CN100427592C - Method for establishing mouse full-genome mutoation ES cell bank - Google Patents
Method for establishing mouse full-genome mutoation ES cell bank Download PDFInfo
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Abstract
The present invention provides an establishment method which utilizes a bidirectional bi-selective gene trapping technology for establishing a large-scale mutation mouse embryo stem cell base. The method comprises the following steps: step a, a gene trapping carrier is guided into an embryonic stem cell, and the gene trapping carrier has the following structure from 5' to 3': SA-ST-DS-S1-P1-P2-S2-pA-P3-S3-SD-Ap-TS-AS; step b, embryonic stem cells with a screening genetic marker are screened; step c, the embryonic stem cells with a screening genetic marker are combined, and a mutation embryonic stem cell base is obtained. The present invention also provides an identification method for mutation gene sites. The method of the present invention establishes a basis for obtaining gene mutation mice in a large-scale and high-flux mode.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to a kind of method of setting up the mouse full genome mutated embryonic stem cell bank.
Background technology
Human genome and some other important animal-plant gene group complete sequence determination or will be finished in the recent period, simultaneously more species gene group order-checking plan is carried out.Yet the first step of organism genome functions is just understood in these work, because most gene function is not known as yet, quick accumulation along with the sequence data of magnanimity, people are faced with the huge challenge of biological function how to identify these sequence data representatives, and the analyzing gene group sequence that develops into of information biology infers that the function of gene provides unprecedented facility.
Yet, experimental study, particularly gene functional research in vivo remains the most important and indispensable means that obtain gene function information, and the gene of inside and outside and the experimental study of genome functions and information biology have constituted two wings of functional genomics research rapid development together.Research to the human genome function is the current project of carrying out that presses for most, thereby the gene relevant by analysis of disease family location provides important approach for the function of understanding Human genome, but the resource of this respect is limited after all, the research of a large amount of human gene function can only be passed through indirect relatively approach, particularly can't utilize the mankind itself to carry out intravital research (except some are used for the purpose of disease treatment) basically, be present the most desirable means and utilize model animals to carry out and study in the body of gene function.
Mouse is at present adopted near the model animal of anthropobiology function, the research of carrying out gene knockout in the mouse genome is the important means that discloses the mouse gene function, and this class research simultaneously provides very directly clue for understanding human homologous gene.
People find that for a long time the exogenous reporter gene in the transfered cell nuclear can single copy random integration take place any one site on any karyomit(e), under the regulating and controlling sequence effect of endogenous or allogenic gene, give expression to the fusion gene product of reporter gene product or endogenous gene and reporter gene, and might cause the structure deteriorate and the afunction of endogenous gene.According to this principle; insert the site and produce to genome, label by the report carrier random integration and insert inactivation gene (gene trapping) technology of " catching " finding new gene and disclose its function of suddenling change and arise at the historic moment, become high-throughput, extensive transgenation of setting up the embryonic stem cell level and a kind of technique means easily that produces the genetic modification mouse model.
Mainly be to utilize gene trapping (gene trapping) or chemical mutagen (as ENU etc.) that mouse embryo stem cell (embryonic stem cell) is carried out random mutation at present both at home and abroad, determine corresponding transgenation position by the screening and the assignment of genes gene mapping then, because embryonic stem cell has totipotency, can utilize these cells to set up mouse individuality, thereby can on individual level, study the function of gene with different genes sudden change.Yet, the mutated embryonic stem cell clone progress of this institute's acquisition at present is slow, the real needs of support function genomics research, the major cause that causes this phenomenon are the strategies that the method that the mutant cell clone is identified one by one that everybody generally adopts remains a consuming time and consumption power.
The technology of the present generation mutated embryonic stem cell bank that adopts, for example enhanser is caught (enhancertrap), is promoted son to catch (promoter trap), gene trap (gene trap) technology etc., and certain limitation is arranged in application process.At first, reporter gene or selection expression of gene depend on the regulating and controlling sequence of the endogenous gene of expressing in embryonic stem cell, thereby can not " catch " to the endogenous regulating and controlling sequence or the gene of not expressing at embryonic stem cell; Secondly, the reading frame frameshit might take place and can not be identified in the reporter gene of Cha Ruing after transcribing shearing at random, causes some functional gene to become " fish that has escape the net ".
Therefore, this area presses for and sets up a kind of extensive, method and correlation technique that high-throughput obtains mutant mouse in full genome range.Especially need the capturing carrier of development of new and strategy to improve capture rate.Have only and so could truly allow functional genome research go on fast-developing road, spur the discovery of input and a large amount of new functions of gene of follow-up research.
Summary of the invention
Purpose of the present invention just provides a kind of extensive, correlation technique that high-throughput obtains mutant mouse, promptly a kind of authentication method of setting up mouse full genome mutated embryonic stem cell bank and mutated embryonic stem cell mutational site.The inventive method can obtain to cover the mouse mutated embryonic stem cell bank of 90% above gene and screen platform accordingly in short duration, utilize this system can satisfy people and most of specific genes are carried out the needs studied in the mouse body.
In first aspect present invention, a kind of method of setting up extensive mutated embryonic stem cell bank is provided, comprise step:
(a) gene capturing carrier is imported embryonic stem cell, described gene capturing carrier from 5 ' to 3 ' structure be:
SA-ST-DS-S1-P1-P2-S2-pA-P3-S3-SD-Ap-TS-AS
In the formula:
SA is the receptor sequence of RNA montage;
ST is a forward series connection terminator codon;
DS is the donor sequences of reverse RNA montage;
S1 is the first reverse screening-gene, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or lacZ gene;
P1 is the first reverse promotor,
P2 is second promotor of forward;
S2 is second screening-gene of forward, is selected from down group: hygromycin B resistant gene Hygro, neomycin resistance gene Neo or puromycin resistance gene Puro;
PA is a forward RNA tailing signal sequence;
P3 is the 3rd promotor of forward;
S3 is the three screening gene of forward, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or lacZ gene;
SD is the donor sequences of RNA montage;
Ap is reverse RNA tailing signal sequence;
TS is reverse three terminator codons;
AS is the receptor sequence of reverse RNA montage;
P1, P2 and P3 are the promotors that is selected from down group respectively: CMV, PGK, LTP or hsv-TK;
Condition is S1, S2 and S3 have nothing in common with each other and S1 and S3 in screening-gene encoding sequence GFP that is green fluorescent protein, the encoding sequence EGFP or the lacZ gene of enhanced green fluorescent protein;
(b) screening has the embryonic stem cell of screening-gene mark;
(c) merge the embryonic stem cell that has the screening-gene mark, obtain mutated embryonic stem cell bank.
In another preference, in described gene capturing carrier, S1 is Hygro, and S2 is neo, and S3 is EGFP.
In another preference, the structure of described carrier is:
SA-ST-DS-Hygro-KTp-PGK-Neo-pA-PGK-EGFP-SD-Ap-TS-AS
Wherein, Hygro is reverse hygromycin B resistant gene
KTp is reverse hsv-TK promotor;
PGK is promotor PGK.
In another preference, described embryonic stem cell is mouse, rat, rabbit, the frog or people's a embryonic stem cell.
In another preference, also comprise step in step (b) with (c):
(b ') to its dna sample of every kind of embryo stem cell extracting and RNA sample that step (b) obtains, preserve.
