CN105505879A - Method and culture medium for culturing transgenic animal embryonic cells or transgenic animals - Google Patents
Method and culture medium for culturing transgenic animal embryonic cells or transgenic animals Download PDFInfo
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Abstract
The invention belongs to the field of biotechnologies, and relates to a method and culture medium for culturing transgenic animal embryonic cells or transgenic animals. The method comprises the steps that animal fertilized eggs obtained after gene modification are cultured to the four-cell stage, a zona pellucida is removed, then plasmodesmata are removed, four-cell embryos are separated into four single blastomeres, then the single blastomeres are put back into the zona pellucida with cytoplasm removed, in-vitro culture is carried out, and homozygous gene modified embryos are obtained efficiently. The optimized culture medium is provided at the same time. Based on the embryo blastomeres separation and independent culture technology, blastospheres can be successfully obtained in an optimized embryo culture system, and the efficiency is 30.33%. In addition, the homozygosis targeting efficiency of the obtained regenerated embryos is relatively improved obviously relative to that of unsplit developing embryos, and the homozygous mutation efficiency reaches up to 55.80%.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method and the substratum of cultivating transgenosis monkey.
Technical background
Animal disease model serves keying action in research human diseases pathogenesis and drug screening.The gene editing method setting up animal model mainly contains transgenosis and gene targeting.Namely foreign gene imports or is incorporated in genome by transgenosis by artificial means, and can stablize and pass to the next generation.But depend on viral methods to carry out genetic modification to animal and also have certain limitation, the radom insertion being namely transferred into gene makes mutational site to predict, sudden change quantity is uncontrollable, thus limits the foundation of animal model.And gene targeting refers to by the restructuring of endogenous dna fixed point, change a certain specific gene in genome thus in living organisms, study the function of this gene, the site-specific recombination mediated as traditional homologous recombination technique, recombinase Cre, zinc refer to ribozyme technology (zinc-fingernucleases), TALEN (transcriptionactivator-likeeffectornuclease) technology and in recent years since the CRISPR/Cas9 technology etc. that receives much concern.
Traditional gene targeting depends on embryonic stem cell line, carries out gene targeting in the embryonic stem cell line cultivated first in vitro, then the embryo stem cell transplantation after practicing shooting is entered animal parent, allows it develop into animal with mutator gene.But only have the embryonic stem cell line of mouse to can be used for gene targeting at present.For most of species, genetic modification still depends on the embryonic cell directly using animal, the low and somewhat expensive of efficiency.The efficiency improving genetic modification in embryonic cell successfully sets up the crux of animal disease model.The artificial incision enzymes such as Zinc finger nuclease (ZFN) technology of development in recent years, transcriptional activation increment effector endonuclease (TALEN) technology and gene editing new technology CRISPR/Cas9 make to edit appoints the problem in genome meaning site to be achieved.TALEN (TranscriptionActivator-LikeEffectorNuclease) transcriptional activation increment effector nuclease technology, overcome conventional ZFN method and can not identify arbitrary target gene order, and recognition sequence is often by problems such as upstream and downstream sequence affect, and there is the equal or better activity of ZFN, but TALENs molecular weight of albumen is excessive often makes troubles to molecule manipulation, not only increase with target gene in conjunction with difficulty, build complicated, and also may produce other impact in vivo.
The CRISPR/Cas9 technology developed for nearly 2 years makes in all species, realize genome editor becomes possibility.CRISPR/Cas9 is a kind of new technology being instructed Cas9 albumen to edit gene by RNA and modified deriving from bacterium acquired immunity.Jinek etc. carry out artificial reconstructed this system that makes to CRISPR/Cas9 system can more simply and easily for gene editing (Jineketal., 2013), and can realize that pair of alleles is carried out shearing and obtain homozygous mutation, also can design multiple gRNA to knock out goal gene completely in multiple sites of goal gene.At present, the Cas9 system of RNA mediation successful mediating bacterial, vegetable cell, zebrafish embryo, mouse, human archeocyte, non-human primate cynomolgus monkey embryo, the even mankind can discard genome editor (Choetal., 2013 of embryo etc.; Congetal., 2013; Jineketal., 2013; Malietal., 2013; Niuetal., 2014; Plattetal., 2014; Shanetal., 2013), for the treatment being applied to human inheritance's deficiency disorders future provides hope.
