, the two mass ratio are 1：1) dosage is 25 μ g, is adopted Electricity, which is carried out, with Bio-rad electroporations turns that (parameter is：Voltage 250, capacitance 500, resistance ∞, electric revolving cup diameter 4mm).

It clones in 4 gene trap site of embodiment

As shown in figure 3,1 μ g/ml Dox are added after electricity turns in cell in culture solution, the expression of SB11 transposases is induced, 24 Transfectional cell is screened using 200 μ g/ml G418 after hour, replaces a subculture daily, screening is for 1 week, to survival After mES monoclonals expand culture, chooses different cell clones progress positive colony screenings and insertion point analysis, positive-selecting draw Object sequence：

Primers F：5'-GACCACCAAGCGAAACATCG-3'(SEQ ID No.3)；

Primer R：5'-ATATCACGGGTAGCCAACGC-3'(SEQ ID No.4).

Insertion point analysis is carried out using Splinker PCR methods, the segment of acquisition is sequenced, database compares Analysis obtains capture gene loci.The primer is as follows：

Long stand：5'-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGT

GTCGACACTAGTGG-3'(SEQ ID No.5)；

Short Sau3aI：5'-GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA-3' (SEQ ID No.6)；

Splink1：5'-CGAAGAGTAACCGTTGCTAGGAGAGACC-3'(SEQ ID No.7)；

Splink2：5'-GTGGCTGAATGAGACTGGTGTCGAC-3'(SEQ ID No.8)；

L2：5'-TTAAAGGCACAGTCAACTTAGTGTATGTAAACTTCTG-3'(SEQ ID No.9)；

L3：5'-TGAAAAACGAGTTTTAATGACTCCAACTTAAG-3'(SEQ ID No.10)；

R1：5'-CTAACTGACCTAAGACAGGGAATTTTTACTAGGATT-3'(SEQ ID No.11)；

R2：5'-TAAGGTGTATGTAAACTTCCGACTTCAACTG-3'(SEQ ID No.12).

Table 1 is the gene trap Locus Analysis in Shoots figure of the embodiment of the present invention 4

Embodiment 5 captures gene expression analysis

Gene expression analysis includes fusion transcript analysis, quantitative fluorescent PCR and Southern blot analyses.We sieve It selects in obtained cell clone there are one being inserted into the 3rd introne of syngap1 genes (Fig. 4 A), we are by the clone It is named as syngap1^+/-Cell clone, using primer X (Fig. 4 B), including：

F：5'-ACACCCCCTCTGATCCGCG-3'(SEQ ID No.13)；

R', 5 '-CGGCCGCTCGTACGTCACTAATTC-3 ' (SEQ ID No.14) and primer sequence carry out transcript and melt Close expression analysis, primer X confirms in capturing carrier that SA-EXON has occurred with the exon 3 of syngap1 genes and merges transcription and (scheme 4C).Then we have quantified the expression of endogenous gene syngap1, and quantitative primer sequence is as follows：

F：5'-ACACCCCCTCTGATCCGCG-3'(SEQ ID No.13)；

R：5 '-TCCATGCTGTACTGTTTCCCCCC-3 ' (SEQ ID No.15) have found in syngap1^+/-Cell clone In, the expression quantity of syngap1 genes has dropped nearly 50% or so (Fig. 4 D).To syngap1^+/-The genome of cell clone carries out BglII, EcoRI digestion are analyzed with Southern blot, and finding the capturing carrier, there are one copy, the results in genome (Fig. 4 E) consistent with the expression quantity of syngap1 genes.It these results suggest that in ES cells, which can be effective right Endogenous gene carries out striking drop mutation.

6 cellular omnipotency of embodiment is analyzed.

