CN1970749A - Process for preparing prion protein gene-free domestic animals - Google Patents

Abstract

The invention discloses a making method of knocked livestock of protein gene, which is characterized by the following: losing activity for protein gene; making livestock possess relative disease of protein.

Description

A kind of method for preparing prion protein gene-free domestic animals

Technical field

The invention belongs to bioengineering field, particularly, the present invention relates to a kind of method for preparing prion protein gene-free domestic animals.

Background technology

Ruan's protein disease (prion diseases), (TransmissibleSpongiform Encephalopathies TSE), is the fatal central nervous system degenerative disease of a class to be Transmissible spongiform encephalopathy again.The principal feature of this disease is death, the brain vacuolation of neurocyte and the star spongiocyte hyperplasia of following etc., and these will cause dyskinesia, dementia, finally cause the death of infected animal.For everybody familiar Ruan's protein disease mainly contains the sheep that occurs on the sheep and goat disease (scrapie) of scratching where it itches, occur in ox mad cow disease (Bovine Spongiform Encephalopathy on one's body, BSE, " mad cow disease " that promptly is commonly called as), and occur in Ku Lushi disease (Kuru) on the person, creutzfeldt-Jacob disease (Creutzfeldt-Jakobdisease, CJD), Gerstmann-Straussler-Sheinker disease (GSS), lethality insomnia syndromes (fatal familial insomnia, FFI), novel creutzfeldt-Jacob disease (Variant Creutzfeldt-Jakobdisease, vCJD) etc. (Prusiner SB:Prions.Proc Natl Acad Sci USA 1998,95:13363-13383).

Now existing a lot of evidences show, occurring in ox mad cow disease on one's body can give the people and cause a kind of novel creutzfeldt-Jacob disease (vCJD) on the person by certain route infection, because Ruan's protein disease does not still have effective treatment means so far, up to the present this disease has caused over one hundred people's death (mainly in Britain), and then caused that in the world huge psychology is panic, bury or burning disposal after causing millions of oxen to be butchered, caused enormous economic loss.

A large amount of evidences shows that the pathogenic agent (English is prion, and pathotype prion also can translate into protein virus in mammiferous TSE disease) of Ruan's protein disease is only by PrP ^ScForm PrP ^ScBe the PrPC (PrP of normal expression in the cell ^C) isomer (conformer), this isomer be rich in beta sheet and have the opposing protease digestion ability.PrP ^CBe an exact function the unknown be anchored on glycoprotein (Prusiner SB:Novelproteinaceous infectious particles cause scrapie.Science 1982,216:136-144 on the cytolemma by GPI (glycosylphosphatidylinositol); Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB:Synthetic mammalian prions.Science 2004,305:673-676).The TSE pathogenic agent that proposes according to doctor Prusiner only contains the theory of protein " protein-only ", as the PrP as pathogenic agent ^ScCan cause PrP after entering in the body ^CBe transformed into PrP ^ScThereby, make PrP ^ScObtain propagation, this theory is predicted simultaneously if do not express PrP in the animal body ^C, will make PrP ^ScCan not breed in vivo, thereby make this animal have the ability of opposing TSE pathogenic infection.This theory is by two prion protein gene-free (Prnp independently ^-/-) confirmation fully of mouse species institute, two (Prnp ^-/-) the mouse species ability that not only has an opposing TSE pathogenic infection also can both the normal development and (the Bueler H that raises up seed, Fischer M, Lang Y, Bluethmann H, Lipp HP, DeArmond SJ, Prusiner SB, Aguet M, Weissmann C:Normal development and behaviour of mice lacking the neuronal cell-surface PrPprotein.Nature 1992,356:577-582).

Sheep scratch where it itches disease be a kind of the spontaneous on one's body generation of sheep or goat simultaneously can be between flock of sheep mutual infectious Ruan's protein disease, also be first kind and successfully propagate Ruan's protein disease to rodent by the artificial inoculation method, thereby sheep scratch where it itches disease be often used as the TSE disease research prototype (Chandler R L:Encephalopathy in mice produced by inoculation with scrapie brain material.Lancet 1961,1:1378-1379).Can not cause tangible harmful effect though in mouse, knock out its prion protein gene, not evidence suggests that knocking out its prion protein gene in sheep or goat also has identical phenotype knocking out mouse itself.In addition, PRNP gene single-gene knocks out with the dual-gene acquisition that knocks out sheep or goat and will help carrying out PrP ^CThe research of definite physiological function, also extremely valuable to pathogenetic research of Ruan's protein disease.At last, the acquisition that the PRNP Gene Double knocks out milk goat will have direct application aspect the biological medicine company, because according to " protein-only " theory, the PRNP Gene Double knocks out goat will have the ability that opposing TSE disease pathogen infects, and the pharmaceutical protein that utilizes these animals to produce as bio-reactor will be accepted by the human consumer than being easier to.