The develop rapidly of the gene targeting such as transgenic technology and TALEN, CRISPR/Cas9 makes the foundation of animal disease model no longer be confined to the model animals of minority.But these technology also all exist at present transgenation is fitted together to the weak points such as polymorphism, gene knock-in homologous recombination efficiency are lower.For the chimeric effect problem of genetic modification, small animal model goes down to posterity because growth cycle is shorter by continuous hybridization and obtains Mutants homozygous.But for large animal, as the primate comparatively close with the mankind, its sexual maturity time just needs 4 to 5 years, pregnant time reaches 165 days, and still lacking effective monkey embryonic stem cell line at present, the existence being therefore fitted together to effect makes to set up the large animal models such as homozygous gene sudden change monkey and has huge challenge.
The generation of chimeric mutational effect may due to the gene targeting RNA of injection or albumen still continuous expression after zygote embryos cleave, or transgenic virus fails stablize transgene group at one cell embryos thus cause the gene of a part of cell in embryonic cell division growth course to be modified period, part cell still maintains the chimerism of wild-type status, and in adorned cell, also may produce mosaic type mutant (Plattetal., 2014 with different genes saltant type; Sungetal., 2014; Kimetal., 2014).Such as, when single fertilized egg cell splits into four cells, the type that each cytogene is modified and degree can differ to some extent, and the embryonic tissue formed and the animal of new life can be carried different genetic modification types or be had the genetic modification of (strand or double-stranded DNA) in various degree in different cell or tissues.In addition, in embryonic cell fission process, the DNA damage repair mechanism of self or the non-identity etc. of the non-homogeneous recombinational repair of DNA also can affect gene targeting efficiency and chimeric effect.Therefore, improve or invention can to overcome the method for the mosaic defect effect of these genetic modification technology most important to the animal disease model efficiently setting up homozygous mutation.
Embryo separating refers to and by artificial means pre-implantation embryos is divided into two or more part, after segmentation, each part can continue to grow for complete embryo under suitable culture condition, the number that the application of this technology can increase effective embryo number in embryo transfer, improve pregnancy rate, increases clone offspring.At present this technology a lot of animal as (Astonetal., 2008 such as mouse, rat, pig, sheep, horses; SchrammandPaprocki, 2004; Tarkowskietal., 2010) widely apply in Mammals, and be also confirmed in primate.The research in past finds, the embryo of 2 to 8 of rhesus monkey cell stages is carried out blastomere separation, and the blastomere of separation is put into sky zona pellucida and grow, restructuring embryo can Successful development to hatched blastocyst stage (Mitalipovetal., 2002).(the Chanetal. such as Chan, 2000) be also four parts in 2000 to rhesus monkey 8 eight cell stage embryo separating, successfully obtain a monkey Tetra come by 1/4 fetal development after wherein two blastomeres migrate to acceptor, the blastomere demonstrated after body early embryo separation can develop into normal fetus and newborn monkey.
But in above-mentioned experiment, by the isolated blastomere of the embryo of 4-8 cell all containing 2 different embryonic cells.Adopt such blastomere still can prepare and modify somatic embryo and new born animal with different genes.This is because mosaic problem existing in genetic modification technology causes, the genetic modification efficiency that namely the gene targeting molecule injected of one cell embryos stage is uneven in different cell in fission process.Because the expression of transgenosis DNA and Cas9mRNA and effect greatly may weaken in 4 cell stage stages, continue division from the isolated single blastomere of 4 cell stage and the embryo that formed unicellularly carries identical genetic modification type each.But, not yet studies have reported that at present and be separated by four cell stage embryo in primate and can the single blastomere that come successfully hatch the single blastomere of originating to blastocyst stage or this four cell stage embryo in vitro and transplant success and grow vervet.
