CN106676075A - PG-haESCs (parthenogenetic-haploid embryonic stem cells) as well as preparation and application thereof - Google Patents
PG-haESCs (parthenogenetic-haploid embryonic stem cells) as well as preparation and application thereof Download PDFInfo
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- CN106676075A CN106676075A CN201510765622.8A CN201510765622A CN106676075A CN 106676075 A CN106676075 A CN 106676075A CN 201510765622 A CN201510765622 A CN 201510765622A CN 106676075 A CN106676075 A CN 106676075A
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Abstract
The invention relates to PG-haESCs (parthenogenetic-haploid embryonic stem cells) as well as preparation and an application thereof. H19 DMR and IG-DMR of the PG-haESCs are knocked out. The invention further provides a preparation method of the PG-haESCs and the application of the PG-haESCs in construction of gene-modified semi-cloned animals and gene-modified semi-cloned animal libraries. The PG-haESCs can be injected into ova, survival SC mice can be obtained stably, the PG-haESCs can be effectively used for polygenic inheritance, and animals modified with polygenic inheritance can be further obtained.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of lonely female monoploid embryo stem cell and its prepare with should
With.
Background technology
The recessive gene screening of full-length genome level is to identify that gene plays a kind of pole of function in particular biological process
Its effective and powerful method.This strategy is successfully used in unicellular lower eukaryote such as saccharomyces cerevisiae, nematode.Feeding
In newborn animal, due to the dliploid property of genome, difficulty (Shi, L., Yang, H., and that this functional screening seems abnormal
Li,J.(2012).Haploid embryonic stem cells:an ideal tool for mammalian genetic
analyses.Protein & cell 3,806-810).(RNAi) means have been disturbed by targetting the RNA of mRNA level in-site at present
Jing becomes carries out the preferred plan that gene function loses screening in the range of cells of mamma animals full-length genome.However, this method is normal
The expression of the effective suppressors of Chang Wufa, while also there is effect of missing the target (Kaelin, W.G., Jr. (2012) .Molecular
biology.Use and abuse of RNAi to study mammalian gene function.Science 337,
421-422).Recently, from bacterium CRISPR-Cas9 systems Successful utilization in mouse and the gene of people's cell level
In the screening of afunction.However, the full-length genome level of CRISPR-Cas9 mediations is screened and is only used for cellular level, and incite somebody to action
Research is confined to the phenotype analytical of cellular level.For this purpose, just as at present in unicellular lower eukaryote as yeast nematode is cooked,
How to realize carrying out efficient, extensive, afunction screening to the wider array of biological process of scope in mammlian system
Approach.
Mammal monoploid embryo stem cell (haESCs) acquisition (Elling, U., Taubenschmid, J.,
Wirnsberger,G.,O'Malley,R.,Demers,S.P.,Vanhaelen,Q.,Shukalyuk,A.I.,Schmauss,
G.,Schramek,D.,Schnuetgen,F.,et al.(2011).Forward and reverse genetics
through derivation of haploid mouse embryonic stem cells.Cell Stem Cell 9,
563-574；Leeb,M.,and Wutz,A.(2011).Derivation of haploid embryonic stem cells
From mouse embryos.Nature 479,131-134), provide a kind of highly desirable instrument for genetic analysis.
The potentiality of development of lonely female monoploid embryo stem cell (PG-haESCs) is by replacing ovum in embryonated egg with it
The reconstructed embryo that genome is obtained is verified.However, its birth efficiency is extremely low (290 pieces of reconstructed embryos only obtain 2 mouse), and
And only 1 mouse grow up it is adult.That is, current research shows lonely female monoploid embryo stem cell (PG-haESCs)
It is infeasible that the method injected by ovum produces half Cloning of mouse.
The content of the invention
In the presence of in order to solve the problems, such as prior art, it is an object of the invention to provide one kind can improve half clone moving
The lonely female monoploid embryo stem cell of thing birth rate and its application.
The reason for causing birth rate very likely is that the imprinting status of lonely female monoploid itself occur in that exception.By reality
Test, surprisingly it has been found that, the otherness of two marking clusters of H19-Igf2 and Dlk1-Dio3 methylated into region (DMR) knocks out can be with
Start against lonely female monoploid embryo stem cell.The lonely female monoploid embryo stem cell that this two DMR are knocked out is named as
DKO-PG-haESCs.This DKO-PG-haESCs can efficiently obtain half Cloning of mouse (about 15.50% SC mouse
Birth rate), and it is many by lonely female monoploid embryo stem cell carrying out still being able to after genetic manipulation stably producing in vitro
Half cloned animal of genetic modification.People is more allowed to be excitedly that this DKO-PG-haESCs only needs to turn on cellular level in vitro
The sgRNA libraries of the stable expression of dye and Cas9 just effectively one-step method can obtain substantial amounts of mutant animals.These experimental results
Show that DKO-PG-haESCs can realize base using by way of as an intermediate carrying sgRNA libraries in individual level
The mutation of cause, therefore can further apply in animal individual level based on the Large-scale Screening of gene mutation.
To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：
A first aspect of the present invention provides a kind of lonely female monoploid embryo stem cell, and the lonely female monoploid embryo is dry thin
H19DMR the and IG-DMR genes of born of the same parents are knocked.
A second aspect of the present invention provides the preparation method of the lonely female monoploid embryo stem cell, including by lonely female list
The H19DMR and IG-DMR of times body embryonic stem cell is knocked out and is obtained the lonely female monoploid embryo stem cell.
A third aspect of the present invention provides the purposes of the lonely female monoploid embryo stem cell, is to change for building gene
Half cloned animal made.
A fourth aspect of the present invention provides a kind of method of the structure cloned animal of genetic modification half, by H19DMR and IG-
The lonely female monoploid embryo stem cell of the double knockouts of DMR is combined half clone embryos of acquisition with egg cell, is cultivated half clone embryos and is obtained
Obtain half cloned animal.
Fifth aspect present invention provides a kind of genetic modification animal, is built by preceding method and is obtained, or for aforementioned side
The generative propagation offspring of half cloned animal that method builds.
Sixth aspect present invention provides a kind of lonely female monoploid embryo stem cell of present invention and sgRNA slow virus library
The method for building the cloned animal library of genetic modification half.
Seventh aspect present invention provides a kind of cloned animal library of genetic modification half, is to build to obtain using preceding method
.