Because the successful foundation of mouse ES cells, gene targeting has become the genomic conventional means of accurate transformation mouse.Although through huge effort, up to the present the ES cell with any domestic animal of reproductive system potentiality of development does not all build up, this makes that accurately transformation domestic animal genome is all difficult in the quite a long time.In recent years, fast development along with the animal cloning technology, can prepare transgenic animal and whole process and closely similar (the Schnieke AE of ES cytogene target practice through infectious fetal fibroblast by nuclear transplantation, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH:Human factor IX transgenic sheep producedby transfer of nuclei from transfected fetal fibroblasts.Science 1997,278:2130-2133).Finally, reported the result of study of a milestone significance people such as McCreath in 2000, they are inserted into COLlAl (α 1 (I) procollagen) site (the McCreath KJ etc. of sheep by the mammary gland specifically expressing framework of the alpha1 Anti-trypsin gene that will contain the people of the gene targeting success of the fetal fibroblast of sheep, Production of gene-targeted sheep by nuclear transfer fromcultured somatic cells.Nature 2000,405:1066-1069.).After this, α (1 by similar techniques route knocking out of several laboratory successes arranged again pig and sheep, 3) galactosyltransferase gene (Dai Y, Deng, Targeted disruption of the alpha 1,3-galactosyltransferase gene incloned pigs.Nat Biotechnol 2002,20:251-255; Lai L, etc., Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science 2002,295:1089-1092).

Up to now, also there are not PRNP gene monoallelic or diallele to knock out the report of goat in the prior art.

Summary of the invention

The purpose of this invention is to provide a kind of method for preparing prion protein gene-free domestic animals.

In a first aspect of the present invention, provide a kind of method for preparing prion protein gene-free domestic animals may further comprise the steps:

(a) by gene targeting one construction fixed point is introduced the domestic animal fetal fibroblast, described construction contains two prion protein gene fragment and at least one selected marker's fragments as the homologous recombination homology arm;

(b) screening obtains the domestic animal fetal fibroblast that prion protein gene is knocked out;

(c) the domestic animal fetal fibroblast that step (b) is obtained prepares the domestic animal of prion protein gene-free as nuclear donor.

In another preference, the preparation method of the domestic animal that described PrPC monoallelic knocks out comprises the steps: that (1) makes up a kind of DNA targeting vector that can knock out prion protein gene by homologous recombination; (2) the DNA targeting vector that builds is transfected into the cell of domestic animal; (3) cells transfected is carried out drug screening, obtain the drug resistance cell clone; (4) the drug resistance cell clone that obtains is identified, picked out the cell clone of homologous recombination; (5) the cell clone cell of homologous recombination is transplanted in the domestic animal ovocyte of stoning and is formed reconstructed embryo; (6) cultivate this reconstructed embryo and grow mulberry fruit or blastula stage; (7) reconstructed embryo that will grow mulberry fruit or blastula stage is transplanted to the acceptor domestic animal intrauterine of synchronization of estrus; (8) gestation, childbirth produces the domestic animal of gene knockouts such as PrPC list.

In another preference, also comprise step (d): the domestic animal that utilizes acquired PrPC monoallelic to knock out breeds, and obtains the domestic animal that the PrPC diallele knocks out.

In another preference, also comprise step (d): the domestic animal fetal fibroblast that utilizes prion protein gene to be knocked out carries out second allelic target practice of PrPC, obtain the domestic animal fetal fibroblast cell that the PrPC diallele knocks out, utilize this cell to carry out animal cloning again, obtain the domestic animal that the PrPC diallele knocks out.

In another preference, described marker gene is a resistant gene.

In another preference, described marker gene is selected from down group: neomycin resistance gene, hygromycin gene, puromycin resistance gene.

In another preference, described marker gene is selected from down group: neomycin resistance gene (neomycin), hygromycin gene (hygromycin), puromycin resistance gene (puromycin).

In another preference, also contain negative selection markers gene in the upstream of 5 ' homology arm sequence or the downstream of 3 ' homology arm sequence.

In another preference, described domestic animal is ox, sheep, goat, rabbit, pig.

In another preference, described animal is a goat.

In another preference, the length of each homology arm sequence is 1-20kb.

In a second aspect of the present invention, a kind of tissue or cell of prion protein gene-free is provided, described tissue or cell are from the domestic animal of the prion protein gene-free that adopts described method preparation.

In a third aspect of the present invention, provide a kind of preparation domestic animal fibroblastic method, the fibroblastic prion protein gene of described domestic animal is knocked out, and the method comprising the steps of:

(a) by gene targeting one construction fixed point is introduced the domestic animal fetal fibroblast, described construction contains two prion protein gene fragment and at least one selected marker's fragments as the homologous recombination homology arm;

(b) screening obtains the domestic animal fetal fibroblast that prion protein gene is knocked out.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1. goat wild-type PRNP gene, GTPrP targeting vector and hit after PRNP gene synoptic diagram.At three exons of clear this gene of wild-type PRNP gene synoptic diagram acceptance of the bid, represent with black box.Indicated simultaneously in the PRNP gene synoptic diagram after hitting and be used for the primer that PCR identifies and the probe of Southern hybridization.This synoptic diagram has indicated simultaneously with the expection DNA stripe size of carrying out Southern hybridization behind the different digestion with restriction enzyme.Restriction endonuclease sites: Bg represents BglI; Xb represents XbaI; Sc represents ScaI; Ba represents BamHI; Sa represents SalI.