Summary of the invention
The object of the invention is that providing a kind of can reduce the chimeric embryo culture method of effect and the preparation method of transgenic animal.
Another object of the present invention is to provide a kind of substratum being applicable to aforesaid method.
Invention is achieved through the following technical solutions above-mentioned purpose.
First, invention provides a kind of embryo culture method that can reduce chimeric effect.
For solving the chimeric effect problem in current Animal Model, the invention provides a kind of method significantly improving genetic modification precision, that is: the fertilised non-human eggs after genetic modification is cultivated to four cell stages, remove zona pellucida, remove cell plasmodesmata again, 4 cell stages are separated into 4 single blastomeres, again single blastomere cell is put back in the zona pellucida removing kytoplasm further, carry out vitro culture, thus improve the homozygosity of genetic modification embryo.
The preparation method of further transgenic animal, then, when above-mentioned single blastomere can be cultivated to two cells to four cell stage, select suitable embryo and transplant, to obtain the genetically modified animal model isozygotied.
For reducing mosaic and improving genetic modification precision, the present invention utilizes kapillary, in conjunction with the method for trysinization, the animal embryo (such as cynomolgus monkey embryo) of four cell stages is separated into four single blastomeres alternatively, again these four single blastomere cells are put back into respectively in four empty zona pellucidas, are cultivated to morula or blastaea under the condition of in vitro culture optimized.Meanwhile, the present invention adopts singe-cell PCR technology alternatively, can analyze transgenation in each single blastomere cell or modified types and degree.When single blastomere after embryo separating is grown to four cells or eight cell stages, significantly can increase the homogeny of genetic modification type and degree in each cell.The embryo utilizing the method for the invention to obtain can be used for transplanting, and will carry identical gene mutation type in every individual cells of the new born animal obtained, thus can set up more can simulate because of transgenation cause the animal model of disease.
Embodiment is following (flow process is as Fig. 6) alternatively:
1., after will implementing transgeneic procedure, the animal embryo being developed to 4 cells is separated into 4 single blastomeres, and is positioned in sky zona pellucida respectively;
2. embryo is proceeded in the culture system of optimization;
3. from the embryo being again developed to 4 cells, take out a blastomere;
4. use the PCR condition optimized to carry out gene type assay;
5. the embryo being applicable to transplanting is filtered out according to PCR result;
6. embryo transfer, the transgenic animal that output is isozygotied.
As an exemplary embodiment, the present invention by based on the unicellular growth of cynomolgus monkey zygote after CRISPR/Cas9 genetic modification to four cell stages, remove zona pellucida by acid tyrode or basic protein enzymic digestion, and not containing Ca
2+, Mg
2+in the pancreatin of ion, cell plasmodesmata is removed in digestion, kapillary mechanical separation 4 cell stage blastomere, again single blastomere cell is put back into remove kytoplasm zona pellucida in, optimize condition of in vitro culture under: the first, these blastomeres broken up again can Successful development to blastaea; Secondly, singe-cell PCR technical evaluation gene targeting homozygous mutation rate can be utilized until growth to separating single blastomere during four cell stages; 3rd, when the blastomere broken up is grown to two cells to four cell stage, select suitable embryo and transplant, to obtain the target practice genetic modification monkey model that isozygotys.
The single blastomere that four cell stage embryo are successfully separated and come by the present invention successfully hatches the embryo for normal development, embryo that in each cell, genetic modification type is consistent can be obtained and enter receptor with this embryo transfer, thus can significantly reduce the mosaic problem of carrying different mutation type in each cell caused because fertilized egg cell divides fast, setting up on genetically modified animal model and will be significant.