Compared with prior art, the present invention has the advantages that：
(1) existing research shows：The PG-haESCs set up from monoploid blastaea can not obtain half by IPAHCI technologies
Cloned animal, and present invention discover that the double PG-haESCs for knocking out of H19 and IG-DMR are expelled in ovum can be with about
15.50% stabilised efficiency ground obtains the SC mouse of survival.Importantly, it has also been found that DKO-PG-haESCs can be effective
Ground further obtains carrying the SC mouse of multiple-factor inheritance modification for multiple-factor inheritance operation by ICPHCI.Furthermore, with reference to
SgRNA libraries, DKO-PG-haESCs can be efficiently used for the mutant mice that a step produces heterozygosis and homozygosis, be in animal individual
Realize that the screening based on gene knockout provides further evidence in level.
(2) half Cloning of mouse can efficiently be obtained using the double PG-haESCs for knocking out of H19 of the present invention and IG-DMR, this
Appearance avoids the need for being done by the way that sperm injection is set up into lonely hero monoploid embryo to non-nucleus egg mother cell by way of this complexity
Clone is cultivating half cloned animal.
Description of the drawings
Figure 1A：Obtain PG-haESCs；Upper left：The ovum of lonely female activation comprises only a protokaryon；Upper right：Lonely female blastaea；It is left
Under：The lonely female monoploid embryo stem cell line (being named as PGH-1) set up by the enrichments of FACS repeatedly；Bottom right：By lonely female list
The embryonic stem cell form that times stomatoblastula is set up；Asterisk is represented in the ovum for activating and comprises only a protokaryon；Arrow is represented to be used for
Set up the blastaea of embryonic stem cell line.
Figure 1B：The methylation level of Snrpn and Peg1DMR in two plants of PG-haESCs；Stain and white point represent respectively methyl
Change and unmethylated CpG sites.
Fig. 1 C：PG-haESCs obtains the ideograph of half Cloning of mouse by ICPHCI；pha：After activation；PPN：Injection
The false protokaryon that PG-haESCs is formed；PB：Polar body；PPB：False polar body.
Fig. 1 D：The genotyping of DKO-PG-haESCs；These clones are that H19- is knocked out in WT-PG-haESCs
DMR and IG-DMR；H19-DMR is knocked out there are two sgRNA that different size of band is because 3.8kb H19-DMR upstream and downstream
Because CRISPR-Cas9 causes random DNA damage reparation so as to cause H19-DMR to have different DNA sequence dnas after knocking out.
Fig. 1 E：DKO-PG-haESCs obtains half Cloning of mouse by ICPHCI, and that what is represented in figure is H19△DMR-IG△DMR-
PGH-1 clones.
Fig. 1 F：The SC dams in DKO-PG-haESCs sources and its offspring.
Fig. 1 G：The genotype identification of offspring produced by the SC dams post-coitums in DKO-PG-haESCs sources；Carry IG-
The offspring of DMR knockouts or the double knockouts of H19-DMR and IG-DMR before birth or after birth can death；H19 is knocked out and difference is occurred
Size is because that mother of offspring is derived from two containing half produced by the different size of DKO-PG-haESCs of H19-DMR
Clone's dams.
Fig. 2A：The PG-haESCs for being set up is summarized.
Fig. 2 B：The flow cytometer showed (FACS) of PG-haESCs clones.
Fig. 2 C：The lonely female monoploid embryo stem cell set up from lonely female blastaea.
Fig. 3 A：Female imprinted gene (Snrpn, Peg1, Igf2 and Peg3) and male imprinted gene (H19, Gtl2)
Bigwig track。
Fig. 4 A：The methylation analysis of PG-haESCs imprinted genes, PG-haESCs's males imprinted gene (H19 and Gtl2)
Methylation state.
Fig. 4 B：The methylation analysis of PG-haESCs imprinted genes, PG-haESCs males imprinted gene (Snrpn and Peg1)
Methylation state, PGH-3 and PGH-4 its very early phase algebraically just lost its female sexual imprinting.
Fig. 4 C：The methylation analysis of PG-haESCs imprinted genes, Snrpn and Peg1 in PGH-2 and PGH-5 clones
Methylate change, PGH-2 and PGH-5 clones gradually lose its female sexual imprinting with passing on.
Fig. 5 A：The ideograph of DKO-PG-haESCs is obtained using CRISPR-Cas9 technologies.
Fig. 5 B：Set up the summary that H19-DMR or IG-DMR or both knock out clone.
Fig. 5 C：The clone that H19-DMR for being set up or IG-DMR or both is knocked out；Upper figure：The monoploid set up is thin
Born of the same parents system airflow classification figure；Figure below：The haploid cell system form set up.
Fig. 5 D：The clone that H19-DMR or IG-DMR is knocked out carries out genotype identification.
Fig. 5 E：The Snrpn methylation states of DKO-PG-haESCs clones.
Fig. 6：Carry PG-haESCs half Cloning of mouse of generation that H19-DMR or IG-DMR or both are knocked out；Wherein Fig. 6 A:
H19△DMR- PGH (left side) and IG△DMR- PGH (the right) clones obtain SC mouse by ICPHCI；H19-DMR or IG-DMR strike
The SC mouse that the PG-haESCs for removing is obtained carry out genotype identification；Fig. 6 B:H19△DMR-IG△DMR- PGH cells are obtained by ICPHCI
Obtain SC mouse；The left side：Newborn SC mouse；The right：Adult SC mouse；Fig. 6 C:The SC mouse that DKO-PG-haESCs is obtained are carried out
Genotype identification；Fig. 6 D：The methylation state of adult SC mouse Snrpn and Peg1DMRs.
Specific embodiment
Present invention firstly provides a kind of lonely female monoploid embryo stem cell, the lonely female monoploid embryo stem cell
H19DMR and IG-DMR are knocked.
The whole gene group of the lonely female monoploid embryo stem cell derives from egg mother cell, self answering with stem cell
Ability processed and versatility, can replace sperm that the development completely of embryo support is combined with egg mother cell.
The H19DMR is referred to：One section of methylation differential region (DMR in H19-Igf2 marking clusters：
differentially methylated region).The particular location of H19DMR can be according to the existing survey that methylates with sequence
The method such as sequence or homologous sequence analyses and prediction is clear and definite.Known people H19DMR is positioned at chromosome 11p15.5 areas, mouse H19DMR then positions
In No. 7 distal chromosomes, between two genes of H19 and Igf2,2kb to the 4kb positions of the upstream of h19 gene.H19DMR
It is in methylation state on paternal allele, causes CTCF albumen to be attached to this section and methylate on region, makes H19 downstreams
Enhancer need not go beyond this obstacle of CTCF, and then reach the expression of the Igf2 for strengthening upstream and reduce the expression of H19.
And on maternal allele it is in demethylation state, CTCF albumen can be attached to this section of unmethylated region, therefore H19
The enhancer in downstream can only strengthen the expression of H19, but the Igf2 of upstream cannot be regulated and controled.