The pcr analysis of Fig. 2 .G418 drug resistance cell clone.Indicated the numbering of cell clone above each swimming lane, (a), (b), (c) primer of Shi Yonging is respectively P1/P4, P1/P2 and P3/P5.Concrete primer location is referring to Fig. 1.(d) the Southern hybridization analysis of the PCR product of P1/P4 primer, used probe are 5 ' homology arms of 1.9kb targeting vector.

The Southern hybridization analysis of Fig. 3 .PCR positive cell clone GTPrP74.The probe that uses is respectively the 5 ' homology arm (a) and the 1.1kb neomycin resistance gene fragment (b) of 1.9kb targeting vector.Restriction endonuclease sites: Bg represents BglI; Xb represents XbaI; Sc represents ScaI; Ba represents BamHI.

Fig. 4. the genomic dna analysis of clone goat.(a) pcr analysis, the primer of use is P1/P4.(b) Southern hybridization analysis, genomic dna is hybridized as probe with 5 ' homology arm of 1.9kb targeting vector after BglI or XbaI digestion.(c) the PRNP monoallelic of five healthy survivals knocks out (PRNP ^+/-) goat (45B, 50A, 50B, 8A, 14A).

Fig. 5 .PRNP ^+/-The Western blot of the PrPC expression level of 46A clone goat detects.Use wild-type (WT) lamb and PRNP ^+/-The brain homogenate of 46A sheep detects, used PrPC PrP ^CAntibody is mouse monoclonal antibody, has also used the antibody of Actin muscle (actin) to equate with the Tot Prot that guarantees each electrophoresis road in contrast simultaneously.

Embodiment

The inventor is through extensive and deep research, discovery can knock out the prion protein gene of domestic animal on cell levels, the domestic animal for preparing prion protein gene-free then by the method for somatic cell clone, the domestic animal prion protein gene functionally inactive that obtains, the PrPC of expressive function not, thereby the ability that can give domestic animal opposing PrPC class relative disease.Finished the present invention based on this.

In brief, technological line of the present invention is that by the method for gene knockout, with specific target practice construction transfection domestic animal fetal fibroblast, fixed point knocks out PrPC specific site sequence, sets up prion protein gene and is knocked out clone.Utilize nuclear transfer technology, obtain the transgene clone domestic animal that PrPC knocks out.

The construction of gene targeting of the present invention contains following element successively from 5 ' → 3 ': 5 ' homology arm sequence, selected marker and 3 ' homology arm sequence.The effect of each element is as follows:

5 ' homology arm sequence and 3 ' homology arm sequence: be used for therebetween sequence is integrated into karyomit(e) by homologous recombination;

Selected marker: be used to screen the transformant that reorganization takes place.The transformant that produces reorganization is because of containing token-based thereby being selected.

In preference, described construction also contains negative selection markers gene in the upstream of 5 ' homology arm sequence or the downstream of 3 ' homology arm sequence, and its effect is a transformant of getting rid of non-homogeneous recombinant type.When homologous recombination takes place, the negative selection markers gene that is positioned at the homology arm outside will can not be integrated into karyomit(e); And when nonhomologous reorganization took place, the negative selection markers gene that is positioned at the homology arm outside will be integrated into karyomit(e).Do not contain the transformant of bearing the selection markers gene by screening, just obtained to take place the transformant of predetermined fixed point homologous recombination.Can be used for negative selection markers gene of the present invention and comprise (but being not limited to) suicide gene, as thymidine kinase (TK) gene etc.

More specifically, main technological route of the present invention is as follows:

1. from the tissue of domestic animal, extract genomic dna.At first, utilize the tissue of this fetus to carry out the extraction of genomic dna simultaneously from pregnant 35-45 days fetus isolated cell.

2. from this genomic dna, clone prion protein gene (PRNP).Method one: the genomic dna that utilizes extraction to obtain carries out the foundation of genomic library, utilizes the prion protein gene design hybridization probe of known array animal, obtains prion protein gene.Method two: directly utilize and extract the PCR clone that the genomic dna that obtains carries out prion protein gene.

3. make up a kind of DNA targeting vector that can knock out prion protein gene by homologous recombination, the optional neomycin resistance gene of selectable marker gene, hygromycin gene, puromycin resistance gene, in a preference of the present invention, select neomycin resistance gene (neo) as selectable marker gene.

4. with the cell of this domestic animal of prion protein gene DNA targeting vector transfection of building.Before transfection, prion protein gene DNA targeting vector is carried out enzyme and cut, obtain linearizing DNA targeting vector.The preferred electrotransfection of transfection method.

5. cells transfected is carried out medicine (G418) screening, obtain the drug resistance cell clone.

6. the drug resistance cell clone that obtains is identified, picked out the cell clone of homologous recombination.At first utilize PCR method that all drug resistance cell clones are carried out preliminary screening, filter out the possible cell clone that hits.Then these PCR being accredited as the positive cells clone and carrying out Southern hybridization, to prove conclusively the real cell clone that hits, promptly is the cell of prion protein gene-free.