The present invention can adopt kapillary to realize cynomolgus monkey four cell stage embryo after CRISPR/Cas9 genetic modification to be separated into four single blastomeres in conjunction with the mode of trysinization alternatively.
Further preferably, the present invention optimize culture condition under by they in vitro Successful development be morula or blastaea.
Invention adopts the culture condition optimized to be favourable, and is necessary to a certain extent.
The single blastomere cell that cultivator work is separated is discrepant with the cell cultivated under state of nature.This is because have mutual material/signal transmission by Cell tracking between embryonic blastomeres, the single blastomere separated loses the interaction with other blastomeres, more independent growth is that complete embryo is more difficult.
Invention particularly provides substratum and the culture condition of this single blastomere cell through separating of a kind of applicable cultivation.
Substratum provided by the present invention is:
As a kind of exemplary embodiment, the present invention adds glucose and vitamins C further on the basis of traditional HECM9 substratum.
As the particularly preferred scheme of one, the glucose added is D-glucose.
Alternatively, glucose and vitamins C content is in the medium divided into: 0.5 ~ 5mM, 0.5 ~ 10mM.
Preferably, glucose and vitamins C content is in the medium divided into: 1 ~ 4mM, 1 ~ 9mM; More preferably 1 ~ 3mM, 1 ~ 8mM; More preferably 1.5 ~ 2.5mM, 3 ~ 6mM; Most preferably 1.5 ~ 2.5mM, 4 ~ 6mM.
D-glucose and ascorbic interpolation can improve blastocyst rate greatly.In particularly preferred scheme, both are added into content is respectively 1.5 ~ 2.5mM, 4 ~ 6mM.
As a rule, add glucose in the medium or vitamins C not rare, but as a rule, the common substratum adding these two kinds of materials is actually rare.Further, as a rule, when basic medium has carbon source, glucose whether is added or vitamins C can't cause great effect to cultivation results.But, in the present invention, because the cultivated single blastomere separated loses the interaction of normal embryonic cells and other blastomeres, lose mutual material/signal transmission, more independent growth is that complete embryo is more difficult.For cynomolgus monkey embryo, under normal circumstances, adopt conventional HECM9 substratum can reach more than 30% close to 40% blastocyst rate (as Fig. 7 control group), but, when cultivating the single blastomere after separation with conventional H ECM9 substratum, morula ratio and blastocyst rate, but all close to 0 (Fig. 3), in other words, adopt conventional H ECM9 substratum cannot cultivate the isolated single blastomere of cynomolgus monkey embryo at all.
And the substratum after adopting the present invention to improve, then can reach the morula ratio of more than 60% and the blastocyst rate of 30.33%, reach the quite level close to cellar culture normal embryonic cells.Glucose and ascorbic being added on serve vital effect here, this be contriver make first there is breakthrough discovery.
It should be noted that, add D-glucose and the not further blastocyst rate (Fig. 7) improving improved culture medium and cynomolgus monkey normal embryonic cells is cultivated of vitamins C in a larger amount, on the contrary, when addition is increased to 3mM D-glucose and 8mM vitamins C, the scheme that the blastocyst rate contrast of normal embryonic cells is optimum declines on the contrary to some extent.
Preferred scheme controls suitable culture condition further.
Cultivating gas ratio is 74-90% nitrogen, and 2-6% carbonic acid gas, 5-20% oxygen, culture temperature is 34 ~ 40 degrees Celsius, preferably 37 degrees Celsius.
Until these single blastomere embryos after embryo separating again grow be four cells or eight cell stages time, accurately can analyze transgenation in each single blastomere cell or modified types and degree by singe-cell PCR technical evaluation, find by differentiation and development and its genetic modification type of four cells of coming is basically identical again after same embryos cleave.Described method in the present invention significantly can increase the homogeny of genetic modification type and degree in each cell, thus reduces the mosaic because producing in the unicellular fission process of zygote.Therefore, the embryo utilizing the method for the invention to obtain can be used for transplanting, and will carry identical gene mutation type in every individual cells of the new born animal obtained, thus be conducive to efficient foundation more can simulate because of transgenation cause the animal model of disease.
