CN101117633B - Nucleus transplantation method - Google Patents
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Abstract
The present invention discloses a method for the nuclear transplantation. The method is characterized in that donor cells and recipent oocytes of non-human mammals of the same mitochondria haplotype are nuclear transferred. The method in the present invention can remarkably improve fusion, cleavage and blastocyst rates of the reconstructed embryo, thus remarkably improving the nuclear transplantation efficiency.
Description
Technical field
The invention belongs to technical field of bioengineering, specifically, is about a kind of nuclear transfer method.
Background technology
Body-cell neucleus transplanting (somatic cell nuclear transfer, SCNT) technology has been widely used in the production of cloned animal and transgenic animal, the cloned animal of multiple different plant species (the Chesne P that comes out one after another, Adenot PG., Viglietta C., et al.Nat Biotechnol.2002,20:366-369; Woods GL., White KL., Vanderwall DK., et al.Science2003,301:1063).Compare with the transgenic animal preparation method of routine, clone technology has special advantages, and this is because clone technology has advanceed to cell levels with screening operation, thereby has shortened excellent kind of animal reproduction cycle greatly.At present, inefficiency is to limit the bottleneck of this technology.
Plastosome (mitochondrion, mt) be not only the maximum organoid of content in the cytoplasm, the more important thing is because mitochondrial high mutation rate, cause the difference that reaches Mitochondrial DNA (mtDNA) sequence between same population Different Individual between different population, show different haplotypes, this species diversity can cause the difference of biological character, for example in milk cow, can cause difference (the Tamassia M. of milk yield and Fertility, Heyman Y., Lavergne Y., et al.Reproduction2003,126:629-637; Sutrno, Cummins JM., Greeff J., et al.Theriogenology2002,57:1603-1610; MannenH., Kojima T., Oyama K., et al.J.Anim.Sci.1998,76:36-41).
Recent years, ovocyte plastosome haplotype becomes the research focus day by day in the animal reproduction field.Traditional haplotype notion is meant uses the specific endonuclease bamhi general layout that produces after the specific fragment of specific limited endonuclease digestion.Research from external Embryo Production (IVP) and body-cell neucleus transplanting illustrates that all external embryo's growth is influenced by maternal mtDNA haplotype obviously, and difference (Tamassia M. with ovocyte ATP and mtDNA copy number, Nuttinck F., Reynier P, et al.Biol.Reprod.2004,71:697-704; Bruggerhoff K., Zakhartchenko V., Wenigerkind H., et al.Biol.Reprod.2002,66:367-373; Hiendleder S., Prelle K., Bruggerhoff K., et al.Biol.Reprod.2004,70:1196-1205).
The applicant discloses a kind of same body-cell neucleus transplanting method in its application number is 200410016219.7 Chinese invention patent application, this method is that supply nucleome cell and the enucleation oocyte of getting same individuality carry out nuclear transplantation, and the result shows that clone's blastaea all is significantly increased on quality and quantity.The contriver associates thus, the embryo who obtains by conventional nuclear transplantation method (allosome nuclear transplantation) at present, owing to contain different acceptors and donor mtDNA haplotype, these haplotypes may influence the developmental potency of reconstructed embryo.The contriver finds in the research in early stage, the ovocyte of some type helps the growth of clone embryos, may to be more suitable for fetal development relevant with some mtDNA haplotype in the kytoplasm, its center coding factor and some mtDNA haplotype compatibility mutually may have been brought into play vital role, thereby help the surviving of clone embryos of certain line plastochondria type.
Summary of the invention
In order to verify above-mentioned supposition, the contriver carries out haplotype analysis by the PCR-RFLP technology to the Mitochondrial DNA of laboratory animal, the Mitochondrial DNA haplotype of different experiments animal is divided into 4 types, utilize haplotype identical supply nucleome cell and acceptor ovocyte to carry out nuclear transplantation then, found that the developmental potency that adopts the identical nuclear transplantation of haplotype can effectively improve reconstructed embryo.
Therefore, purpose of the present invention just is to provide a kind of nuclear transfer method, and supply the nucleome cell and the acceptor ovocyte of the non-human mammal that this method alternative line mitochondrial DNA haplotype is identical carry out nuclear transplantation.