In another preference, in step (b '), also comprise: for extractive dna sample and RNA sample, measure genomic dna sequence and cDNA sequence that carrier inserts the both sides, site, according to clear and definite each mutated embryonic stem cell clone gene mutational site of sequence information and native gene functionally inactive situation, and judge whether newly gene of mutator gene, and then set up the information database of sudden change ES cell.
In a second aspect of the present invention, a kind of mutated embryonic stem cell bank is provided, the embryonic stem cell that described embryonic stem cell bank contains is the cell of transgenation, and described cell bank prepares in order to the below method:
(a) gene capturing carrier is imported embryonic stem cell, described gene capturing carrier from 5 ' to 3 ' structure be:
SA-ST-DS-S1-P1-P2-S2-pA-P3-S3-SD-Ap-TS-AS
Each symbol definition as mentioned above;
(b) screening has the embryonic stem cell of screening-gene mark;
(c) merge the embryonic stem cell that has the screening-gene mark, obtain mutated embryonic stem cell bank.
In another preference, described cell bank contains ten thousand mutated embryonic stem cell clones of 5-30.
In another preference, described cell bank is divided into 500-1000 inferior storehouse, and 100-300 mutated embryonic stem cell clone contained in each inferior storehouse.
In another preference, described embryonic stem cell is mouse, rat, rabbit, the frog or people's a embryonic stem cell.
In a third aspect of the present invention, a kind of method of setting up mutated embryonic stem cell gene mutation site information database of the present invention is provided, comprise step:
(a) its dna sample of every kind of mutated embryonic stem cell extracting and the RNA sample to obtaining among the present invention;
(b) use the method carrier of PCR and RACE to insert the genomic dna sequence and the cDNA sequence of both sides, site;
(c), and judge whether newly gene of mutator gene according to clear and definite each mutated embryonic stem cell clone gene mutational site of sequence information and native gene functionally inactive situation;
(d) classification gathers (c) gained mutant clon information, the corresponding gene mutation site information database of mutated embryonic stem cell bank among foundation and the present invention.
In a fourth aspect of the present invention, the purposes of mutated embryonic of the present invention in cell bank is provided, it be used to produce the specific gene sudden change the genetic modification mouse model, be used to prepare the genomic dna and the cDNA of mutated embryonic stem cell bank.Perhaps, described mutated embryonic stem cell bank is divided into 500-1000 inferior storehouse, and 100-300 mutated embryonic stem cell clone contained in each inferior storehouse, and the inferior storehouse of each cell all is used to prepare inferior storehouse cDNA.Perhaps, described mutated embryonic stem cell bank is used to screen the embryonic stem cell clone of specific gene sudden change.
Embodiment
The inventor is through deeply and extensive studies is optimized the gene capturing carrier system, made up two-way gene capturing carrier system.This two-way gene capturing carrier system makes reporter gene or selects expression of gene to break away from dependency to the endogenous regulating and controlling sequence, can two-wayly be captured in functional gene 3. ends and polyadenylic acid tailing signal that embryonic stem cell is expressed or do not expressed, thereby can obtain a large amount of insertion mutant clons in the mouse embryo stem cell level.
In the present invention, utilize most gene to have the characteristics of polyA signal and gene asymmetric transcription, on embryonic stem cell, carried out the gene trap of two-way gene PolyA.
If gene capturing carrier is inserted in some genes, then the screening-gene that provides of this carrier and the PolyA signal bits inserting gene and provide are named a person for a particular job and are given this embryonic stem cell and have the characteristic that the screening-gene expression product is had; If gene capturing carrier is not inserted in the gene region, screening-gene is not owing to self carry the signal of PolyA, and therefore the RNA that transcribes can not stably exist and translate, and this causes embryonic stem cell not possess the characteristic of selection markers.Therefore, the incident that certain gene is inserted into gene capturing carrier has taken place in the embryonic stem cell clone that obtains through screening.Because the segmental insertion of external source can destroy the function of original gene, therefore utilize this method to set up in a short time and have the mutated embryonic stem cell bank that the huge cell clone of quantity is formed.The mutated embryonic stem cell banks formed of 150,000 cell clones for example, on average each gene is suddenlyd change 5 times.The mutation type that these mutated embryonic stem cell banks had will cover the mouse genomic gene more than 90% in theory.
Particularly, the principle that the inventive method adopts polyA to catch, used gene capturing carrier from 5 ' to 3 ' structure as follows:
(a) gene capturing carrier is imported embryonic stem cell, described gene capturing carrier from 5 ' to 3 ' structure be:
SA-ST-DS-S1-P1-P2-S2-pA-P3-S3-SD-Ap-TS-AS
In the formula:
SA is the receptor sequence of RNA montage;
ST is a forward series connection terminator codon;
DS is the donor sequences of reverse RNA montage;
S1 is the first reverse screening-gene, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or lacZ gene;
P1 is the first reverse promotor,
P2 is second promotor of forward;
S2 is second screening-gene of forward, is selected from down group: hygromycin B resistant gene Hygro, neomycin resistance gene Neo or puromycin resistance gene Puro;
PA is a forward RNA tailing signal sequence;
P3 is the 3rd promotor of forward;
S3 is the three screening gene of forward, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or lacZ gene;
SD is the donor sequences of RNA montage;
Ap is reverse RNA tailing signal sequence;
TS is reverse three terminator codons;
AS is the receptor sequence of reverse RNA montage;
P1, P2 and P3 are the promotors that is selected from down group respectively: CMV, PGK, LTP or hsv-TK;
Condition is S1, and S2 and S3 have nothing in common with each other, and a screening-gene is arranged among S1 and the S3 is the screening-gene of non-lethality, as the encoding sequence GFP of green fluorescent protein, the encoding sequence EGFP or the lacZ gene of enhanced green fluorescent protein.Another screening-gene and S2 are preferably the screening-gene of lethality among S1 and the S3, as Totomycin B resistant gene Hygro, neomycin resistance gene Neo or puromycin resistance gene Puro.
Plasmid construction of the present invention can carry out with ordinary method.Usually, maternal plasmid is retroviral vector or other cloning vector commonly used, for example cloning vector commonly used such as pBluescript, UC18/19
Screening-gene can be drug resistant gene such as Neo, Puro, Hygro, Pac, or reporter gene such as GFP etc.Be used for of the present invention first and the three screening gene need remove polyA signal area after the gene terminator codon.
Each screening-gene can be used in to have the active promotor of gene promoter and transcribes in the embryonic stem cell, as promotors such as CMV, PGK, hsv-TK, LTP.
Carrier 5 ' end is connected with the receptor sequence (SA) of forward RNA montage and the donor sequences (DS) of reverse RNA montage, and 3 of carrier ' end is connected with the donor sequences (SD) of forward RNA montage and the receptor sequence (AS) of reverse RNA montage.Be applicable to that SA of the present invention, SD, AS and DS element are not particularly limited, and can select various SA known in the art, SD, AS and DS element for use.
Gene capturing carrier imports to and can utilize ordinary method to carry out in the embryonic stem cell, infects method or electrotransfer method as pseudovirus.