The present invention has following beneficial effect:
1. genetic modification precision significantly increases (use the homozygous animal embryo efficiency 2.85% that traditional method obtains, this efficiency can be increased to 55.8% by technical scheme of the present invention);
2. single blastomere ectogeneticly to improve (ectogenesis to the efficiency of morula and blastaea is increased to 63.67% and 30.33% respectively, and this is breakthrough, the single blastomere of primate can be cultivated as blastaea before this there are no report).
Accompanying drawing explanation
To be that cynomolgus monkey 4 cell stage single blastomere is external cultivate schema to Fig. 1 again.
Fig. 2 is the external cultivation results again of cynomolgus monkey 4 cell stage single blastomere, and 4 division single blastomere vitro culture are grown blastaea and hatch.
Fig. 3 is the cultivation results of improved culture medium of the present invention to cynomolgus monkey single blastomere cell, the single blastomere of 63.67% can be cultivated densification (morula ratio) by the substratum after optimization, and the single blastomere of 30.33% can be grown to blastocyst stage.
Fig. 4 is fetal development statistics.
Fig. 5 is the homogeny that PCR and enzyme cut qualification single blastomere restructuring embryo gene targeting; Wherein
(A) divide the unicellular target practice qualification result from normal 4-8 cell stage, target practice site is pink1 Exon 2;
(B) be separated through single blastomere the unicellular target practice qualification result be separated after cultivation develops into new 4 cells again by normal 4 cell stages, target practice site is pink1 Exon 2; Hpy188I enzyme cuts qualification target practice gene mutation type;
(C) PCR and Hpy188I enzyme is cut qualification statistics and is shown, normal 4-8 cell stage and cultivate the genetic modification homogeny difference very remarkable (* * * P<0.001) of new 4 cell stages of growth again.In 31 normal 4-8 cell stages, only have 1 embryo's 4 single blastomeres to practice shooting and come to the same thing, double chain mutation or homozygous mutation (Homo-mutation) probability are 2.85%, and mostly are strand sudden change; And being separated in 37 the restructuring embryos obtained by 13 fetal tissues, PCR and enzyme are cut and are identified that the single blastomere obtaining 18 embryos is practiced shooting identical, and homozygous mutation probability, up to 55.8%, is 19.57 times of fetal tissues;
Note: double chain mutation is labeled as black arrow, heterozygosis (strand) mutation markers is grey arrow.
Fig. 6 is that the total figure of experiment flow chooses the raising of single cage, age, body weight, the donor monkey that physiological period is suitable carries out injection of hormone superovulation, the mature egg in the MII period obtained is fertilized by single-semen injection, after fertilization injection Cas9/gRNA modifies gene, when vitro culture is grown to 4 cell stage, in conjunction with pancreatin, 4 cell stages are separated into single blastomere cell by self-control glass capillary, then put into empty zona pellucida relaying supervention to educate, grow to 4 new cell stages, new 4 cell stages of part identify whether be target practice of isozygotying by carrying out singe-cell PCR in separation, part is transplanted in suitable acceptor, conceived through 165 Gestation periods, to expect that can bear homozygous gene modifies monkey model animal.
Fig. 7 is (not being separated the embryo of the blastomere) blastocyst rate under different culture media condition.* expression and control group contrast and have significant difference (t-checks); * represents to contrast with control group to have pole significant difference.
Embodiment
The invention will be further described below, but embodiments of the present invention are not limited to following embodiment introduction, and all equivalent changes of doing according to principle of the present invention or theory or accommodation all should be considered as the category of the present invention's protection.