7. the cell of the prion protein gene-free that obtains is transplanted in the domestic animal ovocyte of stoning, is formed reconstructed embryo.External or this reconstructed embryo of culturing in vivo makes it grow mulberry fruit/blastula stage.Be transplanted to the intrauterine of the acceptor domestic animal of synchronization of estrus with growing to the reconstructed embryo of blastula stage or morula stage.Acceptor after the transplanting carries out pregnancy check with B ultrasonic at 1-2 during the monthly age, the pregnancy that is judged to be of pregnant capsule occurs.Gestation adopts the spontaneous labor or the lambing of cutting open the belly to after date.The clone domestic animal that obtains is carried out the molecular biology identification of prion protein gene-free.

8. the sexual propagation of the domestic animal that knocks out with the PrPC monoallelic (PRNP+/-), the domestic animal that acquisition PrPC diallele knocks out (PRNP-/-), the cell that perhaps utilizes the PrPC monoallelic to knock out domestic animal carries out second allelic target practice of PrPC, obtain the cell that the PrPC diallele knocks out, utilize this cell to carry out animal cloning again and obtain the domestic animal that diallele knocks out (PRNP-/-).

Major advantage of the present invention is:

(1) adopt method of the present invention can obtain a kind of domestic animal of prion protein gene-free, described domestic animal has the ability of opposing PrPC class relative disease (sheep scratch where it itches disease, mad cow disease (being commonly called as mad cow disease) etc.).The acquisition of described domestic animal has great scientific research value and using value.

(2) can utilize described domestic animal to carry out the fundamental research of PrPC class disease-related, further understand endogenous PrPC (PrP as research object ^C) in vivo normal physiological function, provide scientific basis for preventing and treating PrPC class disease.Having the PrPC diallele knocks out the cell of domestic animal and has the cell that PrPC list equipotential knocks out domestic animal and also can be used for the fundamental research of PrPC class disease in the cytobiology level.

(3) concrete application facet, the domestic animal that the PrPC diallele knocks out infects as mad cow disease because have opposing PrPC class disease, can be used to prevent that this type of disease from wider spreading, alleviating the feared state of mind of the common people to such disease simultaneously.

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:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The structure of the clone of goat prion protein gene and goat prion protein gene targeting vector

1, former generation the goat fetal fibroblast cultivation and sex identification thereof

Obtain skin or the ear tissue of 35 days goat fetus of birth, with containing 0.25% pancreatin, the Digestive system of 1mM EDTA digested about 15 minutes in 37 ℃ of water-baths then.

Postdigestive individual cells suspension is centrifugal, after removing supernatant, cell mass is resuspended in the glutamine (GIBCO) that contains 2mM, the Sodium.alpha.-ketopropionate of 1mM (SIGMA), 1 * nonessential amino acid (SIGMA), Prostatropin (the bFGF of 2ng/ml, SIGMA), 10% foetal calf serum (HyClone), the penicillin of 100units/ml, the Glasgow minimum medium of the Streptomycin sulphate of 100 μ g/ml (SIGMA) (GMEM, SIGMA) in, at last cell suspension is assigned in the culturing bottle and in CO2gas incubator, cultivated a couple of days, when cell density reaches 80% left and right sides that it is frozen.

The sex of goat fetal fibroblast system identifies that by the pcr amplification sry gene PCR the primer sequence is as follows:

SRY16f:5 '-CAATCGTATGCTTCTGCTATGTTC-3 ' (SEQ ID NO:1); With

SRY654r：5’-CAATGTTACCCTATCGTGGCC-3’(SEQ ID NO：2)。

As a result, obtained milk goat fetal fibroblast from 35 ages in days, female through being accredited as, be numbered GFF88.

2, the structure of targeting vector

PrP ^CThis gene comprises three exons by euchromosome single-gene in the animal body (PRNP gene) encoded protein to be one, and whole PrP ^CThe coding region all in the 3rd exon.Though the PRNP gene complete sequence of whole goat is report not also, the PRNP gene of sheep is by the clone of success and carried out detail analysis.The PRNP gene of sheep comprises three exons, total length reaches 21kb, wherein the whole coding region of 770bp all is positioned at the 3rd exon (Lee IY, Westaway D, Smit AF, Wang K, Seto J, Chen L, Acharya C, Ankener M, Baskin D, Cooper C, Yao H, Prusiner SB, Hood LE:Complete genomic sequence and analysis of the prion protein generegion from three mammalian species.Genome Res 1998,8:1022-1037).The inventor has made up promotor defect type targeting vector GTPrP (Fig. 1) goat PRNP gene has been practiced shooting.