After importing embryonic stem cell, two-way gene capturing carrier of the present invention is inserted into 4 kinds of situations in the gene karyomit(e), be respectively:
1) is inserted into 5 ' control region of gene, causes gene control region to destroy and can not the normal transcription downstream gene;
2) be inserted in the gene extron subregion,, then cause protein-coding region to be interrupted if this exon is the encoding histone zone;
3) be inserted in the gene intron, in RNA montage process, GFP or Hygro gene will be fused into a mRNA molecule with the upstream and downstream exon of this intron, if the exon in this intron downstream is an albumen coded sequence, then cause protein-coding region to be interrupted;
4) be inserted into gene not in the exon of proteins encoded, if be inserted in the non-coding region exon of gene 5 ' end, then the RNA of Chan Shenging will only translate the frame of GFP or Hygro gene, the gene that is inserted into can not be translated, if be inserted in the non-coding region of gene 3 ' end, then the function to gene may not influence.
No matter be which kind of situation, if gene capturing carrier is inserted in some genes, then the screening-gene that provides of this carrier and the polyA signal bits inserting gene and provide are named a person for a particular job and are given this embryonic stem cell and have the characteristic (green fluorescence or hygromycin resistance etc.) that the screening-gene expression product has; If gene capturing carrier is not inserted in the gene region, screening-gene is not owing to self carry the signal of polyA, and therefore the RNA that transcribes can not stably exist and translate.So the embryonic stem cell clone who survives through screening, certain gene all having taken place be inserted into the incident of gene capturing carrier, so all has been the embryonic stem cell of sudden change.
After gene capturing carrier imported to embryonic stem cell, available ordinary method screening and cultivate the embryonic stem cell that has inserted gene capturing carrier.Its process, step and operational condition all are as known in the art.
In the present invention, term " merging " refers to the embryonic stem cell of each survival is put together, and constitutes mutated embryonic stem cell bank.Can to be (1) mix the embryonic stem cell of a plurality of survivals in this placement, constitutes inferior storehouse; (2) genomic dna and/or the cDNA data with the embryonic stem cell of each survival are placed on information database together, and embryonic stem cell of each survival remains and deposits separately; (3) combination of above-mentioned laying method.
For the embryonic stem cell of each sudden change that filters out, the embryonic stem cell bank of sudden change of the present invention will just have been obtained after its merging.Can obtain to contain ten thousand of 5-30 at short notice with the inventive method, preferably 10-20 is ten thousand, more preferably about 150,000 mutated embryonic stem cells clone's cell bank.
In another preference, cell bank of the present invention will further be divided into 500-1000 inferior storehouse, and each inferior storehouse has 150-300 kind embryo stem cell clone.At each inferior storehouse, mixing cDNA with the corresponding embryonic stem cell of preparation, utilize these cDNA, can adopt PCR or other method to determine the Ya Ku at needed mutated embryonic stem cell clone place easily, in corresponding inferior storehouse, each embryonic stem cell clone is screened then, obtain corresponding mutated embryonic stem cell clone.
A kind of embryonic stem cell clone's of preferred acquisition specific gene sudden change method is, sequence according to specific gene, screen the cDNA sample in inferior storehouse with PCR, candidate's the embryonic stem cell clone that specific gene inserts sudden change that contains is limited in certain (several) individual inferior storehouse.Then, for each the embryonic stem cell clone in the inferior storehouse, filter out the embryonic stem cell clone who contains specific gene insertion sudden change accordingly with identical PCR method.
On the other hand, can utilize corresponding DNA and the cDNA of each embryonic stem cell clone, adopt the method for PCR and RACE to determine that carrier inserts genomic site and the corresponding mutator gene of each embryonic stem cell clone, sets up and the corresponding gene mutation site information database in embryonic stem cell mutant cell storehouse.According to the research needs, the mutant clon of selecting wherein to have important biomolecule function carries out the mouse species that microinjection can obtain this gene insertion sudden change.Adopt this strategy will greatly improve gene recognition and gene knockout mice Study of model efficient, reduce research cost, easily form " industrialization " research general layout.
The major advantage of the inventive method is:
(1) compares with the method that traditional usefulness homologous recombination mode is carried out gene knockout, the inventive method has efficient, economic characteristics, particularly having exempted need be at the complicated processes of each gene constructed gene targeting carrier, thereby obtains mutant mouse and lay a good foundation for extensive, high-throughput.
(2) after capturing carrier of the present invention inserts genome, can two-wayly catch the gene of transcribing, therefore significantly improve capture rate by positive and negative two chains of genome.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the structure of gene capturing carrier
Maternal plasmid is pBluescript, and screening-gene is as follows:
First screening-gene is the screening-gene of the PolyA signal area after the removal gene terminator codon, does not promptly have the hygromycin gene B (Hygro) of PolyA signal;
Second screening-gene is the complete neomycin resistance gene Neo with polyA signal;
The three screening gene is the screening-gene of the PolyA signal area after the removal gene terminator codon, does not promptly have the EGFP gene of PolyA signal.
First and the three screening gene drive and to transcribe because of starting active promotor PGK, hsv-TK by tool strong basis in the ES cell respectively, the two direction is opposite.
First and three screening gene 3 ' end be connected with the donor sequences (SD) of RNA montage respectively, 5 ' end is connected with the receptor sequence (SA) and the series connection termination codon of RNA montage.