Embodiment 1 transgenic protocol
Build the sgRNA carrier of the CRISPR/Cas9 containing PINK1 gene and ASPM gene target sequence.Described sgRNA expression vector, expression containing a T7 promotor control sgRNA, the specific target sequence of 20bpsgRNA can be inserted after with BbsI restriction digest, the recognition site of this specific target sequence is positioned at PINK1 gene Second Exon, ASPM gene the 3rd exon and the tenth exon.Gene editing can be carried out and the sgRNA sequence of less PINK1 and the ASPM gene of effect of missing the target to screen, it is low and meet the target sequence site of CRISPR/Cas9 in conjunction with genome signature (5 '-G (19N)-NGG3 ') or (5 '-CCN (19N)-C-3 '), as table 1 that applicant utilizes the Blast software of NCBI on the PINK1 gene of macaque and the ASPM gene order of cynomolgus monkey, screened effect of missing the target respectively.
A. chemical synthetic oligonucleotide chain, anamorphic zone glues the target spot of end:
During synthesis, positive-sense strand: (N represents arbitrary DNA base to 5 '-CACC-GN19 (containing first G)-3 ', as ATCG.N19 represents arbitrary 19 DNA base sequences)
Antisense strand: (N represents arbitrary DNA base to 5 '-AAAC-GN19 (containing first G, reverse complemental)-3 ', as ATCG.N19 represents arbitrary 19 DNA base sequences)
B. positive-sense strand, antisense strand annealing, forms the fragment of band BbsI sticky end, annealing system:
Oligo110ul(10um)
Oligo210ul(10um)
NEBbuffer35ul
Deionization H2O25ul
Cumulative volume 50ul
Cycle of annealing: 98 degree, 10min, naturally cools to room temperature, connects
C. fragment of annealing above is connected to the T7-sgRNA carrier after being cut by BbsI enzyme
D. transform, selected clone pcr identifies, order-checking verifies qualification result further.
E. by above-mentioned constructed sgRNA target sequence carrier according to reference method, in-vitro transcription synthesis RNA.Cas9 plasmid vector glue after the linearizing of PmeI enzyme is reclaimed, in-vitro transcription synthesis cas9mRNA simultaneously.
F. detect that in the sgRNA of above-mentioned synthesis, PINK2-2 and ASPM3-3, ASPM-Exon10 cutting DNA efficiency is higher by cas9 albumen In vitro digestion experiment (Shaoetal., 2014).
Secondly, PINK2-2sgRNA or ASPM3-3 of above-mentioned synthesis, ASPM-Exon10 and Cas9mRNA are total to microinjection in cynomolgus monkey zygote embryo.
Table 1.
Embodiment 2 is separated out single blastomere
1. the ovum obtained by super row is carried out single-semen injection (ICSI) and treats that embryo occurs two protokaryon, confirms fertilization, 8-10h after single-semen injection, and Cas9/gRNA is in fertilized egg cell's matter in injection.
2. cultivate within 1-2 days, treat that fetal development is to four cells, removes zona pellucida by the pronase digestion 45-60 of 0.5% (w/v) second at conventional H ECM9 substratum (what use before blastomere separation is not improved culture medium).
3. the embryo removing zona pellucida transferred in 0.05% pancreatin drips, four cell stages of zona-free are separated, are washed twice to eliminate the impact of residual pancreatin on blastomere with HECM9 by digestion 2-3 minute.
The cultivation of embodiment 3 single blastomere and differentiation and development
1. blastomere is put in the empty zona pellucida of well in advance by list, is transferred in improvement HECM9 substratum to cultivate respectively can again grow to 4 cells for 1-2 days.
(1) conventional medium, namely traditional HECM9 substratum, have employed formula as shown in table 2 in this exemplary embodiment;
(2) HECM9 substratum is improved: the D-glucose adding 2 mmoles in table 2 is filled a prescription, the vitamins C of 5 mmoles.
The potential of hydrogen PH7.1 of substratum; Osmotic pressure 285.