Immunofluorescence dyeing detects the expression of ES marker gene SSEA-1, OCT-4 gene, and fluorescence developing result is shown SSEA-1, OCT-4 are in the syngap1^+/-There is expression in cell clone, it was demonstrated that syngap1^+/-Cell clone has mouse embryonic stem The all-round activity of cell.Specific operation process is as follows,

(1) in 24 orifice plates, PBS is washed cell growth；

(2) 4% paraformaldehydes fix 20 minutes, and PBS is washed；

(3) 0.5%Triton X-100 permeable membranes 10 minutes, 0.1%BSA is washed；

(4) 37 DEG C are closed 30 minutes, (10% lowlenthal serum+0.1%Triton X-100+1%BSA of confining liquid)；

(5) 20 μ g/ml (Abcam, UK) of 20 μ g/ml (R&D Systems, USA) of incubation primary antibody SSEA-1 and OCT-4,4 DEG C overnight；

(6) 0.1%BSA is washed；

(7) 37 DEG C are incubated secondary antibody 30 minutes, sheep anti mouse-Cy3 and goat-anti rabbit-FITC (BOSTER, China)；

(8) 0.1%BSA is washed；

(9) room temperature redyes DAPI 5 minutes；

(10) fluorescence microscope is taken pictures (Leica DMI6000B/DMi8).

Quantitative detection, quantitative PCR result are carried out to the markup gene of mouse embryo stem cell using fluorescence quantifying PCR method Show syngap1^+/-Stem cell marker genes SSEA-1 in cell clone, OCT-4, Rex-1, NANOG gene expressions are not by shadow It rings, it was demonstrated that syngap1^+/-Cell clone has mouse embryo stem cell all-round active (Fig. 5 B).Required primer sequence is as follows：

ssea1f3：5'-CGGACCGACTCGGATGTC-3'(SEQ ID No.16)；

ssea1r3：5'-CCGACTCAGCTGGTGGTAGTAA-3'(SEQ ID No.17)；

oct4f：5'-TTCCCTCTGTTCCCGTCACT-3'(SEQ ID No.18)；

oct4r：5'-TGGTGCCTCAGTTTGAATGC-3'(SEQ ID No.19)；

Rex-1f：5'-CCGTATCAGTGCACGTTCGA-3'(SEQ ID No.20)；

Rex-1r：TGGGTGCGCAAGTTGAAA-3'(SEQ ID No.21)；

Nanog f：5'-CCTCATCAATGCCTGCAGTTT-3'(SEQ ID No.22)；

Nanog r：5'-CTCAGTAGCAGACCCTTGTAAGCA-3'(SEQ ID No.23)；

GAPDH f：5'-ACATCATCCCTGCATCCACT-3'(SEQ ID No.24)；

GAPDH r：5'-AGATCCACGACGGACACATT-3'(SEQ ID No.25)；

It after Mouse Embryonic Stem Cell Culture 5 days, is detected according to alkaline phosphatase detecting reagent box specification, detection knot Fruit shows syngap1^+/-With alkaline phosphatase activities, and in negative control MEF cells and alkaline phosphatase activities are not detected (Fig. 5 C).

Karyotyping shows syngap1^+/-Cell clone has normal XY types chromosome (Fig. 5 D), illustrates that we obtain The ES cells clone of gene mutation has higher reproduction chimaeric adenovirus ability, further to prepare genetic modification using it Mouse is laid a good foundation.Chromosome and property analytic process are as follows：

One 90% or more the T25 bottle for covering with cell merged is taken, colchicine is added within 3 hours in advance；

(1) 37 DEG C of warm bath KCl hypotonic medium, pre- cold methanol-glacial acetic acid fixer；

(2) vitellophag, piping and druming is uniform, is transferred to 60mm culture dishes, and 37 DEG C gently tilt culture dish after adherent 1 hour, take Supernatant moves into 15ml centrifuge tubes；

(3) 12000rpm is centrifuged 5 minutes；

(4) supernatant is removed, a small amount of raffinate mixing is stayed, 8ml warm bath KCl hypotonic mediums are added, turn upside down mixing, 37 DEG C of warm bath 30 minutes, intermediate mixing up and down was primary；

(5) the pre- cold methanols of addition 1ml-glacial acetic acid fixer terminates hypotonic, and turn upside down mixing, and 1400rpm centrifuges 3 points Clock；