The plasmid construction method is carried out routinely, thereby obtains the following gene capturing carrier of structure:
SA-ST-DS-Hygro-KTp-PGK-Neo-pA-PGK-EGFP-SD-Ap-TS-AS
The sequence of whole gene capturing carrier is as follows:
ccgcggttta?aggagaccaa?tagaaactgg?gcttgtcgag?acagagaaga?ctcttgcgtt 60
tctgataggc?acctattggt?cttactgaca?tccactttgc?ctttctctcc?acaggatagc 120
taactgagat?ccccctccta?cttacctcag?tcagtcagtt?agcctccccc?atctcccgat 180
ccggacgagt?gctggggcgt?cggtttccac?tatcggcgag?tacttctaca?cagccatcgg 240
tccagacggc?cgcgcttctg?cgggcgattt?gtgtacgccc?gacagtcccg?gctccggatc 300
ggacgattgc?gtcgcatcga?ccctgcgccc?aagctgcatc?atcgaaattg?ccgtcaacca 360
agctctgata?gagttggtca?agaccaatgc?ggagcatata?cgcccggagc?cgcggcgatc 420
ctgcaagctc?cggatgcctc?cgctcgaagt?agcgcgtctg?ctgctccata?caagccaacc 480
acggcctcca?gaagaagatg?ttggcgacct?cgtattggga?atccccgaac?atcgcctcgc 540
tccagtcaat?gaccgctgtt?atgcggccat?tgtccgtcag?gacattgttg?gagccgaaat 600
ccgcgtgcac?gaggtgccgg?acttcggggc?agtcctcggc?ccaaagcatc?agctcatcga 660
gagcctgcgc?gacggacgca?ctgacggtgt?cgtccatcac?agtttgccag?tgatacacat 720
ggggatcagc?aatcgcgcat?atgaaatcac?gccatgtagt?gtattgaccg?attccttgcg 780
gtccgaatgg?gccgaacccg?ctcgtctggc?taagatcggc?cgcagcgatc?gcatccatgg 840
cctccgcgac?cggctgcaga?acagcgggca?gttcggtttc?aggcaggtct?tgcaacgtga 900
caccctgtgc?acggcgggag?atgcaatagg?tcaggctctc?gctgaattcc?ccaatgtcaa 960
gcacttccgg?aatcgggagc?gcggccgatg?caaagtgccg?ataaacataa?cgatctttgt 1020
agaaaccatc?ggcgcagcta?tttacccgca?ggacatatcc?acgccctcct?acatcgaagc 1080
tgaaagcacg?agattcttcg?ccctccgaga?gctgcatcag?gtcggagacg?ctgtcgaact 1140
tttcgatcag?aaacttctcg?acagacgtcg?cggtgagttc?aggctttttc?atatctcatt 1200
gccccccggg?atctgcggca?cgctgttgac?gctgttaagc?gggtcgctgc?agggtcgctc 1260
ggtgttcgag?gccacacgcg?tcaccttaat?atgcgaagtg?gacctcggac?cgcgccgccc 1320
cgactgcatc?tgcgtgttcg?aattcgccaa?tgacaagacg?ctgggcgggg?tttgtgtcat 1380
catagaacta?aagacatgca?aatatatttc?ttccggggac?accgccagca?aacgcgagca 1440
acgggccacg?gggatgaagc?aggctcgagg?gcccctgcag?gtcaattcta?ccgggtaggg 1500
gaggcgcttt?tcccaaggca?gtctggagca?tgcgctttag?cagccccgct?ggcacttggc 1560
gctacacaag?tggcctctgg?cctcgcacac?attccacatc?caccggtagc?gccaaccggc 1620
tccgttcttt?ggtggcccct?tcgcgccacc?ttctactcct?cccctagtca?ggaagttccc 1680
ccccgccccg?cagctcgcgt?cgtgcaggac?gtgacaaatg?gaagtagcac?gtctcactag 1740
tctcgtgcag?atggacagca?ccgctgagca?atggaagcgg?gtaggccttt?ggggcagcgg 1800
ccaatagcag?ctttgctcct?tcgctttctg?ggctcagagg?ctgggaaggg?gtgggtccgg 1860
gggcgggctc?aggggcgggc?tcaggggcgg?ggcgggcgcg?aaggtcctcc?cgaggcccgg 1920
cattctcgca?cgcttcaaaa?gcgcacgtct?gccgcgctgt?tctcctcttc?ctcatctccg 1980
ggcctttcga?cctgcagcca?atatgggatc?ggccattgaa?caagatggat?tgcacgcagg 2040
ttctccggcc?gcttgggtgg?agaggctatt?cggctatgac?tgggcacaac?agacaatcgg 2100
ctgctctgat?gccgccgtgt?tccggctgtc?agcgcagggg?cgcccggttc?tttttgtcaa 2160
gaccgacctg?tccggtgccc?tgaatgaact?gcaggacgag?gcagcgcggc?tatcgtggct 2220
ggccacgacg?ggcgttcctt?gcgcagctgt?gctcgacgtt?gtcactgaag?cgggaaggga 2280
ctggctgcta?ttgggcgaag?tgccggggca?ggatctcctg?tcatctcacc?ttgctcctgc 2340
cgagaaagta?tccatcatgg?ctgatgcaat?gcggcggctg?catacgcttg?atccggctac 2400
ctgcccattc?gaccaccaag?cgaaacatcg?catcgagcga?gcacgtactc?ggatggaagc 2460
cggtcttgtc?gatcaggatg?atctggacga?agagcatcag?gggctcgcgc?cagccgaact 2520
gttcgccagg?ctcaaggcgc?gcatgcccga?cggcgatgat?ctcgtcgtga?cccatggcga 2580
tgcctgcttg?ccgaatatca?tggtggaaaa?tggccgcttt?tctggattca?tcgactgtgg 2640
ccggctgggt?gtggcggacc?gctatcagga?catagcgttg?gctacccgtg?atattgctga 2700
agagcttggc?ggcgaatggg?ctgaccgctt?cctcgtgctt?tacggtatcg?ccgctcccga 2760
ttcgcagcgc?atcgccttct?atcgccttct?tgacgagttc?ttctgagggg?atcgatccgt 2820
cctgtaagtc?tgcagaaatt?gatgatctat?taaacaataa?agatgtccac?taaaatggaa 2880
gtttttcctg?tcatactttg?ttaagaaggg?tgagaacaga?gtacctacat?tttgaatgga 2940
aggattggag?ctacgggggt?gggggtgggg?tgggattaga?taaatgcctg?ctctttactg 3000
aaggctcttt?actattgctt?tatgataatg?tttcatagtt?ggatatcata?atttaaacaa 3060
gcaaaaccaa?attaagggcc?agctcattcc?tcccactcat?gatctataga?tctatagatc 3120
tctcgtggga?tcattgtttt?tctcttgatt?cccactttgt?ggttctaagt?actgtggttt 3180
ccaaatgtgt?cagtttcata?gcctgaagaa?cgagatcagc?agcctctgtt?ccacatacac 3240
ttcattctca?gtattgtttt?gccaagttct?aattccatca?gaagctgact?ctagaggatc 3300
cccgggtacc?cttttcccaa?ggcagtctgg?agcatgcgct?ttagcagccc?cgctggcact 3360
tggcgctaca?caagtggcct?ctggcctcgc?acacattcca?catccaccgg?tagcgccaac 3420
cggctccgtt?ctttggtggc?cccttcgcgc?caccttctac?tcctccccta?gtcaggaagt 3480
tcccccccgc?cccgcagctc?gcgtcgtgca?ggacgtgaca?aatggaagta?gcacgtctca 3540
ctagtctcgt?gcagatggac?agcaccgctg?agcaatggaa?gcgggtaggc?ctttggggca 3600
gcggccaata?gcagctttgc?tccttcgctt?tctgggctca?gaggctggga?aggggtgggt 3660
ccgggggcgg?gctcaggggc?gggctcaggg?gcggggcggg?cgcgaaggtc?ctcccgaggc 3720
ccggcattct?cgcacgcttc?aaaagcgcac?gtctgccgcg?ctgttctcct?cttcctcatc 3780
tccgggcctt?tcgacctgca?gccaatcgat?atggtgagca?agggcgagga?gctgttcacc 3840
ggggtggtgc?ccatcctggt?cgagctggac?ggcgacgtaa?acggccacaa?gttcagcgtg 3900
tccggcgagg?gcgagggcga?tgccacctac?ggcaagctga?ccctgaagtt?catctgcacc 3960
accggcaagc?tgcccgtgcc?ctggcccacc?ctcgtgacca?ccctgaccta?cggcgtgcag?4020
tgcttcagcc?gctaccccga?ccacatgaag?cagcacgact?tcttcaagtc?cgccatgccc?4080
gaaggctacg?tccaggagcg?caccatcttc?ttcaaggacg?acggcaacta?caagacccgc?4140
gccgaggtga?agttcgaggg?cgacaccctg?gtgaaccgca?tcgagctgaa?gggcatcgac?4200
ttcaaggagg?acggcaacat?cctggggcac?aagctggagt?acaactacaa?cagccacaac?4260
gtctatatca?tggccgacaa?gcagaagaac?ggcatcaagg?tgaacttcaa?gatccgccac?4320
aacatcgagg?acggcagcgt?gcagctcgcc?gaccactacc?agcagaacac?ccccatcggc?4380
gacggccccg?tgctgctgcc?cgacaaccac?tacctgagca?cccagtccgc?cctgagcaaa?4440
gaccccaacg?agaagcgcga?tcacatggtc?ctgctggagt?tcgtgaccgc?cgccgggatc?4500
actctcggca?tggacgagct?gtacaagtaa?gtaagtggat?ggatccttac?cacatttgta?4560
gaggttttac?ttgctttaaa?aaacctccca?catctccccc?tgaacctgaa?acataaaatg?4620
aatgcaattg?ttgttgttaa?cttgtttatt?gcagcttata?atggttacaa?ataaagcaat?4680
agcatcacaa?atttcacaaa?taaagcattt?ttttcactgc?attctagttg?tggtttgtcc?4740
aaactcatca?atgtatctta?tcatgtctgt?ctagattagt?cagtcagagc?ttatcgatac?4800
cgtcgatccc?cactggaaag?accgcgaaga?gtttgtcctc?aaccgcgagc?tgtggaaaaa?4860
aaagggacag?gataagtatg?acatcatcaa?ggaaaccctg?gactactgcg?ccctacagat?4920
ctgcagcccg?ggggatccac?tagttctagc?ctcgaaattc?ctgcaggtcg?agggacctaa?4980
taacaagctt 4990(SEQ?ID?NO：1)
Wherein, each position of components is as follows in the sequence:
SA：100-115
ST：116-132
DS：145-139
Hygromycin gene Hygro:1192-140
TK promotor: 1463-1201
PGK：1507-2002，3305-3810
NEO：2003-2806
pA：2809-3302
EGFP：3811-4530
SD：4531-4545
Ap：4770-4546
TS：4790-4771
AS：4990-4773。
Embodiment 2: the foundation of mutated embryonic stem cell bank
In the present embodiment, gene capturing carrier imports to the method that can adopt electrotransfer in the ES cell.