Particularly, nuclear transfer method of the present invention may further comprise the steps:
A, choose the non-human mammal of a certain Mitochondrial DNA haplotype, become skin flbroblast or cumulus cell as for the nucleome cell with it;
B, same a kind of Mammals that alternative line mitochondrial DNA haplotype is identical, with its ovocyte as recipient cell;
C, the stoning of acceptor ovocyte;
D, will move in the tenuigenin of enucleation oocyte, and make nucleus incorporate ovocyte, form reconstructed embryo for the nucleus of nucleome cell.
Homotype of the present invention (the Mitochondrial DNA haplotype is identical) nuclear transfer method can effectively improve fusion rate, spilting of an egg rate and the blastaea rate of reconstructed embryo, compares abnormal shape (Mitochondrial DNA haplotype difference) nuclear transplantation, and the blastaea rate has improved about 1.5 times.
Nucleus and cytoplasmic interaction and coordination are the bases of performance biological function, and in the evolution of long period of time process, different nuclear backgrounds has formed the Mitochondrial DNA of the specific haplotype that adapts with it.Therefore, utilize donor cell with identical haplotype and ovocyte can effectively solve in the nuclear transfer technology incompatible between caryoplasm, thereby improve nuclear transplantation efficient.
Embodiment
The invention will be further described below in conjunction with specific embodiment.Should be understood that following examples only to be used to the present invention is described and be not used in the scope of the present invention that limits.
PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) is meant and adopts round pcr amplification target DNA fragment, then with fragment digestion with restriction enzyme to be detected, restriction enzyme identification is also cut special sequence, product after afterwards enzyme being cut carries out electrophoresis, compares the otherness of different sources gene order according to the size of dna fragmentation.
The present invention utilizes the PCR-RFLP technology exactly, and the Mitochondrial DNA of the different oxen of increasing carries out rflp analysis then, selects the fragment with difference in length, according to restriction enzyme mapping it is divided into four types of A, B, C, D.The cell that selection wire mitochondrial DNA haplotype is identical carries out nuclear transplantation then, found that fusion rate, spilting of an egg rate and the blastaea rate of cell all is significantly improved.
Embodiment 1, the total length Mitochondrial DNA amplification
Extract different young ox peripheral blood samples respectively, anticoagulant heparin, phenol-chloroform separates extracting DNA, is template with extractive DNA, is primer with following H1, H2, four fragments of H3, H4 respectively, the Mitochondrial DNA of the different oxen of increasing:
H1：S：5’-CTGCAGTCTCACCATCAACC-3’；
A：5’-GTGTAGATGCTTGCATGTAAGT-3’；
H2：S：5’-TTATCCGTTGGTCTTAGGAA-3’；
A：5’GCGGCATGGTAATTAAGCTC-3’；
H3：S：5’-TTATCACAATCCAGAACTGAC-3’；
A：5’-CTAGTGAGAGTGAGGAGATATG-3’；
H4：S：5’-TGTGCATGTGACACGTATCC-3’；
A：5’-AAGCGATTGCTTACTAGTCGG-3’；
Concrete amplification condition is as follows:
H1:94 ℃ of pre-sex change 5min enters circulation; Cycling condition is: 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min, 35 circulations; Last 72 ℃ are extended 10min.
H2:94 ℃ of pre-sex change 5min enters circulation; Cycling condition is: 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 4min, 35 circulations; Last 72 ℃ are extended 10min.
H3:94 ℃ of pre-sex change 5min enters circulation; Cycling condition is: 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 4min, 35 circulations; Last 72 ℃ are extended 10min.
H4:94 ℃ of pre-sex change 5min enters circulation; Cycling condition is: 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 5min, 35 circulations; Last 72 ℃ are extended 10min.
All carry out 2% agarose gel electrophoresis after amplification is finished,, guarantee consistent with expection to detect the PCR product.
The pcr amplification product that obtains carries out purifying by PCR product purification test kit (Takara) product description (production number DV807A).
The amplified production of purifying carries out enzyme with restriction enzyme respectively to be cut, specific as follows:
Wherein BamH I cuts in 30 ℃ of enzymes and spends the night, and all the other are all cut at 37 ℃ of enzymes and spend the night.
Enzyme is cut the product electrophoresis, and (0.8～2% agarose, 120V is 1h) to do further analysis.