(6) supernatant is removed, stays a small amount of raffinate that cell is resuspended, the pre- cold methanols of 10ml-glacial acetic acid fixer is added, turns upside down mixed It is even, 20 minutes are placed at room temperature for, intermediate mixing up and down is primary；

(7) 1200rpm is centrifuged 5 minutes, repeats step (7) 2 times, is placed at room temperature for 10 minutes for the second time；

(8) waiting time takes appropriate bubble in the glass slide of absolute ethyl alcohol, and distilled water is washed, and slide is put into the burning equipped with boiling water In cup, glass slide is heated；

(9) 1200rpm is centrifuged 5 minutes, removes supernatant, stays a small amount of raffinate that cell is resuspended；

(10) glass slide being put in ground, the appropriate blotting paper of underlay takes appropriate cell suspension, certain altitude to drip, with After take glass slide micro- roasting on alcolhol burner, be dried overnight glass slide；

(11) Giemsa is dyed 1 hour, is developed a film, is taken pictures under the microscope.

Sequence table

<110>Inst. of Hydrobiology, Chinese Academy of Sciences

<120>It is a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application

<130>Nothing

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5968

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 60

ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccctataca 120

gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta 180

ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt 240

gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat 300

tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt 360

aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttgc tagcgattgc 420

agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt tgacttccct 480

gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc cgatgatgaa 540

attgccgcac tggttgttag caacgtagcc ggtatgtgaa tgatgaatta gtgacgtacg 600

agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 660

taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 720

ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 780

caaataaagc atttttttca ctgcgaattc tttttatttt attttatttt taattttttt 840

ttgacgtcga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcccag 900

gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 960

ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 1020

gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 1080

ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 1140

ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 1200

ggagcttgga tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 1260

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 1320

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 1380

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 1440

gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 1500

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 1560

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 1620

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 1680

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 1740

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 1800

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 1860

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1920

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1980

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 2040

gagttcttct gagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc 2100

actagtgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag 2160

gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 2220

gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 2280

ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 2340

gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 2400

ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 2460

agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 2520

tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 2580

ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 2640

accacgggga cgtggttttc ctttgaaaaa cacgatgata atatcgccac aaccatggac 2700

catgcaccat gtacaaaccc ccccaaaccg aaggtgagtt gatctttaag ctttttacat 2760

tttcagctcg catatatcaa ttcgaacgtt taattagaat gtttaaataa agctagatta 2820

aatgattagg ctcagttacc ggtctttttt ttctcattta cgtagatcta gcttgtggaa 2880

ggctactcga aatgtttgac ccaagttaaa caatttaaag gcaatgctac caaatactaa 2940

ttgagtgtat gtaaacttct gacccactgg gaatgtgatg aaagaaataa aagctgaaat 3000

gaatcattct ctctactatt attctgatat ttcacattct taaaataaag tggtgatcct 3060

aactgaccta agacagggaa tttttactag gattaaatgt caggaattgt gaaaaagtga 3120

gtttaaatgt atttggctaa ggtgtatgta aacttccgac ttcaactgta tagggatcct 3180

ctagctagag tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg 3240

gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 3300

gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 3360

atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3420

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3480

tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 3540

agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 3600

aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 3660

gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 3720

tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 3780

cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 3840

ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 3900

cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 3960

atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4020

agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4080

gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 4140

gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4200

tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4260

agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4320

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 4380

aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 4440

aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 4500

ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 4560

gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 4620

aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 4680

ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 4740

tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 4800

ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 4860

cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 4920

agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 4980

gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5040

gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5100

acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 5160

acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 5220

agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 5280

aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 5340

gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 5400

tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5460

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5520

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5580

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 5640

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5700

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5760

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 5820

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat 5880

tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 5940

cgccagctgg cgaaaggggg atgtgctg 5968

<210> 2

<211> 6673

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 60

ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccgctagca 120

tatatgacgt cataacttcg tatagcatac attatacgaa gttatgttga cattgattat 180

tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 240

tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 300

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 360

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 420

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 480

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 540

ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 600

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 660

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 720

gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga 780

gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg 840

gccccgaatt caccatgtct agactggaca agagcaaagt cataaacggc gctctggaat 900

tactcaatgg agtcggtatc gaaggcctga cgacaaggaa actcgctcaa aagctgggag 960

ttgagcagcc taccctgtac tggcacgtga agaacaagcg ggccctgctc gatgccctgc 1020

caatcgagat gctggacagg catcataccc acttctgccc cctggaaggc gagtcatggc 1080

aagactttct gcggaacaac gccaagtcat tccgctgtgc tctcctctca catcgcgacg 1140

gggctaaagt gcatctcggc acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc 1200

agctcgcgtt cctgtgtcag caaggcttct ccctggagaa cgcactgtac gctctgtccg 1260

ccgtgggcca ctttacactg ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag 1320