The IG-DMR is referred to：One section of methylation differential region (DMR in Dlk-Dio3 marking clusters：
differentially methylated region).The particular location of IG DMR can be according to the existing survey that methylates with sequence
The method such as sequence or homologous sequence analyses and prediction is clear and definite.The IG-DMR of known mouse is located on No. 12 chromosomes, in being in marking cluster
One section of repetitive sequence of 4.15kb between Dlk1 and Gtl22 gene, people's is then located on No. 14 chromosomes (14q32.2).IG-
In paternal allele, DNA methylation in this section of region to DMR there is, the gene Gtl2 of this marking cluster and some
MircroRNA is not expressed, but gene Rtl1, Dlk1 and Dio3 are then expressed.In maternal allele, this section of region does not occur
DNA methylation (demethylation state), therefore Gtl2 and some mircroRNA are expressed, but gene Rtl1, Dlk1 and
Dio3 is not then expressed.
Further, the lonely female monoploid embryo stem cell is in addition to H19DMR and IG-DMR are knocked out, also through it
His genetic modification.
Genetic modification specifically refer to by the means of biological or chemical or physics make gene compare transformation before send out in structure
Changing.This change is primarily referred to as the change of base-pair composition, the including but not limited to replacement of one or more base-pairs, increasing
Add, lack the change for causing.
The lonely female monoploid embryo source of human stem cell, in mammal, can be people or non-human mammal.Preferably,
In rodent, such as rabbit, mouse, mouse can be mouse, rat to the lonely female monoploid embryo source of human stem cell.Preferably implementing
In example, the lonely female monoploid embryo source of human stem cell is in mouse.
Compare the lonely female monoploid embryo stem cell that H19DMR and IG-DMR is not knocked out simultaneously, the lonely female monoploid of the present invention
The birth rate that embryonic stem cell builds half cloned animal is higher.
Present invention also offers the preparation method of the lonely female monoploid embryo stem cell, including by lonely female monoploid embryo
The H19DMR and IG-DMR of stem cell is knocked out and is obtained the lonely female monoploid embryo stem cell.
The H19DMR and IG-DMR can be knocked out using existing gene editing method.In a preferred embodiment, adopt
The genetic manipulation mediated with CRISPR/Cas9 knocks out H19DMR and IG-DMR.Also gene knockout can be realized using additive method, and
The mode being not limited to cited by embodiment.
Based on the knockout of H19DMR, the complete sequence of H19DMR is removed from chromosomal DNA；Based on the knockout of IG-DMR,
The complete sequence of IG-DMR is removed from chromosomal DNA.
In one embodiment, the lonely female monoploid embryo stem cell that direct construction H19DMR and IG-DMR is knocked out simultaneously.
Further, other genetic modifications of the lonely female monoploid embryo stem cell also Jing.
Other described genetic modifications refer to the genetic modification in addition to the double knockouts of H19DMR and IG-DMR.Described other bases
Because transformation can be the transformation or the transformation for multiple interesting target genes of single target gene.Interesting target gene is simultaneously
It is not specific to, can sets and transform according to research needs.For example, it may be one, two, more than three target gene changes
Make.It is because the double lonely female monoploid embryo stem cells for knocking out of H19DMR of the present invention and IG-DMR can pass in vitro therefore theoretical
On constantly genetic modification can be carried out to it, for the transformation quantity of target gene can be operated as required, without especially limit
System.
The genetic modification includes but is not limited to the knocking in of target gene, knockout of target gene etc..Target gene strikes
Enter, the knockout of target gene can be completed using technologies such as gene targeting, homologous recombinations, including but not limited to based on ZFN (zinc
Finger nuclease), (Regularity is spaced the short palindrome to be repeated for TALEN (transcriptional activation sample effector nuclease) and CRISPR/Cas9
Technology) etc. genetic manipulation.
It is all to can be achieved on the double knockouts of H19DMR and IG-DMR and the other biological technological means of other gene mutations
For build H19DMR and IG-DMR it is double knock out and Jing other genetic modifications lonely female monoploid embryo stem cell.
Present invention also offers the purposes of the lonely female monoploid embryo stem cell, is for building the half of genetic modification gram
Grand animal.
Further, substitute sperm using the lonely female monoploid embryo stem cell to change for building gene as fertilization medium
The animal made.
Present invention also offers a kind of method for building the cloned animal of genetic modification half, by the double knockouts of H19DMR and IG-DMR
Lonely female monoploid embryo stem cell is combined half clone embryos of acquisition with egg mother cell, is cultivated the half clone embryos acquisition half and is cloned
Animal.
Generally, the egg mother cell and the lonely female monoploid embryo source of human stem cell be in species, preferably
, it is allogenic animal.
Half clone embryos specifically can be with the double lonely female monoploid embryo stem cells for knocking out of H19DMR and IG-DMR as ICPHCI
Donor, using ICPHCI methods obtain half clone embryos.
Further, the method that can adopt embryo transfer cultivates half gram of the half clone embryos acquisition in suitable parent
Grand animal.
In a preferred embodiment, the suitable parent selects false pregnancy ICR dams.
Further, other genetic modifications of the lonely female monoploid embryo stem cell Jing.
Present invention also offers a kind of genetic modification animal, is built by preceding method and obtained, or build for preceding method
Half cloned animal generative propagation offspring.
Half cloned animal of the present invention can be non-human mammal.Preferably, half cloned animal is dynamic for grinding tooth
Thing, such as rabbit, mouse.In a preferred embodiment, half cloned animal is mouse.
Present invention also offers the method for building the cloned animal library of genetic modification half, comprises the following steps：
1) the lonely female monoploid embryo of the infestation with virus particles present invention prepared with sgRNA slow virus Library plasmid is dry thin
Born of the same parents, obtain the lonely female monoploid embryo stem cell library for carrying sgRNA libraries；
2) using the lonely female monoploid embryo stem cell in the lonely female monoploid embryo stem cell bank for carrying sgRNA libraries as
The donor of ICPHCI, using the carrier and/or the mRNA of Cas9 of expression Cas9, using ICPHCI methods half clone embryos text is obtained
Storehouse；
3) embryo cultivated in the half clone embryos library obtains half cloned animal library.
Slow virus carrier of the sgRNA slow virus Library plasmid comprising several expression difference sgRNA.SgRNA slow virus
Library can be built using prior art, can also purchase existing sgRNA slow virus Library plasmid.Specifically, will can be directed to
SgRNA designed by different genes is cloned into slow virus carrier acquisition.The sgRNA can be according to interested to oneself gene
It is designed.
In a preferred embodiment, the sgRNA slow virus library for commercially available mouse full-length genome for adopting.