Embodiment 2, the analysis of endonuclease bamhi and Mitochondrial DNA classification
The fragment that has difference in length in the various enzyme rflp analysis is gathered, the results are shown in following table 1.
The fragment that table 1, restriction endonuclease rflp analysis have difference in length gathers
Annotate :+corresponding enzyme restriction enzyme site is arranged or have the extra restriction enzyme site of this enzyme;
-no corresponding enzyme restriction enzyme site or do not have the extra restriction enzyme site of this enzyme.
Analyze the difference of endonuclease bamhi, according to different individuality sources, the plastosome haplotypes of different oxen is divided into four types of A, B, C, D, four types restriction enzyme mapping is as shown in table 2:
Table 2, plastosome haplotype restriction enzyme mapping
| NlaIIIa(H1) | NlaIIIb(H1) | HpaII(H1) | HpaII(H2) | Pst?I(H2) | AvaII(H3) | BamH?I(H3) | BglII(H4) | |
| A | - | - | - | - | + | + | - | + |
| B | - | - | + | - | + | + | - | + |
| C | + | - | - | + | - | - | + | - |
| D | - | + | + | - | + | + | - | + |
Embodiment 3, the acceptor ovocyte preparation
The young ox of haplotype A type is carried out live egg-fetching, and concrete grammar sees that application number is 200510023510.1 Chinese invention patent application.
To collect liquid and pour into and choose in the ovum cup, (DPBS contains 8g NaCl in 1 liter of deionized water, 0.2g KCl, 1.44g Na with no calcium magnesium Du Shi phosphoric acid buffer
2HPO
4, 0.24g KH
2PO
430g BSA and 2000u heparin) flushing, sort out ovocyte complex body (cumulus oocyte complexes, COCs) move into culture dish, under stereoscopic microscope, COCs is carried out classification, the selection kytoplasm is even, cumulus cell is arranged closely on every side, hold the ovocyte more than three layers, put into ripe liquid (TCM-199 (Gibco, Grand Island NY) adds the short ovarian follicle of 10% foetal calf serum (FBS), 10 μ g/ml prolan Bs, 1 μ g/ml estradiol and 1 μ g/ml and generates plain) the middle 15～30h of cultivation.
Embodiment 4, for the preparation of nucleome cell
Young ox inoblast or cumulus cell that the above Mitochondrial DNA haplotype of learning from else's experience is respectively classified are cultivated with the DMEM/F12 (Gibco company, production number is respectively 11039-021) that contains 10%FBS.
1, cell former be commissioned to train foster
1. fibroblastic former be commissioned to train foster
Get the new born bovine ear tissue, place 0.9% physiological saline that contains two anti-(2% mycillins), ear tissue is after the tincture of iodine and 75% alcohol-pickled sterilization, stroke-physiological saline solution is washed 3～5 times, shreds, and adds 0.25% trypsinase, 37 ℃ of digestion 2h, add the DMEM/F12 substratum contain 10%FBS, it is foster that 37 ℃, 5%CO2, saturated humidity incubator Central Plains are commissioned to train, and the adherent back of observation of cell is changed substratum and continued to cultivate under the inverted microscope.
2. cumulus cell former is commissioned to train foster
Ripe 20h hour COCs (seeing embodiment 3) is put into the DPBS of the no calcium magnesium that contains 0.5% Unidasa, and digestion 1～2min simultaneously with inhaling embryonic tube pressure-vaccum repeatedly, sloughs the cumulus cell of outer loose, expansion.
Collect the cumulus cell of this part, move among the DPBS of no calcium magnesium as early as possible, the centrifugal 5min of 1200rpm abandons supernatant; Add 100 μ l0.25% pancreatin piping and druming 1min, add DMEM/F12+10%FBS (1 volume FBS+9 volume DMEM/F12 nutrient solution) and stop digestion, the centrifugal 5min of 1000rpm, DMEM/F12 nutrient solution washing 2 times.Use the DMEM/F12+10%FBS suspension cell, piping and druming fully dispels cell gently, is seeded to 25cm
2(inoculum density is about 1 * 10 to culturing bottle
5, include DMEM/F12+10%FBS nutrient solution 5ml), in 38.5 ℃, 5%CO
2, cultivate in the incubator of saturated humidity.