aggaaagaga gacacctacc accgattcta tgcccccact tctgagacaa gcaattgagc 1380

tgttcgaccg gcagggagcc gaacctgcct tccttttcgg cctggaacta atcatatgtg 1440

gcctggagaa acagctaaag tgcgaaagcg gcgggccggc cgacgccctt gacgattttg 1500

acttagacat gctcccagcc gatgcccttg acgactttga ccttgatatg ctgcctgctg 1560

acgctcttga cgattttgac cttgacatgc tccccgggta actaagtaag gatccagaca 1620

tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 1680

ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 1740

aagttgctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 1800

gccctttcgt cttcactcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc 1860

gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 1920

cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg 1980

aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact 2040

ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga 2100

aaagtgaaag tcgagctcgg tacccgggtc gagtaggcgt gtacggtggg aggcctatat 2160

aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga 2220

cctccataga agacaccggg accgatccag cctcctagga ccggtgccac catgggaaaa 2280

tcaaaagaaa tcagccaaga cctcagaaaa aaaattgtag acctccacaa gtctggttca 2340

tccttgggag caatttccaa acgcctgaaa gtaccacgtt catctgtaca aacaatagta 2400

cgcaagtata aacaccatgg gaccacgcag ccgtcatacc gctcaggaag gagacgcgtt 2460

ctgtctccta gagatgaacg tactttggtg cgaaaagtgc aaatcaatcc cagaacaaca 2520

gcaaaggacc ttgtgaagat gctggaggaa acaggtacaa aagtatctat atccacagta 2580

aaacgagtcc tatatcgaca taacctgaaa ggccgctcag caaggaagaa gccactgctc 2640

caaaaccgac ataagaaagc cagactacgg tttgcaagag cacatgggga caaagatcgt 2700

actttttgga gaaatgtcct ctggtctgat gaaacaaaaa tagaactgtt tggccataat 2760

gaccatcgtt atgtttggag gaagaagggg gaggcttgca agccgaagaa caccatccca 2820

accgtgaagc acgggggtgg cagcatcatg ttgtgggggt gctttgctgc aggagggact 2880

ggtgcacttc acaaaataga tggcatcatg aggaaggaaa attatgtgga tatattgaag 2940

caacatctca agacatcagt caggaagtta aagcttggtc gcaaatgggt cttccaacaa 3000

gacaatgacc ccaagcatac ttccaaacac gtgagaaaat ggcttaagga caacaaagtc 3060

aaggtattgg agtggccatc acaaagccct gacctcaatc ctatagaaaa tttgtgggca 3120

gaactgaaaa agcgtgtgcg agcaaggagg cctacaaacc tgactcagtt acaccagctc 3180

tgtcaggagg aatgggccaa aattcaccca acttattgtg ggaagcttgt ggaaggctac 3240

ccgaaacgtt tgacccaagt taaacaattt aaaggcaatg ctaccaaata ctaggcggcc 3300

gcctatcgat aaacggactg ttaccacttc acgccgactc aactgcgcag agaaaaactt 3360

caaacgacaa cattggcatg gcttttgtta tttttggcgc ttgactcagg atctaaaaac 3420

tggaacggcg aaggtgacgg caatgttttg gcaaataagc atccccgaag ttctacaatg 3480

catctgagga ctcaatgttt tttttttttt ttttttcttt agtcattcca aatgtttgtt 3540

aaatgcattg ttccgaaact tatttgcctc tatgaaggct gcccagtaat tgggagcata 3600

cttaacattg tagtattgta tgtaaattat gtaacaaaac aatgactggg tttttgtact 3660

ttcagcctta atcttgggtt tttttttttt ttttttggtt ccaaaaaact aagagctctt 3720

taccattcaa gatgtaaagg tttccattcc ccctgggcat attgaaaaag ctgtgtggaa 3780

cgtggcggtg ccagacattt ggtggggcca acctgtacac tgactaattc aaataaaagg 3840

gacagatcat aacttcgtat agcatacatt atacgaagtt atccgcgggg atatagatct 3900

ctcgaggggg ggcccggtac ccagcttttg ttccctttag tgagggttaa tttcgagctt 3960

ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4020

caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4080

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4140

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4200

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4260

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4320

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4380

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4440

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4500

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4560

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4620

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4680

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4740

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4800

cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4860

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4920

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4980

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5040

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5100

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5160

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5220

aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5280

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5340

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5400

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5460