Concrete building process is as follows: the left side homology arm of 1.9kb and the right side homology arm of 4.5kb are to increase from the genomic dna of GFF88 fetal fibroblast by PCR method, and its primer is to design according to the sequence of the PRNP gene of sheep (GenBank number is U67922).1.9kb the amplimer of left side homology arm is PrPLF:5 '-GAATCCGCGGTGTCGACTCTCTAGTCCATGATCGTTCCTC-3 ' (SEQ ID NO:3), its 5 ' end has SacII and SalI restriction enzyme site; And PrPLR:5 '-TGTTCAATGGCCGATCCCATGATGACTTCTCTGCAAAATAAAG-3 ' (SEQID NO:4), its start code region sequence complementation terminal and neo.1.1kb the segmental amplimer of neo-pA be neoF:5 '-CTTTATTTTGCAGAGAAGTCATCATGGGATCGGCCATTGAACA-3 ' (SEQ ID NO:5), the end sequence complementation of its 5 ' end sequence and left side homology arm; And neoR:5 '-GAATGCGGCCGCAGTACTCCCCAGCTGGTTCTTTCCG-3 ' (SEQ ID NO:6), its 5 ' end contains NotI site and ScaI site.These two fragments connect the dna fragmentation that obtains a 3.0kb by PCR.This fragment is connected in pBluescript IISK (STRATAGENE) carrier after SacII and NotI enzyme are cut.The amplimer of right side homology arm is PrPRF:5 '-ATAAGCGGCCGCGGATCCAGACTATGAGGACCGTTACTATCGTG-3 ' (SEQ ID NO:7), and its 5 ' end has NotI and BamHI restriction enzyme site; And PrPRR:5 '-CCGCTCGAGGTCGACGTATCATTCACTTCGGCTCTGTAAA-3 ' (SEQ IDNO:8), its 5 ' end band XhoI and SalI restriction enzyme site, this PCR product through Not I be connected with the segmental pBluescript II of neo-pA SK carrier (Stratagene company) with the above-mentioned left side homology arm that has after Xho I enzyme is cut, finish the structure of whole carrier.

In the GTPrP targeting vector that makes up, the neo-pA fragment of 1.1kb is placed in after the initiator codon of PRNP gene.If between GTPrP targeting vector that changes over to and the endogenous PRNP gene homologous recombination takes place, the PRNP gene coding region that has about 430bp is deleted, should can be replaced by the neo-pA fragment of 1.1kb in the zone simultaneously.

Embodiment 2

Utilize the GTPrP targeting vector to knock out goat PRNP gene

Carrier GTPrP changes it GFF88 fetal fibroblast system of the third generation that goes down to posterity over to after the SalI linearizing by electrotransfection.About 50,000 ten thousand fetal fibroblasts are mixed with the linearizing GTPRP carrier of 10 μ g, transfer to the electric revolving cup (Bio-Rad) of 0.4cm, by electroporation Gene pulserII (Bio-Rad) it is imposed 220v, the culture dish that several contain the GMEM nutrient solution is then assigned to cell in the pulse of 950 μ F.Adding G418 after 48 hours is 250 μ g/ml to final concentration.

About about 10 days, as seen there is cell clone to form.Wherein well-grown cell clone is chosen, and transfers to 96 orifice bores.When covering with soon, the cutting out partial cell is used for PCR to be identified, remaining cell continues to go down to posterity and is respectively applied for frozen and the extracting genomic dna is identified as Southern hybridization up to obtaining enough cells.

The inventor at first uses 3 groups of different primers that the cell clone that produces is carried out pcr analysis so that filter out the cell clone (targeted colonies) that hits, and the outside of a primer at homology arm arranged respectively in every group of primer.The particular location of each primer is seen Fig. 1.Concrete PCR process is as follows: the cell of separating is added lysis buffer (40mM Tris-HCl, pH8.0,0.9%Triton X-100,0.9%NonidetP-40,0.4mg/ml proteinase K), in 65 degree water-baths, digested 30 minutes after the cracking, in 95 degree water-baths, handled 10 minutes at last, make proteolytic enzyme (proteinase) K inactivation.Utilize TAKARA LA system (available from TAKARA company) to carry out PCR, in the reaction system of 20 μ l, add the above-mentioned cell pyrolysis liquid of 2 μ l as template.

The PCR primer sequence is as follows:

P1，5’-CACAGCCAGGCATTCAGAAAC-3’(SEQ ID NO：9)；

P2，5’-CCACCATGATATTCGGCAAG-3’(SEQ ID NO：10)；

P3，5’-CGCCTTCTTGACGAGTTCTTC-3’(SEQ ID NO：11)；

P4，5’-CACGATAGTAACGGTCCTCATAGTC-3’(SEQ ID NO：12)；

P5，5’-GTATGATGCAGGGAAACCAAAG-3’(SEQ ID NO：13)。

The PCR loop parameter is as follows: 94 ℃, and 30 seconds; 62 ℃, 30 seconds; 72 ℃, 3.5 minutes (to P1/P2 and P1/P4 primer) or 5 minutes (to the P3/P5 primer), totally 30 circulations.If utilize the DNA that from tissue or blood, extracts to identify as carry out PCR as template, then only need the DNA that adds 1 μ l purifying to get final product as template, other parameter is identical.

Further utilize the Southern hybridizing method to identify to separating from the genomic dna of cultured cells or goat tissue.Sample at first spends the night in the middle cracking of following lysate (10mM Tris-HCl, pH8.0,50mMEDTA, 10mM NaCl, 0.5%SDS, 0.4mg/ml proteinase K), carries out the conventional extracting of genomic dna then.Use following three groups of different restriction endonucleases (BglI, XbaI or ScaI/BamHI) that the genomic dna that obtains is carried out enzyme and cut, then enzyme is cut product and carried out agarose gel electrophoresis, transfer on the nylon membrane again.Use the probe (particular location see Fig. 1) of two kinds of dna fragmentations: the neo fragment of fragment of the left side homology arm that is equivalent to targeting vector of a 1.9kb (called after 5 ' arm) and 1.1kb as hybridization.