Specifically, step 2) may be selected from it is following arbitrary：
Method A：
To the plasmid of the further transfection expression Cas9 in lonely female monoploid embryo stem cell library for carrying sgRNA libraries, will
The lonely female monoploid embryo stem cell of acquisition obtains half clone embryos as the donor of ICPHCI using ICPHCI methods.
Method B：
Using carry sgRNA libraries lonely female monoploid embryo stem cell library lonely female monoploid embryo stem cell as
The donor of ICPHCI, using ICPHCI methods mature egg is injected, then obtains half clone's embryo to the ovum injection Cas9mRNA of reconstruct
Tire.
Method C：
To the plasmid of the further transfection expression Cas9 in lonely female monoploid embryo stem cell library for carrying sgRNA libraries, will
The lonely female monoploid embryo stem cell of acquisition injects mature egg as the donor of ICPHCI using ICPHCI methods, then to reconstruct
Ovum injection Cas9mRNA obtain half clone embryos, then Jing embryo transfers obtain half cloned animal.
The plasmid of Cas9 is expressed described in said method A and C can enter expression plasmid structure by the gene cloning of expression Cas9.
For example, the plasmid of the expression Cas9 is pX330-mCherry plasmids.The plasmid that can be used for construction expression Cas9 is not limited to
PX330 plasmids.The expression plasmid only needs to the expression for being adapted to carry out foreign gene in mammalian cell.
Present invention also offers another kind of method for building the cloned animal library of genetic modification half, comprises the following steps：
1) lentiviral particle prepared with the lentiviral particle and sgRNA slow virus library of expressing Cas9 infects the present invention's
Lonely female monoploid embryo stem cell, obtains the lonely female monoploid embryo stem cell for carrying sgRNA libraries and Cas9 continuous expressions
Library；
2) with the lonely female list in the lonely female monoploid embryo stem cell library for carrying sgRNA libraries and Cas9 continuous expressions
Times body embryonic stem cell obtains half clone embryos library as the donor of ICPHCI using ICPHCI methods；
3) embryo cultivated in the half clone embryos library obtains half cloned animal library.
The virion of the expression Cas9 can adopt prior art, and the encoding gene of expression Cas9 is cloned into slow virus
Then carrier carries out slow virus packaging and obtains, and the slow virus carrier for expressing Cas9 is commercially available.
Half cloned animal of the half cloned animal library comprising several gene mutations.The animal can be heterozygote or
The mutant animals of diallele mutation.
Further, the method that can adopt embryo transfer cultivates half gram of the half clone embryos acquisition in suitable parent
Grand animal.
For example, the suitable parent can select false pregnancy ICR dams.
Present invention also offers a kind of cloned animal library of genetic modification half, is to build to obtain using preceding method.
The cloned animal library of genetic modification half of the present invention can carry out the genetic screening of gene in subordinate's individual level.
Half cloned animal library of the present invention can be non-human mammal library.Preferably, half cloned animal
Library is rodent library, such as rabbit library, mouse library.In a preferred embodiment, the half cloned animal library is mouse
Library.
Embodiments of the present invention are illustrated below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands easily other advantages and effect of the present invention.The present invention can also pass through concrete realities different in addition
The mode of applying is carried out or applies, the every details in this specification can also based on different viewpoints with application, without departing from
Various modifications and changes are carried out under the spirit of the present invention.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can select between point and two end points.Unless otherwise defined, the present invention used in all technologies and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the concrete grammar used in embodiment, equipment,
Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, can also use and this
Any method of the similar or equivalent prior art of method, equipment described in inventive embodiments, material, equipment and material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method using this technology lead
The conventional molecular biology in domain, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.The perfect explanation in existing document of these technologies.
Write a Chinese character in simplified form：
PG-haESCs：Lonely female monoploid embryo stem cell
DKO-PG-haESCs：The double lonely female monoploid embryo stem cells for knocking out of H19DMR and IG-DMR
1. experiment material and method
1.1 materials and reagent
Cell culture medium (DMEM), hyclone (FBS), serum substitute (KSR), pancreatin, Opti-MEM, DPBS,
Lipofectamine 2000 is purchased from Life Technologies companies；Restriction enzyme, T4 ligases are purchased from NEB companies；
Taq enzyme and dNTPs are purchased from TaKaRa companies；CDNA reverse transcription reagent box, fluorescent quantitation reagent SYBR-Green are purchased from TOYOBO
Company；Oligonucleotide is synthesized by Shanghai generay companies.
HEPES-CZB nutrient solutions：
H-CZB Stock 98.5ml, Hepes.2Na (sigma, CAT#H0763) or ICN 520mg or Hepes
(sigma,CAT#H4034)476mg、NHCO342mg、CaCl2·2H2O 100×stock 1ml、Pyruvate 3.0mg、
200 × stock of Glutamin 0.5ml adjust pH to 7.4, mix and filter
H-CZB stock：
CZB stock 500ml,PVA(sigma,P8136)50mg、CZB stock:H2O 985ml、NaCl(sigma,
CAT#S5886) 4760mg、KCL(sigma,CAT#P5405) 360mg、MgSO4·7H2O(sigma,CAT#M1880)
290mg、EDTA·2Na(sigma,CAT#E6635) 40mg、Na-Lactate(sigma,CAT#L7900) 5.3ml、D-
Glucose(sigma,CAT#G6152) 1000mg、KH2PO4(sigma,CAT#P5655) 160mg
Activation liquid：10mM Sr2+,5ng/ml Trichostatin A(TSA)
KSOM nutrient solutions (KSOM+AA with glucose)：millipore,CAT#MR-106-D
ESC culture mediums：
DMEM (millipore, CAT#SLM-220-M) 75%, 20% serum substitute KSR (Gibco, CAT#10828-
028), 1,500U/ml LIF (Millopre, CAT#ESG1107), 3M CHIR99021 (Stemgent, CAT#04-0004) and
1M PD0325901(Stemgent,CAT#04-0006)
Acid Tyrode solution：sigma,CAT#T1788
CZB nutrient solutions：
CZB stock 99ML、CaCl2·2H2O 100×stock 1ml、Pyruvate 3.0mg、Glutamin 200
×stock 0.5ml、BSA 500mg
pX330-mCherry：
Px330 (addgene) plasmids carry out digestion with NotI, then will expand from pmCherry-C1 (Clontech)
The px330 plasmids that good CMV-mcherry-pA fragments are inserted into digestion are obtained.
Amplification the primer：
mCherry-F：ATTTGCGGCCGCATAGTAATCAATTACGGG(SEQ ID NO.5)
mCherry-R：ATTTGCGGCCGCATGCAGTGAAAAAAATGC(SEQ ID NO.6)
The sgRNA slow virus library of mouse：Addgene companies
The virus particle of Cas9：Addgene companies provide
The mRNA of Cas9:
With reference to Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F.,
and Jaenisch,R.(2013).One-step generation of mice carrying mutations in
Multiple genes by CRISPR/Cas-mediated genome engineering.Cell 153,910-918.'s
Record and obtain.