Adopt Bio-Rad electricity conversion instrument 500 μ F/240V in the present embodiment, 1.2 * 10
7Cell transfecting 30-40ug linearizing gene capturing carrier plasmid.After 24-36 hour, use the substratum that contains G418 (0.3mg/ml) that above-mentioned transfectional cell is cultivated with mono-clonal and screen.Pick out the green fluorescence cell clone after 6-7 days from the opposing clone, frozen after the enlarged culturing, cracking prepares DNA, RNA.All the other opposing clones continue screening after 7-10 days with the substratum that contains hygromycin B (0.15mg/ml), and the opposing clone that will survive chooses, and frozen after the enlarged culturing, cracking prepares DNA, RNA.
The ES cell cultures is undertaken by standard method.The trophocyte has the mouse embryo fibroblasts of Hygro-Neo gene for the former foster commentaries on classics of handling with mitomycin of being commissioned to train.From liquid nitrogen container, take out a frozen ES cell, drop into 37 ℃ of water-baths rapidly; Put into the 15ml Falcon centrifuge tube of being with 10ml ES nutrient solution with aseptic pasteur pipe sucking-off cell, 1000rpm is centrifugal 5 minutes under the room temperature; Abandon supernatant, cell washs once with 10ml 1xPBS, the same centrifugal removal supernatant; With an amount of nutrient solution suspension cell again, seed cells in the trophocyte's who is covered with the mitomycin processing the 100mm culture dish.Cell places 37 ℃, 5%CO
2Cell culture incubator in, keep enough humidity, renew bright nutrient solution every day, long to proper density until the ES cell, this process needs 2-3 days usually.The ES cell that is in logarithmic phase is changed nutrient solution at results precontract 4h, with twice of PBS rinsing, add 2ml 0.05% trypsinase/EDTA solution, digest 3-5min under the room temperature, add 10ml ES cell culture fluid and stop digestion, blow and beat into single cell suspension, move in the 15ml centrifuge tube with suction pipe, the centrifugal 5min collecting cell of 1000rpm is with 10ml PBS re-suspended cell and counting.Collecting cell after centrifugal removes supernatant, adds an amount of PBS and makes cell density reach about 1.2 * 10
7/ ml.Get in the above-mentioned ES cell suspension of 0.8ml and to add 0.1ml and contain Ca
2+And Mg
2+PBS (containing about 40 μ g) through linearizing gene trap plasmid, be added to behind the mixing in the aseptic electroporation cup.Under the condition of normal temperature,, after the electrical parameter of 500 μ F carries out monopulse percussion, be suspended in again in the plate of 3.0ml ES inoculation of medium to 2 100mm and cultivate with 240V.Carry out G418 (0.3-0.6mg/ml) drug screening behind the 24-36h, change nutrient solution every day.After 5 days, change liquid every other day, ES cell positive clonal growth after 6-7 days the ES cell clone grow up to macroscopic cell colony, can the picking green fluorescence clone.Method is as follows: inhale and remove nutrient solution, with 10ml PBS rinsing one time, add 5ml PBS again; At microscopically, with the Tip absorption positive colony of 0.2ml, put into the about 5min of 96 orifice plates (concave bottom) digestion that contains 20 μ l, 0.1% trypsinase/0.04%EDTA, piping and druming gently disperses cell.Be transferred to collagenzation and also added in 96 orifice plates of ES cell culture medium, put in the incubator and cultivate.Changed liquid in second day, treat that cell covers with 60%-80% after, reach 48 orifice plates and cultivate, after treating that cell covers with 60%-80%, get wherein 3/4 cell cryopreservation, remaining 1/4 is seeded to two only in 48 orifice plates (non-trophoblast) of collagenzation, is used for extracting genomic dna and RNA after cell 100% covers with.Residue ES cell positive clone contains the substratum continuation cultivation screening of hygromycin B (0.05-0.20mg/ml) in the 100mm culture dish, changes nutrient solution every day.After 5 days, change liquid every other day, after 7-10 days, picking survival opposing clone, a frozen after the enlarged culturing, another part is used to prepare genomic dna, RNA.
Repeat said procedure, the inferior storehouse of the upright mutated embryonic stem cell 500-1000 part of building together, each inferior storehouse contains 100-300 cell clone.
Embodiment 3: the preparation of sudden change ES cell clone genomic dna and cDNA
For each the sudden change ES cell clone among the embodiment 2, from 48 orifice plates that cover with the ES cell, inhale and remove nutrient solution, with the PBS washing once, every hole adds 500 μ l cell pyrolysis liquids (1mg/ml Proteinase K), 56 ℃ of digested overnight, add the 1ml dehydrated alcohol, centrifugal 10min abandons supernatant, 70% ethanol, wash 2 times, be dissolved in 100 μ l ddH
2Standby among the O.The a RNA of corresponding preparation, every hole covers with in 48 orifice plates of ES cell and adds 500 μ l Trizol, and piping and druming is collected preparation RNA in the back several times repeatedly, and method is carried out according to the specification sheets of Trizol (Invitrogen company).Adopt polydT and random primer to obtain cDNA as the primer of reverse transcription.