Experimental result is as follows:

It is that two homology arms of GTPrP increase exactly from this clone that the inventor changes GTPrP over to female GFF88 goat fetal fibroblast by electrotransfection.Through 8-10 days drug screening, 163 cell clones have been chosen.These drug resistance cell clones by three independently pcr analysis detect wherein middle target cell.Using P1 primer and 112 cell clones that are positioned at right side homology arm P4 primer analysis that are arranged in the homology arm outside, left side that 10 cell clones that hit are arranged.This is to decide according to the fragment that has two expection sizes in the PGR product of these cell clones, a 2.8kb fragment, and allelic and 3.5kb fragment from normal PRNP is from the PRNP allelotrope that hits.If between GTPrP targeting vector that changes over to and the endogenous PRNP gene homologous recombination takes place, the PRNP gene coding region that has about 0.4kb is deleted, should can be replaced by the neo-pA fragment of 1.1kb in the zone simultaneously, a new 3.5kb fragment (Fig. 1) when analyzing, will occur with the P1/P4 primer.

Fig. 2 a is the P1/P4 primer analytical results of several representative cell clones.Yet the concentration of two bands during according to electrophoresis can judge that 10 P1/P4 primers analyze and have 8 to be to mix the clone in the positive cell clone.

For the success of further practicing shooting certainly, the inventor has also used other two groups of PCR that these cell clones have been done further checking.10 P1/P4 primers are analyzed in the positive cell clone, it are not done further checking because be to mix the clone for 8, and remaining two cell clones (GTPrP74 and GTPrP78) are all proved positive colony (Fig. 2 b and 2c) by other two groups of PCR.In addition, the product of P1/P4 primer amplification also be transferred on the nylon membrane and with 5 ' the arm probe hybridization that is equivalent to the left side homology arm of 1.9kb, the result has proved that also the new 3.5kb band that is produced by the P1/P4 primer amplification is not a non-specific band and be to be produced by the PRNP allelotrope that hits really.

Occur owing to use pcr analysis may have false positive, so the method for necessary use Southern hybridization is done further affirmation to the positive cell clone of these pcr analysis.This cell clone of GTPrP78 is used for the extracting genomic dna as the hybridization use, so only this cell clone of GTPrP74 has been done the Southern hybridization analysis owing to fail to produce enough cell count in the process of amplification.

The inventor has used three groups of different restriction endonucleases and two different probes that the genomic dna of wild-type GFF88 cell and GTPrP74 cell clone has been carried out detail analysis (Fig. 3 a and 3b).If the grade of PRNP gene is successfully practiced shooting, two BglI sites that are positioned at the 0.4kb coding region of being replaced by the neo-pA fragment will disappear, this makes when the genomic dna of target cell in these is cut 5 ' the arm probe hybridization of back and 1.9kb with the BglI enzyme, except having a 6.1kb from the allelic BglI endonuclease bamhi of normal PRNP, also can occur one new be that (Fig. 1 and Fig. 3 are a) for the BglI endonuclease bamhi of 12.0kb from the allelic size of the PRNP that hits.

Similarly, when using XbaI or two groups of enzymes of ScaI/BamHI enzyme cut the back with 5 ' arm probe hybridization respectively, except the ScaI/BamHI endonuclease bamhi of the XbaI enzyme cutting fragment of endogenous 6.7kb or 4.4kb, (Fig. 3 a) also can to occur the ScaI/BamHI endonuclease bamhi of a new 7.4kb XbaI enzyme cutting fragment or 5.5kb separately.When the neo probe that uses 1.1kb is hybridized, have only the fragment that conforms to the expection size of new generation to be hybridized to come out (Fig. 3 b).This conforms to the successful target practice of PRNP gene, and proof has only the targeting vector of single copy to be integrated in the genome of goat.

Embodiment 3

By goat PRNP+/-nuclear transplantation of cell prepare PRNP+/-goat

In the inventor's research, the confession matter ovocyte that nuclear transplantation is used, interim foster mother, acceptor are all from Sa energy milk goat.

The synchronization of estrus of goat: female goat is by the prostaglandin(PG) (prostaglandin-Cl of injection 0.1mg, PG, Shanghai birth control Science Institute), (luteinizing hormone-releasing hormone LHRH) carries out synchronization of estrus to injection 25 μ g luteinizing hormone-releasing hormones after 24 hours.

The superovulation of she-goat: (follicle stimulation hormone FSH), injects twice every day the intramuscular injection follicle stimulating hormone, injection is 3 days continuously, the total dose of every goat injection FSH is 240IU, at PG of injection in the 3rd day, and LHRH of injection after 24 hours.

The recovery of ovocyte: LRH injection adopted operation to reclaim in 24-30 hour, with collecting ovocyte behind F10 (GIBCO) the nutrient solution flushing uterine tube, Unidasa with 0.2% is removed the granulosa cell that adheres to, and then is incubated at 37.5 ℃, 5%CO with the M16 nutrient solution ₂Incubator in.