1.2 experimental animals
All of animal using flow process be all according to Shanghai life science institute of the Chinese Academy of Sciences it is biochemical with cell animal
Handbook in turn.
B6D2F1 (C57BL/6X DBA2) female mice：Dams and the public mouse mating of DBA2 strains with C57BL/6 strains
The female descendant for obtaining afterwards.
False pregnancy ICR dams：ICR adult rats are purchased from Si Laike, the adult dams of ICR are mated with the ICR public affairs mouse of ligation and is obtained final product
To ICR false pregnancy dams.
The foundation of 1.3 lonely female monoploid embryo stem cell lines
The construction method of lonely female monoploid embryo stem cell line, comprises the steps：
Super row is carried out to B6D2F1 (C57BL/6X DBA2) female mice, the ripe MII ovums collected, in CZB cultures
Cultivate 1 hour in liquid, be then transferred to Sr containing 1mM2+Activation liquid enter line activating.After activation, all of embryo go to containing
The KSOM nutrient solutions of amino acid are cultivated in 37 DEG C under the conditions of 5%CO2.The reconstruct of mulberries or blastocyst stage is reached after 3.5 days
Embryo implantation is in ESC culture mediums.
Zona pellucida carries out digestion removal by Acid Tyrode solution.Each embryo transfer is to being covered with mouse into fiber
In 96 orifice bores of cytotrophoblast, and with containing 20% serum substitute (KSR), 1,500U/ml LIF, 3uM
The ESC culture mediums of CHIR99021 and 1uM PD0325901 are cultivated.After culture 4-5 days, cell clone is passed by pancreatin digestion
To being covered with fresh trophoblastic 96 orifice plate.The further expansion of cell by passing on to 48 orifice plates so that in 6 orifice plates, then
Daily cell is maintained only in 6 holes.In order to sub-elect haploid cell, after the digestion of embryonic stem cell pancreatin, PBS is used
(GIBCO) wash one time, then in the culture medium water-bath 30min containing 15 μ g/ml Hoechst 33342.Subsequently, by streaming
Sorter BD FACS AriaII separation and collections go out the cell of monoploid 1N peak shapes to carry out follow-up and cultivates.
The genetic manipulation of 1.4CRISPR-Cas9 mediations
CRISPR-Cas9 plasmid constructions：The positive oligonucleotide chain and reverse oligonucleotide chain of synthetic sgRNA are entered
Row annealing obtain double chain oligonucleotide chain (sgRNA sequences refer both to the positive oligonucleotides chain-ordering of sgRNA in the present invention) and then
It is attached with BbsI (New England Biolabs) the good pX330-mCherry of digestion.The corresponding plasmid for building according to
Specification is transfected into lonely female monoploid embryo stem cell with Lipofectamine 2000 (Life Technologies).Turn
Contaminate 48 hours afterwards, it is by streaming (FACS AriaII, BD Biosciences) that the monoploid with red fluorescent protein is thin
After born of the same parents sort out, with relatively low density paving.After growth 4-5 days, picking monoclonal carries out follow-up building and is.Finally by
The method that PCR genes of interest is sequenced obtains the clone of corresponding gene mutation.
Such as relating to knocking in for gene, then need the DNA donors for building double-strand.
It is prepared by double-stranded DNA donor：
The sequence to coding EGFP, mCherry or ECFP is expanded, is then accordingly connected to be obtained on pMD19-T carriers
pMD19-T-EGFP/mCherry/ECFP.Subsequently, the left and right homology arm of genes of interest is correspondingly inserted into pMD19-T-
On the carrier of EGFP/mCherry/ECFP.
The preparation of 1.5 viruses and double virus infection for striking lonely female monoploid embryo stem cell (DKO-PG-haESCs)
The sgRNA viral libraries of mouse and the virus particle of Cas9 have been reported (Cong, L., Ran, F.A., Cox, D.,
Lin,S.,Barretto,R.,Habib,N.,Hsu,P.D.,Wu,X.,Jiang,W.,Marraffini,L.A.,et al.
(2013).Multiplexgenome engineering using CRISPR/Cas systems.Science 339,819-
823；Koike-Yusa,H.,Li,Y.,Tan,E.P.,Velasco-Herrera Mdel,C.,and Yusa,K.(2014)
.Genome-wide recessive genetic screening in mammalian cells with a lentiviral
CRISPR-guide RNA library.Nature biotechnology 32,267-273,2014), present invention mouse used
The virus particle of sgRNA viral libraries and Cas9 is provided by Addgene companies.In order to prepare virus, HEK293T is passed in advance
In 10cm culture dishes, use2000Reagent (Invitrogen, Life Technologies) transfects 3ug
Virus particle (sgRNA slow virus library or Cas9 slow virus) and 9 μ g of ViraPower Lentiviral
Packaging Mix slow virus package combinations (Invitrogen) is transfected into HEK293T cells.Transfection is collected after 72 hours
Supernatant, is concentrated with slow virus concentrate Lenti-Concentin virus precipitation solution (SBI),
Subsequently can just be saved in -80 DEG C.
Cas9 slow-virus infections：Contain 10 with 8ug/ml polybrene (Sigma) and packaged Cas9 slow-virus infections8
The cell suspension of individual DKO-AG-haESCs 48 hours, then carries out dosing screening with the blasticidin (Sigma) of 10ug/ml
3 days, remaining drug-resistant clones were the clone of Cas9 slow virus integration.
Infect in sgRNA slow virus library：It is literary with 8ug/ml polybrene (Sigma) and packaged CRISPR-sgRNA
Storehouse slow-virus infection contains 108The cell suspension of individual DKO-AG-haESCs, changes afterwards 1ug/ml puromycin into 48 hours
(Invitrogen) culture medium) carries out drug screening, and the positive obtained after screening 2 days as carries sgRNA libraries slow virus
The clone of integration.
Cas9 slow virus and sgRNA libraries slow virus double infection：
The clone of Cas9 slow virus integration is first prepared, sgRNA slow virus library is then further infected, with 1ug/ml
Puromycin (Invitrogen)) culture medium carry out drug screening, the positive obtained after screening 2 days is Cas9 slow virus
The clone integrated with sgRNA libraries slow virus.