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5520

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5580

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 5640

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 5700

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 5760

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5820

aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 5880

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 5940

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6000

cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6060

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6120

acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 6180

gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 6240

cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 6300

atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 6360

tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 6420

tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 6480

tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 6540

atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg 6600

cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 6660

gggggatgtg ctg 6673

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

gaccaccaag cgaaacatcg 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

atatcacggg tagccaacgc 20

<210> 5

<211> 61

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

cgaagagtaa ccgttgctag gagagaccgt ggctgaatga gactggtgtc gacactagtg 60

g 61

<210> 6

<211> 48

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

gatcccacta gtgtcgacac cagtctctaa tttttttttt caaaaaaa 48

<210> 7

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

cgaagagtaa ccgttgctag gagagacc 28

<210> 8

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

gtggctgaat gagactggtg tcgac 25

<210> 9

<211> 37

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

ttaaaggcac agtcaactta gtgtatgtaa acttctg 37

<210> 10

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

tgaaaaacga gttttaatga ctccaactta ag 32

<210> 11

<211> 36

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

ctaactgacc taagacaggg aatttttact aggatt 36

<210> 12

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

taaggtgtat gtaaacttcc gacttcaact g 31

<210> 13

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

acaccccctc tgatccgcg 19

<210> 14

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

cggccgctcg tacgtcacta attc 24

<210> 15

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

tccatgctgt actgtttccc ccc 23

<210> 16

<211> 18

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

cggaccgact cggatgtc 18

<210> 17

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

ccgactcagc tggtggtagt aa 22

<210> 18

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 18

ttccctctgt tcccgtcact 20

<210> 19

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

tggtgcctca gtttgaatgc 20

<210> 20

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

ccgtatcagt gcacgttcga 20

<210> 21

<211> 18

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

tgggtgcgca agttgaaa 18

<210> 22

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 22

cctcatcaat gcctgcagtt t 21

<210> 23

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

ctcagtagca gacccttgta agca 24

<210> 24

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 24

acatcatccc tgcatccact 20

<210> 25

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 25

agatccacga cggacacatt 20

Claims

1. a kind of polyA gene capturing carrier transposon-mediated based on sleeping beauty, it is characterised in that：The gene capturing carrier Including：SA-EXON-SV40polyA-TT gene mutations element, SV40 promoters, IRES-SD-ARE sequences, riddled basins Neo and FRT sequences, gene press the sequence string of SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE Connection.

2. a kind of derivable transposase expression vector, it is characterised in that：The transposase expression vector include CMV promoter, RtTA and TRE elements, SB11 transposases, β actin polyA sequences, gene press pCMV-rtTA-TRE-SB11- β actin The sequential series of polyA.

3. a kind of gene insertion mutation system for mouse embryo stem cell, it is characterised in that：The gene insertion mutation System includes：

(1) the polyA gene capturing carrier transposon-mediated based on sleeping beauty SB described in claim 1；With

(2) the derivable transposase expression vector described in claim 2.