For the inferior storehouse of each mutated embryonic stem cell that embodiment 2 sets up, a genomic dna of corresponding foundation and cDNA (all sudden change ES cell clone genomic dna mixing/cDNA mix in the inferior storehouse), common phase should prepare 500-1000 part; The preparation method of cells involved RNA and genomic dna is undertaken by standard method. and adopt polydT and random primer to obtain inferior storehouse cDNA. as the primer of reverse transcription
Embodiment 4: the screening of specific gene sudden change ES cell clone
Contain the ES cell clone of specifying transgenation for screening, adopt the method for conventional PCR to carry out, for clone inferior storehouse for green fluorescence, 5 ' end primer design is selected gene capturing carrier 3 ' terminal sequence CTA CCAGCA GAA CAC CCC CAT (SEQ ID NO:2), the selection of 3 ' end primer is waited to study gene and is stopped near the exon sequence in coding back, can be designed to the nest-type PRC primer in case of necessity, for the inferior storehouse of hygromycin B opposing clone, 5 ' end primer design is selected the complementary sequence cgt aca caa atcgcc cgc aga (SEQ ID NO:3) of gene capturing carrier 5 ' terminal sequence, the selection of 3 ' end primer is waited to study gene and is stopped near the exon sequence in coding back, can be designed to the nest-type PRC primer in case of necessity, with above-mentioned primer the cDNA in all inferior storehouses of sudden change ES cell of obtaining among the embodiment 3 is carried out the PCR screening, there is positive products to carry out dna sequence analysis and checking to obtaining, acquisition male cDNA storehouse to checking, get the cDNA (cDNA that each clone retains separately) of all mutant clons in the inferior storehouse of sudden change ES cell of its correspondence, be one group by 10-30 and be mixed into the performing PCR screening, further reduce the scope and carry out the PCR screening up to definite positive clone of single sudden change ES cell clone. the ES cell clone of the sudden change of depositing separately in the corresponding inferior storehouse of recovering, extract RNA after the enlarged culturing, reverse transcription obtains to repeat above-mentioned PCR behind the cDNA, can determine after the checking positive that this clone is required clone.
Embodiment 4: the evaluation in sudden change ES cell clone gene trap site
For the sudden change ES cell clone genomic dna among the embodiment 3, can adopt inverse PCR or joint method PCR to obtain the genome sequence that carrier inserts the both sides, site, determine that carrier inserts the position in the genome.
After adopting Sau3AI or MspI digestion ES cell clone genomic dna in the present embodiment, after dna ligase connects, utilize two pairs of primers to carry out inverse PCR and obtain carrier two terminal sequences native gene group sequence in addition according to carrier two ends sequences Design.For the sudden change ES cell clone RNA among the embodiment 3, the method for available 3 '-RACE and 5 '-RACE obtains the cDNA sequence that carrier inserts the both sides, site.In the present embodiment for green fluorescence clone gained RNA according to EGFP3 ' terminal sequence design primer CTA CCA GCA GAA CAC CCCCAT (SEQ ID NO:4) carry out 3 '-RACE, designing primer ATC AGA GCT TGG TTG ACG GCA (SEQ ID NO:5) according to hygromycin gene 3 ' terminal sequence (carrier 5 ' end SA downstream) carries out 5 '-RACE; For hygromycin B opposing clone gained RNA according to hygromycin gene 3 ' terminal sequence design primer CGTACA CAA ATC GCC CGC AGA (SEQ ID NO:6) carry out 3 '-RACE, according to gene capturing carrier 3 ' terminal sequence (carrier the other end SA downstream) design primer ATC TCC CCC TGA ACC TGAAAC (SEQ ID NO:7) carry out 5 '-RACE.3 '-RACE and 5 '-RACE all carries out with reference to the specification sheets of Clontech test kit.
After identifying and obtaining needed mutated embryonic stem cell clone, can corresponding frozen embryonic stem cell recovery, cultivate and be used to set up gene knockout mice after increasing and maybe can carry out other research.
Embodiment 5: the foundation of sudden change ES cell clone information database
For the sudden change ES cell clone gene capturing carrier both sides genomic dna and the cDNA sequence that obtain among the embodiment 4, carry out bioinformatic analysis, determine the gene that is hunted down, whether its gene function of analyses and prediction is influenced, find its sino-singaporean gene, above information classification is gathered, set up database, and corresponding with sudden change ES cell bank classifying and numbering.
Further can select wherein interested mutant clon to carry out microinjection and can obtain the mouse species that this gene inserts sudden change, study the function of this gene.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Nanfangmoshi Biological Sci-Tech Dev Co., Ltd.
Ruijin Hospital Attached to Shanghai Medical Univ No.2
Shanghai Second Emdical University of Shanghai life science institute of Chinese Academy of Sciences health science center
Shanghai Second Emdical University
＜120〉a kind of method of setting up the mouse full genome mutated embryonic stem cell bank
<130>033914
<160>7
<170>PatentIn?version?3.1
<210>1
<211>4990
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(4990)
＜223〉gene capturing carrier
<400>1
ccgcggttta?aggagaccaa?tagaaactgg?gcttgtcgag?acagagaaga?ctcttgcgtt 60
tctgataggc?acctattggt?cttactgaca?tccactttgc?ctttctctcc?acaggatagc 120
taactgagat?ccccctccta?cttacctcag?tcagtcagtt?agcctccccc?atctcccgat 180
ccggacgagt?gctggggcgt?cggtttccac?tatcggcgag?tacttctaca?cagccatcgg 240
tccagacggc?cgcgcttctg?cgggcgattt?gtgtacgccc?gacagtcccg?gctccggatc 300
ggacgattgc?gtcgcatcga?ccctgcgccc?aagctgcatc?atcgaaattg?ccgtcaacca 360
agctctgata?gagttggtca?agaccaatgc?ggagcatata?cgcccggagc?cgcggcgatc 420
ctgcaagctc?cggatgcctc?cgctcgaagt?agcgcgtctg?ctgctccata?caagccaacc 480
acggcctcca?gaagaagatg?ttggcgacct?cgtattggga?atccccgaac?atcgcctcgc 540
tccagtcaat?gaccgctgtt?atgcggccat?tgtccgtcag?gacattgttg?gagccgaaat 600
ccgcgtgcac?gaggtgccgg?acttcggggc?agtcctcggc?ccaaagcatc?agctcatcga 660
gagcctgcgc?gacggacgca?ctgacggtgt?cgtccatcac?agtttgccag?tgatacacat 720