If DKO-PG-haESCs clones have only infected sgRNA slow virus library, then the cell just table without Cas9
Reach, this when, we needed to carry out the transfection of pX330-mCherry plasmids on cellular level again, to realize the volume of genome
Volume.1.6 sulphite methylate sequencing
1) DNA of mouse and 15ul 2%LMP agarose (low melting-point agarose) are bundled into beads, add 460ul
DNA digestion buffer, add 40ul proteinase K to each sample, 50 DEG C of night incubation sample digestions；
2) after TE washes 3 times, reacted with bisulfite solution and beads, 50 DEG C of incubation 4h-8h；
3) nest-type PRC is carried out by template of beads, PCR primer is attached after being reclaimed with PMD19-T carriers, is turned
Change, coated plate；
4) 10 bacterium colonies of each sample picking are sequenced.
According to EZ DNA methylation Gold kit (ZYMO Research) kits, it is only necessary to get out phase
The DNA that should be measured, then follow-up all processes operated according to the step of kit.The kit for obtaining is reclaimed and produced
Thing, as template performing PCR is entered, and product is reclaimed, is attached with PMD19-T carriers, is converted, coated plate.Choose flat board 10
Bacterium solution is sequenced.
1.7 quantitative fluorescent PCRs
Total amount RNA of cell or organ is extracted with Trizol reagent (Invitrogen), then with the total serum IgE of 1ug
With First Strand cDNA Synthesis kit (TOYOBO) reverse transcription is carried out into cDNA.Real-time fluorescence quantitative PCR reacts
With SYBR Green Realtime PCR Master Mix (TOYOBO), and complete on Bio-Rad CFX96 instruments, per group
Sample sets up 3 repetitions.Expression of all of gene expression dose with house-keeping gene Gapdh is as internal reference.
1.8Cobra Assay
1) sample DNA of 100ng is taken, with the quick restriction enzymes of TaqI (fementas companies) enzyme (T/CGA) is carried out
Cut reaction 15min；
2) row agarose gel electrophoresis are entered, you can.
1.9ICPHCI and embryo transfer build half Cloning of mouse
Lonely female monoploid embryo stem cell injection in endochylema (intracytoplasmic PG-haESCs injection,
ICPHCI)：
To obtain half clone (SC) embryo, by medium treatment 8hs of the PG-haESCs containing 0.05 μ g/ml colchicines
By cells Synchronous to M phases, then to carry out ooecium slurry injection.The PG-haESCs for having digested HEPES-CZB nutrient solutions are washed 3 times,
Then the HEPES-CZB nutrient solutions with 3% (w/v) polyvinylpyrrolidone (polyvinylpyrrolidone, PVP) are resuspended.
With in nucleus injection MII ovums of the Piezo micromanipulations instrument by the PG-haESCs of each M phase.The embryo for having reconstructed first exists
1F is cultivated in CZB nutrient solutions, then with the activation 5-6h of the activation liquid without CB.After activation, all of reconstruct embryo is in KSOM
Nutrient solution in 37 DEG C, 5%CO2Under the conditions of cultivated.ICPHCI embryos cultivate 24h in KSOM nutrient solutions, and just to reach 2 thin
Born of the same parents phase embryo.
Every 15-20 2 cell stages obtained by ICPHCI are moved into 0.5dpc (post-coitum 0.5 day) false pregnancy ICR mouse
Each uterus in.Dams carry out caesarean birth or natural production after pregnant 19.5 days.Caesarean birth is for WT PG-haESCs
Or the reconstruct embryo that the PG-haESCs that knocks out of single DMR is obtained, overdue fetus quickly separates from the uterus of dams
Come.For the embryo obtained by DKO-PG-haECs, the pregnancy dams of 19.5 days can carry out natural production.Dispose birth little
After liquid with mouse, mouse is placed in the incubator of oxygen, and the mouse for surviving then subsequently is brought up by replace-conceive dams.
1.10RNA-seq and gene expression analysis
It is the TreSeq RNA Sample Prep v2Guide according to illumina officials that the RNA-seq of total serum IgE builds storehouse
Carry out.After the completion of learn the Illumina HiSeq that calculation biology research institute calculates Zu Xue centers in the Chinese Academy of Sciences-Ma Pu
Deep sequencing is carried out on 2000 instruments.
The RPKM that gene expression amount is used, specific algorithm be with (Yang, L., Duff, M.O., Graveley, B.R.,
Carmichael,G.G.,and Chen,L.L.(2011).Genomewide characterization of non-
Polyadenylated RNAs.Genome biology 12, R16) it is consistent.
Use in (Yang et al., Genome boil 2011) during the p-value for calculating difference expression gene
The method that waldscore is calculated, selects abs (waldscore)>1.96 (i.e. p-value<0.05), differential expression is then screened
Gene.
1.11RRBS (the apparent bisulfite sequencing of simplification)
RRBS build storehouse be R be according to standard protocol of Illumina officials prepare.Then Illumina is used
HiSeq 2000 be sequenced (Gu, H., Smith, Z.D., Bock, C., Boyle, P., Gnirke, A., and Meissner,
A.(2011). Preparation of reduced representation bisulfite sequencing
libraries for genome-scale DNA methylation profiling.Nature protocols 6,468-
481.).All of sequencing readings compares with the genome of mouse.
1.12 genotype identification methods
The genomic DNA for having extracted enters performing PCR with corresponding primer, and then PCR primer further carries out Ago-Gel
Electrophoretic analysis.
Embodiment 1 builds half Cloning of mouse based on lonely female monoploid embryo stem cell line
A. lonely female monoploid embryo stem cell line is built：
Ovum is originated：B6D2F1 (C57BL/6X DBA2) female mice；Using aforementioned 1.3 method, to B6D2F1
(C57BL/6X DBA2) female mice carries out super row, the ripe MII ovums collected, and then picks out only one of which female pronucleus
Embryo carry out follow-up culture to blastocyst stage.The female blastaea of a total of 76 orphans further containing 2i (PD0325901 and
CHIR-99021 culture in embryonic stem cell culture system).38 plants of embryonic stem cell lines are finally we obtain, wherein
There is 6 plants (being named as PGH-1 to PGH-6) identification to contain monoploid, and be enriched with by the method for airflow classification repeatedly (FACS)
To a high proportion of haploid cell system (as shown in Figure 1A and Fig. 2A-C).
B. half Cloning of mouse is built：
Then, in order to study these lonely female monoploid embryo stem cell lines by be expelled to maturation egg mother cell in can
The development that expires of embryo support, we have carried out the lonely female monoploid embryo stem cell injection of ooecium matter according to above-mentioned 1.9 method
(ICPHCI).Simply, the embryonic stem cell containing a chromosome being enriched with by FACS is expanded with ES cultivating systems
Pass on, the colchicine that 0.05mg/ml is then used before injection processes 8 hours by cells Synchronous to the M phases.Then will be same then,
The haploid cell for walking the M phases is expelled in MII egg mother cells to produce half Cloning of mouse.