2, after growth in about 3～5 days converges, go down to posterity.
3, get the donor cell that the cell of cultivating for 1～5 generation is used as nuclear transplantation.
At the bottom of cell grows to bottle 80～90% o'clock, change nutrient solution into contain 0.5%FBS nutrient solution serum starvation 2～3 days, inducing cell enters the G0/G1 phase.
Before the nuclear transplantation, with 0.25% trysinization attached cell, after the cell DMEM/F12 nutrient solution that digests washing 2 times, add an amount of M199 (Gibco company, production number: 12340-030)+10%FBS blows and beats into the suspension of individual cells repeatedly, is stored in the thermostat container stand-by.
Embodiment 5, nuclear transplantation
With the kernel removing needle that internal diameter is about 20 microns the first polar body of acceptor ovocyte and the egg nucleus of near zone thereof are removed, donorcells nuclear with different Mitochondrial DNA haplotypes injects enucleation oocyte ovum week crack then, fusion parameters with 2.5KV/cm-3V/cm and 6-10 μ s, donor cell nuclear is incorporated ovocyte, obtain clone's reconstructed embryo.
Ovum behind the mixing operation moves among the M199 and washes 3 times, places the M199 that covers paraffin oil to cultivate droplet, in 5%CO
2, 38.5 ℃, cultivate in the incubator of saturated humidity.Behind 30～60min, check the fusion situation under stereoscopic microscope, calculate fusion rate, the result is as shown in table 3.
Reconstructed embryo put into B2+Vero co-culture system (respectively available from French academy of agricultural sciences and cell institute of the Chinese Academy of Sciences, article No. be respectively 220015 and GDC029) in, at 5%CO
2, 38.5 ℃, cultivate in the incubator of saturated humidity.Change nutrient solution every 48h half amount.48h observes the spilting of an egg situation of reconstructed embryo, calculates spilting of an egg rate, cultivates the quantity of the 7th day record blastaea, calculates the blastaea rate, and the result is as shown in table 3.
The comparison of different haplotype combination and nuclear transplantation efficient between table 3, caryoplasm
Annotate: fusion rate=reconstructed embryo merges number/ovocyte sum
Spilting of an egg rate=48 hours reconstructed embryo division number/reconstructed embryos of cultivation merge number
Blastaea rate=7 days gained blastaea number/spilting of an egg numbers of cultivation
According to the result of table 3, add up the nuclear transplantation efficient of (abnormal shape) between nuclear transplantation efficient between the same line mitochondrial DNA haplotype (homotype) and the different Mitochondrial DNA haplotype respectively, the result is as shown in table 4.
The comparison of table 4, dissimilar nuclear transplantation efficient
| Group | The ovum number | Merge number (%) | Spilting of an egg number (%) | Blastaea number (%) |
| Homotype | 1058 | 416(39.3) | 319(76.7) | 81(25.4) |
| Special-shaped | 330 | 109(33.0) | 78(71.6) | 13(16.7) |
| Add up to | 1388 | 525(37.8) | 413(78.7) | 94(22.8) |
From the data of above table 3 as can be seen, the fusion rate basically identical of various combination mode, all about in the of 35%, spilting of an egg rate then is combined as the highest with Mitochondrial DNA haplotype A-A, reached 82.7%, and A-C combined efficiency is minimum, only is 70.5%; Aspect the blastaea rate, the combination between the identical haplotype (A-A and C-C combination) best results has all reached more than 20%, obviously is better than the combination between the different haplotypes.
Comprehensive result relatively can see most clearly that also compare with special-shaped nuclear transplantation, the homotype nuclear transplantation all obviously is being dominant aspect fusion rate and the spilting of an egg rate from table 4, and the advantage of blastaea rate is more outstanding, is about 1.5 times of special-shaped nuclear transplantation.
In sum, adopt identical nucleus and the ovocyte tenuigenin of Mitochondrial DNA haplotype to carry out nuclear transplantation, can effectively improve the efficient of body-cell neucleus transplanting.Adopt method of the present invention can overcome the problem of the unclear nuclear transplantation inefficiency that causes in the prior art because ovocyte source.