Donor cell is the goat somatocyte of PrP gene knockout, and 0.5% foetal calf serum is hungry to be cultivated 5 days with containing when growing into 80% density, then is used for nuclear transplantation (NT) with 0.25% trysinization to individual cells.(cytochalasin B, M16 nutrient solution washing CB) 3 times places this solution to handle again 10-20 minute, then carries out stoning and moves the nuclear operation with containing 2.8mg/ml HEPES and 7.5 μ g/ml cytochalasin Bs before the ovocyte stoning.Single donor cell is expelled under the zona pellucida of non-nucleus egg mother cell, make it be close to cytoplasmic membrane, adopt the method for electricity irritation to merge, merging matrix is the solution that contains 0.3mM N.F,USP MANNITOL, 0.05mM calcium chloride, 0.1mM sal epsom, 0.5%BSA, fusion conditions is DC600-610v/cm, pulse durations 80 μ s, continued stimulus 3 times.Embryo after the fusion cultivates 5 hours in M16 after, in the M16 liquid that contains 5 μ M ionomycins (ionomycin), 7.5 μ g/ml CB, handle 5min, in the M16 liquid that contains 2mM 6-dimethylaminopurine (6-DMAP) and 7.5 μ g/ml CB, handle again to move on in the M16 nutrient solution after 5 hours and cultivate.

With the heavily enough ovum after activating after of short duration cultivation, agarose with 1% carries out twice embedding, embedded block is transplanted in the uterine tube of interim foster mother sheep of synchronization of estrus and is carried out culturing in vivo, after cutting the uterine tube of interim foster mother sheep after 5 days, wash uterine tube to reclaim the NT embryo with F10 (GIBCO) nutrient solution.With the intrauterine of growing that reclaims to mulberry fruit/NT embryo transfer blastula stage to the acceptor sheep, 2-3 piece of every acceptor sheep transplanting embryo.The pregnant situation of acceptor sheep is diagnosed pregnancy receptor term birth, birth clone sheep by B ultrasonic.

Experimental result is as follows:

Be numbered among the GTPrP74 target cell as donor cell, the MII phase of being transplanted to stoning suckle in the ovocyte of mountain, the structure nuclear transfer embryo.Carried out two altogether and examined and approved transplantation experiments (table 1).

In the nuclear transplantation experiment first time, have 66 pieces and grow a mulberry fruit or the embryo transfer to 30 of a blastula stage acceptor goat intrauterine, by ultrasound diagnosis, 10 acceptor sheep gestation are arranged when transplanting back 35 days, wherein 2 pregnancy receptor sheep early abortion is not recovered to the tissue that can be used for DNA analysis; 3 not tangible miscarriage phenomenons of pregnancy receptor sheep do not have the lamb birth yet, and possible fetal tissue dissolving back has been absorbed by parent.The pregnancy of remaining 5 acceptor sheep has been arrived mature, divides 6 clone sheep of giving birth to, and wherein 1 is stillborn foetus, and 3 clone sheep just die off after birth soon, 2 clone sheep survivals, and still growth is normal at present.

In second time nuclear transplantation experiment, 5 clone sheep that have been born, wherein 2 dead in back 48 hours of birth, 3 survivals, healthy growth at present.Table 2 has been listed the principal character of these 11 clone goats.

These clone goats PCR and Southern hybridization evaluation have been carried out.Pcr analysis shows, in 6 clone sheep of the birth of nuclear transplantation for the first time 5 and all 5 clone sheep of the birth of nuclear transplantation for the second time all contain a normal PRNP allelotrope and a PRNP allelotrope (table 2) that hits.Fig. 4 a has just provided the PCR qualification result of 7 clone sheep of birth at first.Still contain 1 non-lamb that hits in 11 clone sheep of birth.

This explanation: through the GTPrP74 cell clone that P1/P4 primer PCR analytical proof mixes the clone still have non-in the target cell existence.The lamb that 3 numberings are respectively the health survival of 8A, 14A and 45B carries out the Southern results of hybridization proves that also these lambs contain normal PRNP allelotrope and a PRNP allelotrope that hits (Fig. 4 b).

All 5 PRNP that continue survival ^+/-Clone sheep all demonstrates normal developmental state in back 2 months of birth (8A and 14A) or 1 month (45B, 50A and 50B) according to these lambs of judgement such as its body weight and individual sizes.

Table 1, nuclear transplantation situation

Grouping *	1	2
			Be transplanted to the lamb of the Number of Foetus birth of embryo number final receptor sheep in the time of several 35 days that embryo number mulberry body that the embryo number of interim acceptor sheep reclaims and blastaea number be transplanted to the final receptor sheep: the lamb number in the sheep that lives (dead sheep) at least one week of surviving	230 151 66 66 30 10 5(1) 2	132 127 59 59 25 9 3 3

^*Between twice nuclear transplantation grouping probably there is one month the timed interval.