Culture condition: gas ratio is the nitrogen of 75%, the carbonic acid gas of 5%, 20% oxygen, culture temperature 37 degrees Celsius
The traditional HECM9 culture medium prescription of table 2.
| Composition | mg/100ml |
| PVA | 10.0 |
| Phenol red | 1.0 |
| CaCl 2·2H 2O | 28.0 |
| MgCl 2.6H 2O | 9.3 |
| NaCl | 664.0 |
| KCl | 22.3 |
| NaHCO 3 | 210.0 |
| Na-Lactate 60％syrup | 65ul |
| 1M HCl | 140ul |
Fig. 1 is shown in by schematic diagram, and cultivation results as shown in Figure 3.When cultivating the single blastomere after separation with conventional H ECM9 substratum, morula ratio and blastocyst rate are all close to 0 (Fig. 3); And adopt the substratum after improvement, then can reach the morula ratio of more than 60% and the blastocyst rate of 30.33%.
2. cultivated in defined medium by normal embryonic cells, defined medium comprises:
(1) control medium, namely traditional HECM9 substratum, have employed formula as shown in table 2 in this exemplary embodiment;
(2) 1mMG+1mMVC substratum: namely add the D-glucose of 1 mmole and the vitamins C of 1 mmole in HECM9
(3) 2mMG+5mMVC substratum: namely add the D-glucose of 2 mmoles and the vitamins C of 5 mmoles in HECM9
(4) 3mMG+8mMVC substratum: namely add the D-glucose of 3 mmoles and the vitamins C of 8 mmoles in HECM9
The potential of hydrogen 7.0-7.5 of substratum; Osmotic pressure is 270-310.
Culture condition is the same.
Cultivation results as shown in Figure 7.
Result display, with the addition of glucose and ascorbic substratum blastocyst rate is former in traditional HECM9 substratum.Especially in foster base, add D-glucose2 mmole (2mM), during VC5 mmole, embryo culture improves best results.
3. the single blastomere separated is carried out singe-cell PCR qualification homozygous mutation rate (in table 3, sequence number is followed successively by SEQIDNO.4-SEQIDNO.16 to pcr amplification primer sequence).
Table 3.
4. the embryonic development again selecting suitable 2-4 cell stage is transplanted.
Embodiment 4
1., by after fertilized embryo vitro culture to the two protokaryon of appearance, Cas9/gRNA is in fertilized egg cell's matter in injection, and Cas9 is with RNA or protein form injection, and concentration is that 200ng/ul, gRNA inject with rna form, and concentration is 200ng/ul.
2. treat that fetal development is to four cells at HECM9 culture medium culturing 1-2 days, remove zona pellucida second by the pronase digestion 45-60 of 0.5% (w/v).
3. the embryo removing zona pellucida transferred in 0.05% pancreatin drips, four cell stages of zona-free are separated, are washed twice to eliminate the impact of residual pancreatin on blastomere with HECM9 by digestion 2-3 minute.
4. blastomere is put in the empty zona pellucida of well in advance by list, be transferred back in the culture system of optimization to cultivate and within 1-2 days, can again grow to 4 cells (in this step, use the HECM9 substratum of improvement, be specially, to add the vitamins C of 5mg/ml, cultivating gas ratio is 90% nitrogen, 5% carbonic acid gas, 5% oxygen, culture temperature is 37 degrees Celsius).
5. the embryo of the above-mentioned cell stage of differentiation and development to 4 is again separated single blastomere by step 3 equally and carry out singe-cell PCR qualification homozygous mutation rate.
6. find its derived embryo according to PCR result, and give suitable receptor by embryo transfer homozygous for genetic modification.