4. a kind of construction method of the polyA gene capturing carrier transposon-mediated based on sleeping beauty SB described in claim 1, It is characterized in that：The construction method of the gene capturing carrier is specific as follows：PT2/polyA-trap carriers are subjected to AatII With SacII double digestions, by SA-EXON-SV40polyA-TT gene mutations element, SV40 promoters, riddled basins Neo, IRES-SD-ARE sequences, are attached, and obtain pT2/SA-EXON-SV40PolyA-TT-SV40-Neo-IRES-SD-ARE loads FRT sequences are then cloned into the both ends SV40-Neo by body by PCR method, obtain the gene capturing carrier pT2/SA- EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE, the nucleic acid sequence of the gene capturing carrier is such as Shown in SEQ ID No.1.

5. a kind of construction method of the derivable transposase expression vector described in claim 2, it is characterised in that：Described turns The construction method of seat expression of enzymes carrier is specific as follows：By AgeI/BsiWI double digestions, TRE-miniCMV segments are cloned into load On body pCMV-rtTA-SB11- β actin polyA, the transposase expression vector pCMV-rtTA-TRE-SB11- β are obtained Actin polyA, the nucleic acid sequence of the carrier is as shown in SEQ ID No.2.

6. a kind of gene insertion mutation method of mouse embryo stem cell, it is characterised in that：Described method includes following steps：

(a) structure of insertion mutation system, including the polyA base transposon-mediated based on sleeping beauty SB described in claim 1 The structure of transposase expression vector described in the claim 2 to play a role by the structure and control gene capturing carrier of capturing carrier It builds；

(d) expression analysis of mutator.

7. the gene insertion mutation method of mouse embryo stem cell according to claim 6, it is characterised in that：Step (b) The genetic transformation that middle mutational vector imports embryonic stem cell is specially electrotransformation, and specific electrotransformation process is as follows：Digestion is collected small Mouse ES cells, quantity 1x10⁷A cell, mutational vector DNA dosages are 25 μ g, and carrying out electricity using Bio-rad electroporations turns, ginseng Number is：Voltage 250, capacitance 500, resistance ∞, electric revolving cup diameter 4mm, wherein：The mutational vector include gene capturing carrier and Transposase expression vector, the two mass ratio 1：1.

8. the gene insertion mutation method of mouse embryo stem cell according to claim 6, it is characterised in that：Insertion mutation Colony screening is analyzed with insertion point, and detailed process is as follows：1 μ g/ml Dox are added after electricity turns in cell in culture solution, induce The expression of SB11 transposases screens transfectional cell using 200 μ g/ml G418 after 24 hours, replaces a subculture daily, It screens for 1 week, after expanding culture to the mES monoclonals of survival, chooses different cell clones and carry out positive colony screenings and insert Angle of striking is analyzed, and the segment of acquisition is sequenced, and database compares analysis and obtains capture gene loci.

9. the application of the gene insertion mutation method described in claim 6~8, it is characterised in that：The method is mainly used for pair Mouse embryo stem cell genome carries out extensive gene insertion mutation, and batch screens unicellular gram of different genes insertion mutation Long Ku obtains the cell clone library for carrying single mutator and having totipotential differentiation ability.

10. the application of the gene insertion mutation method described in claim 9, it is characterised in that：Described pair carries term single gene and dashes forward The cell clone of change carries out totipotential differentiation capability analysis and establishes single cell clone library, specifically includes cell clone embryonic stem cell The detection of expression of marker gene and the batch screening of single cell clone；Using the antibody of molecular marker gene SSEA-1, OCT-4, Immunofluorescence dyeing is carried out, meanwhile, the table of fluorescence quantitative PCR detection molecular marker gene SSEA-1, OCT-4, REX-1, NANOG Up to level, and alkaline phosphatase staining and chromosome karyotype analysis are carried out, determines that the cell clone that the mutation method obtains has Embryonic stem cell totipotency.