ggggatcagc?aatcgcgcat?atgaaatcac?gccatgtagt?gtattgaccg?attccttgcg 780
gtccgaatgg?gccgaacccg?ctcgtctggc?taagatcggc?cgcagcgatc?gcatccatgg 840
cctccgcgac?cggctgcaga?acagcgggca?gttcggtttc?aggcaggtct?tgcaacgtga 900
caccctgtgc?acggcgggag?atgcaatagg?tcaggctctc?gctgaattcc?ccaatgtcaa 960
gcacttccgg?aatcgggagc?gcggccgatg?caaagtgccg?ataaacataa?cgatctttgt 1020
agaaaccatc?ggcgcagcta?tttacccgca?ggacatatcc?acgccctcct?acatcgaagc 1080
tgaaagcacg?agattcttcg?ccctccgaga?gctgcatcag?gtcggagacg?ctgtcgaact 1140
tttcgatcag?aaacttctcg?acagacgtcg?cggtgagttc?aggctttttc?atatctcatt 1200
gccccccggg?atctgcggca?cgctgttgac?gctgttaagc?gggtcgctgc?agggtcgctc 1260
ggtgttcgag?gccacacgcg?tcaccttaat?atgcgaagtg?gacctcggac?cgcgccgccc 1320
cgactgcatc?tgcgtgttcg?aattcgccaa?tgacaagacg?ctgggcgggg?tttgtgtcat 1380
catagaacta?aagacatgca?aatatatttc?ttccggggac?accgccagca?aacgcgagca 1440
acgggccacg?gggatgaagc?aggctcgagg?gcccctgcag?gtcaattcta?ccgggtaggg 1500
gaggcgcttt?tcccaaggca?gtctggagca?tgcgctttag?cagccccgct?ggcacttggc 1560
gctacacaag?tggcctctgg?cctcgcacac?attccacatc?caccggtagc?gccaaccggc 1620
tccgttcttt?ggtggcccct?tcgcgccacc?ttctactcct?cccctagtca?ggaagttccc 1680
ccccgccccg?cagctcgcgt?cgtgcaggac?gtgacaaatg?gaagtagcac?gtctcactag 1740
tctcgtgcag?atggacagca?ccgctgagca?atggaagcgg?gtaggccttt?ggggcagcgg 1800
ccaatagcag?ctttgctcct?tcgctttctg?ggctcagagg?ctgggaaggg?gtgggtccgg 1860
gggcgggctc?aggggcgggc?tcaggggcgg?ggcgggcgcg?aaggtcctcc?cgaggcccgg 1920
cattctcgca?cgcttcaaaa?gcgcacgtct?gccgcgctgt?tctcctcttc?ctcatctccg 1980
ggcctttcga?cctgcagcca?atatgggatc?ggccattgaa?caagatggat?tgcacgcagg 2040
ttctccggcc?gcttgggtgg?agaggctatt?cggctatgac?tgggcacaac?agacaatcgg 2100
ctgctctgat?gccgccgtgt?tccggctgtc?agcgcagggg?cgcccggttc?tttttgtcaa 2160
gaccgacctg?tccggtgccc?tgaatgaact?gcaggacgag?gcagcgcggc?tatcgtggct 2220
ggccacgacg?ggcgttcctt?gcgcagctgt?gctcgacgtt?gtcactgaag?cgggaaggga 2280
ctggctgcta?ttgggcgaag?tgccggggca?ggatctcctg?tcatctcacc?ttgctcctgc 2340
cgagaaagta?tccatcatgg?ctgatgcaat?gcggcggctg?catacgcttg?atccggctac 2400
ctgcccattc?gaccaccaag?cgaaacatcg?catcgagcga?gcacgtactc?ggatggaagc 2460
cggtcttgtc?gatcaggatg?atctggacga?agagcatcag?gggctcgcgc?cagccgaact 2520
gttcgccagg?ctcaaggcgc?gcatgcccga?cggcgatgat?ctcgtcgtga?cccatggcga 2580
tgcctgcttg?ccgaatatca?tggtggaaaa?tggccgcttt?tctggattca?tcgactgtgg 2640
ccggctgggt?gtggcggacc?gctatcagga?catagcgttg?gctacccgtg?atattgctga 2700
agagcttggc?ggcgaatggg?ctgaccgctt?cctcgtgctt?tacggtatcg?ccgctcccga 2760
ttcgcagcgc?atcgccttct?atcgccttct?tgacgagttc?ttctgagggg?atcgatccgt 2820
cctgtaagtc?tgcagaaatt?gatgatctat?taaacaataa?agatgtccac?taaaatggaa 2880
gtttttcctg?tcatactttg?ttaagaaggg?tgagaacaga?gtacctacat?tttgaatgga 2940
aggattggag?ctacgggggt?gggggtgggg?tgggattaga?taaatgcctg?ctctttactg 3000
aaggctcttt?actattgctt?tatgataatg?tttcatagtt?ggatatcata?atttaaacaa 3060
gcaaaaccaa?attaagggcc?agctcattcc?tcccactcat?gatctataga?tctatagatc 3120
tctcgtggga?tcattgtttt?tctcttgatt?cccactttgt?ggttctaagt?actgtggttt 3180
ccaaatgtgt?cagtttcata?gcctgaagaa?cgagatcagc?agcctctgtt?ccacatacac 3240
ttcattctca?gtattgtttt?gccaagttct?aattccatca?gaagctgact?ctagaggatc 3300
cccgggtacc?cttttcccaa?ggcagtctgg?agcatgcgct?ttagcagccc?cgctggcact 3360
tggcgctaca?caagtggcct?ctggcctcgc?acacattcca?catccaccgg?tagcgccaac 3420
cggctccgtt?ctttggtggc?cccttcgcgc?caccttctac?tcctccccta?gtcaggaagt 3480
tcccccccgc?cccgcagctc?gcgtcgtgca?ggacgtgaca?aatggaagta?gcacgtctca 3540
ctagtctcgt?gcagatggac?agcaccgctg?agcaatggaa?gcgggtaggc?ctttggggca 3600
gcggccaata?gcagctttgc?tccttcgctt?tctgggctca?gaggctggga?aggggtgggt 3660
ccgggggcgg?gctcaggggc?gggctcaggg?gcggggcggg?cgcgaaggtc?ctcccgaggc 3720
ccggcattct?cgcacgcttc?aaaagcgcac?gtctgccgcg?ctgttctcct?cttcctcatc 3780
tccgggcctt?tcgacctgca?gccaatcgat?atggtgagca?agggcgagga?gctgttcacc 3840
ggggtggtgc?ccatcctggt?cgagctggac?ggcgacgtaa?acggccacaa?gttcagcgtg 3900
tccggcgagg?gcgagggcga?tgccacctac?ggcaagctga?ccctgaagtt?catctgcacc 3960
accggcaagc?tgcccgtgcc?ctggcccacc?ctcgtgacca?ccctgaccta?cggcgtgcag 4020
tgcttcagcc?gctaccccga?ccacatgaag?cagcacgact?tcttcaagtc?cgccatgccc 4080
gaaggctacg?tccaggagcg?caccatcttc?ttcaaggacg?acggcaacta?caagacccgc 4140
gccgaggtga?agttcgaggg?cgacaccctg?gtgaaccgca?tcgagctgaa?gggcatcgac 4200
ttcaaggagg?acggcaacat?cctggggcac?aagctggagt?acaactacaa?cagccacaac 4260
gtctatatca?tggccgacaa?gcagaagaac?ggcatcaagg?tgaacttcaa?gatccgccac 4320
aacatcgagg?acggcagcgt?gcagctcgcc?gaccactacc?agcagaacac?ccccatcggc 4380
gacggccccg?tgctgctgcc?cgacaaccac?tacctgagca?cccagtccgc?cctgagcaaa 4440
gaccccaacg?agaagcgcga?tcacatggtc?ctgctggagt?tcgtgaccgc?cgccgggatc 4500
actctcggca?tggacgagct?gtacaagtaa?gtaagtggat?ggatccttac?cacatttgta 4560
gaggttttac?ttgctttaaa?aaacctccca?catctccccc?tgaacctgaa?acataaaatg 4620
aatgcaattg?ttgttgttaa?cttgtttatt?gcagcttata?atggttacaa?ataaagcaat 4680
agcatcacaa?atttcacaaa?taaagcattt?ttttcactgc?attctagttg?tggtttgtcc 4740
aaactcatca?atgtatctta?tcatgtctgt?ctagattagt?cagtcagagc?ttatcgatac 4800
cgtcgatccc?cactggaaag?accgcgaaga?gtttgtcctc?aaccgcgagc?tgtggaaaaa 4860
aaagggacag?gataagtatg?acatcatcaa?ggaaaccctg?gactactgcg?ccctacagat 4920
ctgcagcccg?ggggatccac?tagttctagc?ctcgaaattc?ctgcaggtcg?agggacctaa 4980
taacaagctt 4990
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>2
ctaccagcag?aacaccccca?t 21
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
cgtacacaaa?tcgcccgcag?a 21
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>4
ctaccagcag?aacaccccca?t 21
<210>5
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
atcagagctt?ggttgacggc?a 21
<210>6
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
cgtacacaaa?tcgcccgcag?a 21
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>7
atctccccct?gaacctgaaa?c 21
Claims (10)
1. a method of setting up extensive mutated embryonic stem cell bank is characterized in that, comprises step:
(a) gene capturing carrier is imported embryonic stem cell, wherein said embryonic stem cell is the embryonic stem cell of mouse or rat, described gene capturing carrier from 5 ' to 3 ' structure be:
SA-ST-DS-S1-P1-P2-S2-pA-P3-S3-SD-Ap-TS-AS
In the formula:
SA is the receptor sequence of RNA montage;
ST is a forward series connection terminator codon;
DS is the donor sequences of reverse RNA montage;
S1 is the first reverse screening-gene, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or beta galactosidase enzyme lacZ gene;
P1 is the first reverse promotor,
P2 is second promotor of forward;