As a result show：PG-haESCs can not support the normal development of embryo, it is impossible to obtain overdue half Cloning of mouse
(as shown in table S1), that is to say, that the birth efficiency of normal half Cloning of mouse is zero.Analysis reason：It is primarily due to ICPHCI weights
The embryo of structure is the lonely female dliploid embryo of development of cannot expiring in vivo from maternal inhereditary materials only comprising 2 parts.
In order to further assess the epigenetic situation of the lonely female monoploid embryo stem cell line of step A structure, Wo Mentong
Cross bisulfite sequencing analysis detect 2 male H19 genomic imprintings and Gtl2 and 2 female imprinted gene Snrpn with
The methylation state (Fig. 4 A) of Peg1, to prove the lonely female source of haploid cell.However, it has been found that should protect
The DMRs for holding complete methylation state Snrpn and Peg1 but occurs in that the serious loss that methylates (Figure 1B and Fig. 4 B).Enjoyably
It is in the clone of the PG-haESCs of 6 plants of detections, have 4 plants just to lost the marking in phase algebraically very early, and in addition
2 plants although hyper-methylation state is still remain in early stage algebraically, as being passed on for cell gradually lose it and methylate
State (Fig. 4 C).In sum, in the foundation and Process of in vitro of PG-haESCs, source of parents marking site can quickly lose
Its imprinting status.
Embodiment 2 is based respectively on the lonely female monoploid embryo stem cell line of the mono- knockouts of H19-DMR, the lonely female list of the mono- knockouts of IG-DMR
Times body embryonic stem cell line, H19-DMR and IG-DMR be double to be knocked out lonely female monoploid embryo stem cell line and builds half Cloning of mouse
The mono- lonely female monoploid embryos of the mono- knockout of lonely female monoploid embryo stem cell line, IG-DMR that knock out of A.H19-DMR do thin
Born of the same parents system, the double structures for knocking out lonely female monoploid embryo stem cell line of H19-DMR and IG-DMR：
Initial cell：The WT-PG-haESCs clones that embodiment 1 builds.
We respectively separately design sgRNA in the upstream and downstream of 4.15kb IG-DMR and 3.8kb H19-DMR, respectively：
IG-DMR-sgRNA1 sequences：cgtacagagc tccatggcac(SEQ ID NO.1)；
IG-DMR-sgRNA2 sequences：ctgcttagag gtactacgct(SEQ ID NO.2)；
H19-3.8K-sgRNA-1 sequences：catgaactca gaagagactg(SEQ ID NO.3)；
H19-3.8K-sgRNA-2 sequences：aggtgagaac cactgctgag(SEQ ID NO.4).
The oligo of sgRNA is annealed, then by the sgRNA of H19 and IG-DMR respectively with the px330- of BbsI digestions
MCherry plasmids are attached conversion.Correct bacterium solution will be sequenced carries out plasmid extraction, in case follow-up transfection.
Above-mentioned acquisition will be proceeded in WT-PG-haESCs clones and carries the plasmid transfection of sgRNA and Cas9 to 4 plants of PG-
In haESCs systems (Fig. 5 A).The PG-haESCs of expression mCherry represents Successful transfection CRISPR-Cas9 systems, these cells
Spread lower for follow-up Establishment of Cell Line after collection.
91 plants of haploid cell systems are always obtained, wherein there are the 24 plants of haploid cell system knocked out for IG-DMR (names
IG△DMR- PGH), 27 plants of haploid cell system (H19 knocked out for H19-DMR△DMR- PGH) (Fig. 5 B, Fig. 5 C and Fig. 5 D).44 plants
Clone carries knockout (the name H19 of 2 DMRs△DMR-IG△DMR- PGH or DKO-PG-haESCs) (Fig. 1 D).
Full-length genome methylation level analysis is carried out using aforementioned 1.11 method, methylation analysis show：H19-DMR and
The knockout of IG-DMR does not change the methylation state (Fig. 5 E) of haploid cell Snrpn DMR, illustrates PG-haESCs and knockout
The DKO-PG-haESCs of H19-DMR and IG-DMR has a similar express spectra.
B. half Cloning of mouse is built：
Using aforementioned 1.9 method, carried out as donor with the PG-haESCs for carrying 1 DMR or 2 DMR knockout
ICPHCI is tested, and builds half Cloning of mouse.As a result, the PG-haESCs of the mono- knockouts of H19-DMR can be obtained with 2.6% efficiency
Develop overdue SC mouse, and carry the SC mouse (1.4%) of the PG-haESCs of list IG-DMR knockouts then producing ratio normal size
Want more retarded growth type SC mouse (4.1%) (table S1, Fig. 6 A and Fig. 6 B).It is infusive to be：DKO-PG-haESCs leads to
Crossing ICPHCI can have the development of 15.5% half clone embryos to expire, and all of SC mouse are normal (Fig. 1 E and table S1).
It is interesting that building the DKO-PG-haESCs for obtaining using the PG-haESCs for passing on more late period, it obtains the ability of SC mouse
Stronger, this gradually loses consistent (table S1 and Fig. 4 C) with female sexual imprinting is methylated in our observed cell processes.
158 normal SC mouse are generated altogether.These SC mouse can grow up and grow up and breed offspring (Fig. 1 F and Fig. 6 C-
6E).Additionally, the mono- knockouts of H19-DMR or wild type are carried in the offspring of SC mouse can survive, and carrying IG-
The mono- knockouts of DMR or the double offsprings for knocking out of H19-DMR and IG-DMR then can die, and this is the matrilinear inheritance knocked out due to IG-DMR
Can cause mouse before birth or post-natal lethality.In a word, these data are further illustrated：The PG-haESCs of late period algebraically passes through
Two male imprint areas H19-DMR obtain can PG-haESCs and efficiently produce half Cloning of mouse's with the knockout of IG-DMR
Ability.
The potentiality of development of lonely female monoploid embryo stem cell (PG-haESCs) is by replacing ovum in embryonated egg with it
The reconstructed embryo that genome is obtained is verified.However, its birth efficiency is extremely low (290 pieces of reconstructed embryos only obtain 2 mouse), and
And only 1 mouse grow up it is adult.A potential reason is that cell is gradually lost with passing on as observed by us
Its female sexual imprinting (Figure 1B and Fig. 3 A, Fig. 4 A-C), causes have different epigenetic states compared to ovum.Therefore, PG-
The core of haESCs is not appropriate to the genome for replacing ovum.Our research finds that in fact PG-haESCs is with passing on appearance
Its female sexual imprinting easy to lose, height is obtained in that by two male imprint areas H19-DMR with knockout PG-haESCs of IG-DMR
Effect produces the ability of half Cloning of mouse.Our research promotes the Technology application of monoploid embryo stem cell, because setting up ovum
The haploid cell in mother cell source can avoid adopting nuclear transfer technology, and using sperm acquisition monoploid, this process is also suitable
It is loaded down with trivial details.The more interesting application of this technology can be to obtain gene by the ICPHCI technologies of PG-haESCs in other species
The animal of operation, these species are mainly difficult the monoploid embryo stem cell for setting up sperm source, such as monkey.