Four cell stages are successfully separated through the embryo that CRISPR/Cas9 genetic modification is crossed and obtain single blastomere by the present invention, and with the substratum improved, single blastomere to be put into sky zona pellucida can Successful development be complete embryo again, show as 63.67% morula rate and and 30.33% blastocyst rate.This method and past adopt the blastomere containing 2 embryonic cells to carry out vitro culture a great difference.Meanwhile, the survival rate for complete embryo is grown after invention significantly improves embryo separating again.Secondly, the present invention is single blastomere Cell redifferentiation by described embryo separating is individual embryos, and then through the identity of singe-cell PCR technical evaluation energy accurate evaluation genetic modification type and mosaic (Fig. 2).Analytical results shows that the embryo's homozygous mutation probability again breaking up institute's Successful development obviously increases 19.57 times, and the different blastomeres shown as from same embryo all realize gene targeting.Therefore the present invention is conducive to the embryo transfer number improving homozygous mutation, to reduce manpower that primate chimeric embryo transplanting etc. causes, time and economic waste, effectively set up homozygous gene mutant animals model and simulate the great human diseases caused because of transgenation more accurately.
Result shows: the present invention is by embryo separating differentiation technique again, and in the embryo culture system optimized, successfully can obtain blastaea, efficiency is 30.33%; Secondly, the differentiating embryonic again obtained isozygoty target practice efficiency do not split before embryonic development relatively obviously increase, the homozygous mutation efficiency (improving 19.97 times than ameristic embryo) up to 55.80%.Therefore, select the embryo broken up again through embryo separating to carry out transplanting and can significantly improve genetic modification precision and reduce mosaic embryo transfer.
Claims (10)
1. cultivate the method for transgenic animal embryo cell or transgenic animal for one kind, it is characterized in that, by the animal after genetic modification by when cultivating to four cell stages, remove zona pellucida, remove cell plasmodesmata again, 4 cell stages are separated into 4 single blastomeres, again single blastomere cell is put back in the zona pellucida removing kytoplasm further, carry out vitro culture, the animal embryo that rate is high thus acquisition genetic modification isozygotys.
2. the method for transgenic animal as claimed in claim 1, is characterized in that, when growing above-mentioned single blastomere to two cells to four cell stage, select suitable embryo and transplant, to obtain the target practice genetically modified animal model that isozygotys.
3., for cultivating a substratum for transgenic animal embryo cell or transgenic animal, it is characterized in that comprising following composition:
Polyvinyl alcohol 2 ~ 8g/100ml; Phenol red 0.1-2mg/100ml;
Ca
2+6.85～8.22mg/100ml；Mg
2+0.83～1.3mg/100ml；
NaCl500～700mg/100ml；KCl15-30mg/100ml；
NaHCO3120 ~ 300mg/100ml; DL-LACTIC ACID sodium solution 50 ~ 80ul/100m;
Glucose 0.5 ~ 5mM; Vitamins C 0.5 ~ 10mM.
4. for cultivating a substratum for animal embryo cell, it is characterized in that, glucose and vitamins C are added again in the basis of HECM9 substratum, make its final concentration be respectively 0.5 ~ 5mM and 0.5 ~ 10mM.
5. the substratum as described in claim 3 or 4, is characterized in that described glucose is D-glucose.
6. the substratum as described in claim 3 or 4, is characterized in that glucose and ascorbic final concentration are respectively 1 ~ 4mM, 1 ~ 9mM; More preferably 1 ~ 3mM, 1 ~ 8mM; More preferably 1.5 ~ 2.5mM, 3 ~ 6mM; More preferably 1.5 ~ 2.5mM, 4 ~ 6mM.
7. the substratum as described in claim 3 or 4, is characterized in that described transgenic animal are Mammals, preferred non-human mammal, more preferably primate, more preferably monkey.
8. the substratum as described in claim 3 or 4, is characterized in that the single blastomere cell for cultivating described animal embryo cell; More preferably be separated and next single blastomere cell for cultivating transgenic animal four cell stage embryo.
9. utilize a method for the culture medium culturing single blastomere cell as described in claim 3 or 4, it is characterized in that the gas condition cultivated is 74-90% nitrogen, 2-6% carbonic acid gas, 5-20% oxygen.
10. method as claimed in claim 9, is characterized in that culture temperature is 34 ~ 40 degrees Celsius, preferably 37 degrees Celsius.
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