S2 is second screening-gene of forward, is selected from down group: hygromycin B resistant gene Hygro, neomycin resistance gene Neo or puromycin resistance gene Puro;
PA is a forward RNA tailing signal sequence;
P3 is the 3rd promotor of forward;
S3 is the three screening gene of forward, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or beta galactosidase enzyme lacZ gene;
SD is the donor sequences of RNA montage;
Ap is reverse RNA tailing signal sequence;
TS is reverse three terminator codons;
AS is the receptor sequence of reverse RNA montage;
P1, P2 and P3 are the promotors that is selected from down group respectively: cytomegalovirus is early promoter CMV, phosphoglycerokinase promotor PGK or human herpes simplex vicus's thymidine kinase gene promotor hsv-TK at once;
Condition is S1, S2 and S3 have nothing in common with each other and S1 and S3 in screening-gene encoding sequence GFP that is green fluorescent protein, the encoding sequence EGFP or the beta galactosidase enzyme lacZ gene of enhanced green fluorescent protein;
(b) screening has the embryonic stem cell of screening-gene mark;
(c) merge the embryonic stem cell that has the screening-gene mark, obtain mutated embryonic stem cell bank.
2. the method for claim 1 is characterized in that, in the described gene capturing carrier, S1 is Hygro, and S2 is neo, and S3 is EGFP.
3. the method for claim 1 is characterized in that, also comprises step in step (b) with (c):
(b ') to its dna sample of every kind of embryo stem cell extracting and RNA sample that step (b) obtains, preserve.
4. method as claimed in claim 3, it is characterized in that, in step (b '), also comprise: for extractive dna sample and RNA sample, measure genomic dna sequence and cDNA sequence that carrier inserts the both sides, site, according to clear and definite each mutated embryonic stem cell clone gene mutational site of sequence information and native gene functionally inactive situation, and judge whether newly gene of mutator gene, and then set up the information database of sudden change ES cell.
5. a mutated embryonic stem cell bank is characterized in that, the embryonic stem cell that described embryonic stem cell bank contains is the cell of transgenation, and described embryonic stem cell is the embryonic stem cell of mouse or rat, and described cell bank prepares in order to the below method:
(a) gene capturing carrier is imported embryonic stem cell, described gene capturing carrier from 5 ' to 3 ' structure be:
SA-ST-DS-S1-P1-P2-S2-pA-P3-S3-SD-Ap-TS-AS
In the formula:
SA is the receptor sequence of RNA montage;
ST is a forward series connection terminator codon;
DS is the donor sequences of reverse RNA montage;
S1 is the first reverse screening-gene, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or beta galactosidase enzyme lacZ gene;
P1 is the first reverse promotor,
P2 is second promotor of forward;
S2 is second screening-gene of forward, is selected from down group: hygromycin B resistant gene Hygro, neomycin resistance gene Neo or puromycin resistance gene Puro;
PA is a forward RNA tailing signal sequence;
P3 is the 3rd promotor of forward;
S3 is the three screening gene of forward, is selected from down group: the encoding sequence GFP of hygromycin B resistant gene Hygro, neomycin resistance gene Neo, puromycin resistance gene Puro, green fluorescent protein, the encoding sequence EGFP of enhanced green fluorescent protein or beta galactosidase enzyme lacZ gene;
SD is the donor sequences of RNA montage;
Ap is reverse RNA tailing signal sequence;
TS is reverse three terminator codons;
AS is the receptor sequence of reverse RNA montage;
P1, P2 and P3 are the promotors that is selected from down group respectively: cytomegalovirus is early promoter CMV, phosphoglycerokinase promotor PGK or human herpes simplex vicus's thymidine kinase gene promotor hsv-TK at once;
Condition is S1, S2 and S3 have nothing in common with each other and S1 and S3 in screening-gene encoding sequence GFP that is green fluorescent protein, the encoding sequence EGFP or the beta galactosidase enzyme lacZ gene of enhanced green fluorescent protein;
(b) screening has the embryonic stem cell of screening-gene mark;
(c) merge the embryonic stem cell that has the screening-gene mark, obtain mutated embryonic stem cell bank.
6. cell bank as claimed in claim 5 is characterized in that, described cell bank contains ten thousand mutated embryonic stem cell clones of 5-30.
7. the purposes of mutated embryonic stem cell bank as claimed in claim 5 is characterized in that, the stem cell clone in the described embryonic stem cell bank is used to produce the genetic modification mouse model of specific gene sudden change.
8. the purposes of mutated embryonic stem cell bank as claimed in claim 5 is characterized in that, is used to prepare the genomic dna and the cDNA of mutated embryonic stem cell bank.
9. the purposes of mutated embryonic stem cell bank as claimed in claim 5, it is characterized in that, described mutated embryonic stem cell bank is divided into 500-1000 inferior storehouse, and 100-300 mutated embryonic stem cell clone contained in each inferior storehouse, and the inferior storehouse of each cell all is used to prepare inferior storehouse cDNA.
10. the purposes of mutated embryonic stem cell bank as claimed in claim 5 is characterized in that, is used to screen the embryonic stem cell clone of specific gene sudden change.
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| CN108715864A (en) * | 2018-06-06 | 2018-10-30 | 中国科学院水生生物研究所 | It is a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application |
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| US5922601A (en) * | 1995-01-19 | 1999-07-13 | Biotransplant, Inc. | High efficiency gene trap selection of regulated genetic loci |
| CN1375012A (en) * | 1999-07-14 | 2002-10-16 | 株式会社基因转移 | Trap vector and gene trapping method by using the same |
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| US5922601A (en) * | 1995-01-19 | 1999-07-13 | Biotransplant, Inc. | High efficiency gene trap selection of regulated genetic loci |
| CN1375012A (en) * | 1999-07-14 | 2002-10-16 | 株式会社基因转移 | Trap vector and gene trapping method by using the same |
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