Though below only be that example is verified method of the present invention with the ox, but those skilled in the art is according to the content of specification sheets, obviously method of the present invention as can be seen is equally applicable to other non-human mammal, for example pig, sheep and mouse etc., therefore, the homotype nuclear transplantation method at these non-human mammals should belong to scope of the present invention equally.
Claims (5)
1. nuclear transfer method, it is characterized in that, this method is to carry out nuclear transplantation with the identical non-human mammal of plastosome haplotype for nucleome cell and acceptor ovocyte, and the described nucleome cell that supplies derives from different individualities with the acceptor ovocyte, described non-human mammal is an ox, and the classifying method of described plastosome haplotype may further comprise the steps:
Extract different young ox peripheral blood samples respectively, anticoagulant heparin, phenol-chloroform separates extracting DNA, is template with extractive DNA, respectively with following H1, H2, four groups of primers of H3, H4, the Mitochondrial DNA of the different oxen of increasing:
H1：S：5’-CTGCAGTCTCACCATCAACC-3’；
A：5’-GTGTAGATGCTTGCATGTAAGT-3’；
H2：S：5’-TTATCCGTTGGTCTTAGGAA-3’；
A：5’GCGGCATGGTAATTAAGCTC-3’；
H3：S：5’-TTATCACAATCCAGAACTGAC-3’；
A：5’-CTAGTGAGAGTGAGGAGATATG-3’；
H4：S：5’-TGTGCATGTGACACGTATCC-3’；
A：5’-AAGCGATTGCTTACTAGTCGG-3’；
The amplified production of purifying carries out enzyme with restriction enzyme respectively to be cut, specific as follows:
Wherein BamH I cuts in 30 ℃ of enzymes and spends the night, and all the other are all cut at 37 ℃ of enzymes and spend the night;
Enzyme is cut the product electrophoresis, with 0.8～2% agarose electrophoresis 1h under 120V, to do further analysis; The fragment that has difference in length in the various enzyme rflp analysis is gathered, and the result is as follows:
Wherein "+" is for having corresponding enzyme restriction enzyme site or having the extra restriction enzyme site of this enzyme; "-" is for no corresponding enzyme restriction enzyme site or do not have the extra restriction enzyme site of this enzyme;
Analyze the difference of endonuclease bamhi, according to different individuality sources, the plastosome haplotypes of different oxen is divided into four types of A, B, C, D, four types restriction enzyme mapping is as follows:
2. the method for claim 1 is characterized in that, may further comprise the steps:
A, the non-human mammal inoblast of choosing a certain Mitochondrial DNA haplotype or cumulus cell are as supplying the nucleome cell;
B, choose same a kind of mammal ovocyte of same line mitochondrial DNA haplotype as recipient cell;
C, the stoning of acceptor ovocyte;
D, will move in the tenuigenin of enucleation oocyte, and make nucleus incorporate ovocyte, form reconstructed embryo for the nucleus of nucleome cell.
3. method as claimed in claim 2 is characterized in that, is to cultivate 1-5 generation through the DMEM/F12 that adds 10% foetal calf serum as inoblast or cumulus cell for the nucleome cell, and with 2-3 days cell of 0.5% foetal calf serum hunger.
4. method as claimed in claim 2 is characterized in that, described ovocyte stoning is that the egg nucleus of the first polar body of ovocyte and near zone thereof is removed.
5. method as claimed in claim 2 is characterized in that, the fusion parameters that described nucleus incorporates ovocyte is 2.5KV/cm, 6-10 μ s.
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| CN200610029674XA CN101117633B (en) | 2006-08-03 | 2006-08-03 | Nucleus transplantation method |
| PCT/CN2007/002239 WO2008017234A1 (en) | 2006-08-03 | 2007-07-23 | Cell nuclear transfer method |
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| CN200610029674XA CN101117633B (en) | 2006-08-03 | 2006-08-03 | Nucleus transplantation method |
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| AU2003237257A1 (en) * | 2002-05-24 | 2003-12-12 | Advanced Cell Technology, Inc. | A bank of stem cells for transplantation |
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| K Brüggerhoff等.Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up:effect of maternal lineage of oocyte donors.《Biology of reproduction》.2002,第66卷367-373. * |
| M.Tamassia等.In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content,quantity of mitochondrial DNA,and mitochondrial DNA haplogroup.《Biology of reproduction》.2004,第71卷697-704. * |
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