The principal character of table 2, clone sheep

The experiment batch	First						Second batch
												The clone sheep numbering	7A	7B	8A	14A	16A	18A	45A	45B	46A	50A	50B
Final receptor sheep numbering *	PrP7		PrP8	PrP14	PrP16	PrP18	PrP45		PrP46	PrP50
												Period of pregnancy (my god)	149	149	153	154	150	148	148	148	131	145	145
Baby weight (Kg)	2.6	2.0	3.6	3.0	3.0	6.2	3.1	4.8	1.8	2.2	2.8
												Birth healthy state	Live	Live	Live	Live	Extremely	Live	Live	Live	Live	Live	Live
The survival fate	1	3	＞60	＞60	0	1	2	＞30	1	＞30	＞30
												Gene knockout whether	Be	Be	Be	Be	Not	Be	Be	Be	Be	Be	Be

^*Three final receptor sheep (PrP7, Prp45 and PrP50), two lambs that have been born are separately respectively arranged.

Embodiment 4

PRNP+/-the PrPC expression analysis of goat

Western blot analyzes.For cell, collect the back directly with lysate (10mM Tris-HCl, pH7.4,100mM NaCl, 10mM EDTA, 0.5%Nonidet P-40,0.5%sodium deoxycholate) dissolving.For cerebral tissue, utilize the homogenate of making 10% (w/v) with top identical lysate, 1000g is after centrifugal 5 minutes, and supernatant is used for the Western analysis.The protein sample of equivalent is transferred on poly(vinylidene fluoride) (polyvinylidene fluoride (the PVDF)) film with semidrying after 12% SDS-PAGE separation.Earlier with confining liquid (50mM Tris-HCl, pH7.5,150mM NaCl, 10%non-fat dry milk) sealed 1 hour, then use confining liquid overnight incubation under 4 degree of anti-Ruan's protein monoclonal antibody that contains dilution in 1: 500 or the anti-actin monoclonal antibody of diluting at 1: 1000.After washing three times, with containing 1: 1000 the confining liquid of goat anti-mouse IgG incubated at room 2 hours.After the washing, utilize Pierce Corporation's Super Signal WestPico chemiluminescent substrate test kit to develop the color once more.

Experimental result is as follows:

Can make this allelotrope that hits inactivation on expression level in order to prove after the neo-pA fragment of using 1.1kb is replaced the coding region of PRNP gene of 0.4kb, to 1 PRNP that is numbered 46A ^+/-The brain tissue sample of lamb has carried out PrP ^CThe detection of protein expression level.PRNP ^+/-The 46A lamb is to examine and approve in the transplanting second to be given a birth when pregnancy has only 131 days by the acceptor sheep that is numbered PrP46, this lamb at postnatal second day with regard to natural death (seeing Table 2).

Separation is from wild-type goat and PRNP ^+/-The cerebral tissue of 46A goat brain same area at first is made into homogenate, then uses anti-Ruan's protein monoclonal antibody to detect.

The result shows, can detect the PrP that size is approximately 28-36kDa in two sample ^CBand, however compare PRNP with the wild-type goat ^+/-PrP in the 46A goat brain ^CExpression level obviously descends (Fig. 5), and this has illustrated that the PRNP allelotrope that hits is really by functional inactivation.

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 Genon Bioengineering Co., Ltd

＜120〉a kind of method for preparing prion protein gene-free domestic animals

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Claims (8)

1. a method for preparing prion protein gene-free domestic animals is characterized in that, may further comprise the steps:

(a) by gene targeting one construction fixed point is introduced the domestic animal fetal fibroblast, described construction contains two prion protein gene fragment and at least one selected marker's fragments as the homologous recombination homology arm;

(b) screening obtains the domestic animal fetal fibroblast that prion protein gene is knocked out;

(c) the domestic animal fetal fibroblast that step (b) is obtained prepares the domestic animal of prion protein gene-free as nuclear donor.

2. the method for claim 1 is characterized in that, also comprises step (d): the domestic animal that utilizes acquired PrPC monoallelic to knock out breeds, and obtains the domestic animal that the PrPC diallele knocks out.

3. the method for claim 1, it is characterized in that, also comprise step (d): the domestic animal fetal fibroblast that utilizes prion protein gene to be knocked out carries out second allelic target practice of PrPC, obtain the domestic animal fetal fibroblast cell that the PrPC diallele knocks out, utilize this cell to carry out animal cloning again, obtain the domestic animal that the PrPC diallele knocks out.

4. the method for claim 1 is characterized in that, described marker gene is a resistant gene.

5. the method for claim 1 is characterized in that, described domestic animal is ox, sheep, goat, rabbit, pig.

6. the method for claim 1 is characterized in that, the length of each homology arm sequence is 1-20kb.

7. the tissue of a prion protein gene-free or cell is characterized in that, described tissue or cell are from the domestic animal of the prion protein gene-free that adopts the arbitrary described method preparation of claim 1-3.

8. one kind prepares the fibroblastic method of domestic animal, and the fibroblastic prion protein gene of described domestic animal is knocked out, and it is characterized in that the method comprising the steps of:

(a) by gene targeting one construction fixed point is introduced the domestic animal fetal fibroblast, described construction contains two prion protein gene fragment and at least one selected marker's fragments as the homologous recombination homology arm;

(b) screening obtains the domestic animal fetal fibroblast that prion protein gene is knocked out.

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