Table 1：The internal developmental state of ICPHCI embryos collects
a:H19△DMR-PGH Cells versus WT PG-haESCs p<0.05；
b:IG△DMR-PGH Cells versus WT PG-haESCs p<0.05；
c:DKO-PG-haESCs versus WT PG-haESCs p<0.001。
Table S1：The internal developmental state of different PG-haESCs clones ICPHCI embryos collects
Embodiment above is, in order to illustrate embodiment disclosed by the invention, can not to be interpreted as the limit to the present invention
System.Additionally, method, the change of composition in various modifications listed herein and invention, without departing from the scope of the present invention
With for those skilled in the art be obvious on the premise of spirit.Although having combined the various concrete of the present invention
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various modifications obvious for those skilled in the art as above all should include obtaining invention
Within the scope of the invention.
Claims (17)
1. a kind of lonely female monoploid embryo stem cell, the H19DMR and IG-DMR of the lonely female monoploid embryo stem cell are struck
Remove.
2. as claimed in claim 1 lonely female monoploid embryo stem cell, it is characterised in that the lonely female monoploid embryo stem cell
From mammal, more preferably from rodent, mouse is preferably derived from, most preferably from mouse.
3. a kind of preparation method of lonely female monoploid embryo stem cell, including by the H19DMR of lonely female monoploid embryo stem cell and
IG-DMR is knocked out and is obtained the lonely female monoploid embryo stem cell.
4. the preparation method of lonely female monoploid embryo stem cell as claimed in claim 3, it is characterised in that adopt CRISPR/
The genetic manipulation of Cas9 mediations knocks out H19DMR and IG-DMR.
5. the preparation method of lonely female monoploid embryo stem cell as claimed in claim 3, it is characterised in that also include to described
Lonely female monoploid embryo stem cell has carried out the transformation of single or multiple interesting target genes.
6. the purposes of lonely female monoploid embryo stem cell as claimed in claim 1 or 2, is half clone for building genetic modification
Animal.
7. it is a kind of build the cloned animal of genetic modification half method, including：The lonely female list that H19DMR and IG-DMR are knocked times
Body embryonic stem cell is combined half clone embryos of acquisition with egg cell, is cultivated half clone embryos and is obtained half cloned animal.
8. the method for building the cloned animal of genetic modification half as claimed in claim 7, it is characterised in that with H19DMR and IG-DMR
The lonely female monoploid embryo stem cell being knocked is the donor of ICPHCI, and using ICPHCI methods half clone embryos are obtained.
9. the method for building the cloned animal of genetic modification half as claimed in claim 7, it is characterised in that the H19DMR and IG-
The single or multiple interesting target gene of the lonely female monoploid embryo stem cell that DMR is knocked is engineered.
10. genetic modification animal as claimed in claim 7, it is characterised in that half cloned animal is non-human mammal, compared with
Preferably rodent, more preferably mouse, most preferably mouse.
A kind of 11. genetic modification animals, are that half clone for obtaining is built by claim 7-10 any claim methods described
Animal, or the generative propagation offspring of half cloned animal built for claim 7-10 any claim methods described.
A kind of 12. methods for building the cloned animal library of genetic modification half, selected from following arbitrary：
Method one, comprises the following steps：
1) done with the lonely female monoploid embryo described in the infestation with virus particles claim 1 of sgRNA slow virus Library plasmid preparation
Cell, obtains the lonely female monoploid embryo stem cell library for carrying sgRNA libraries；
2) using the lonely female monoploid embryo stem cell in the lonely female monoploid embryo stem cell bank for carrying sgRNA libraries as
The donor of ICPHCI, using the carrier and/or the mRNA of Cas9 of expression Cas9, using ICPHCI methods half clone embryos text is obtained
Storehouse；
3) embryo cultivated in the half clone embryos library obtains half cloned animal library；
Method two, comprises the following steps：
1) with express Cas9 lentiviral particle and sgRNA slow virus library prepare infestation with virus particles claim 1 described in
Lonely female monoploid embryo stem cell, obtain carry sgRNA libraries and Cas9 continuous expressions lonely female monoploid embryo it is dry thin
Born of the same parents library；
2) with the lonely female monoploid in the lonely female monoploid embryo stem cell library for carrying sgRNA libraries and Cas9 continuous expressions
Embryonic stem cell obtains half clone embryos library as the donor of ICPHCI using ICPHCI methods；
3) embryo cultivated in the half clone embryos library obtains half cloned animal library.
13. methods for building the cloned animal library of genetic modification half as claimed in claim 12, it is characterised in that methods described one
In step 2) may be selected from it is following arbitrary：
Method A：
To the plasmid of the further transfection expression Cas9 in lonely female monoploid embryo stem cell library for carrying sgRNA libraries, will obtain
Lonely female monoploid embryo stem cell as ICPHCI donor, using ICPHCI methods obtain half clone embryos.
Method B：
Using carry sgRNA libraries lonely female monoploid embryo stem cell library lonely female monoploid embryo stem cell as ICPHCI
Donor, using ICPHCI methods inject mature egg, then to reconstruct ovum injection Cas9mRNA obtain half clone embryos.
Method C：
To the plasmid of the further transfection expression Cas9 in lonely female monoploid embryo stem cell library for carrying sgRNA libraries, will obtain
Lonely female monoploid embryo stem cell as ICPHCI donor, using ICPHCI methods inject mature egg, then to reconstruct ovum
Son injection Cas9mRNA obtains half clone embryos.
14. methods for building the cloned animal library of genetic modification half as claimed in claim 13, it is characterised in that the expression
The plasmid of Cas9 is pX330-mCherry plasmids.
15. methods for building the cloned animal library of genetic modification half as claimed in claim 12, it is characterised in that described half gram
Animal in grand animal library is the animal that heterozygote and/or diallele are mutated.
16. methods for building the cloned animal library of genetic modification half as claimed in claim 12, it is characterised in that described half gram
Grand animal library is non-human mammal library, is more preferably rodent library, more preferably mouse library, most preferably mouse
Library.
A kind of cloned animal library of 17. genetic modification half, the structure gene by described in claim 12-16 any claim changes
The method for making half cloned animal library builds acquisition.
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| CN107164527A (en) * | 2017-06-29 | 2017-09-15 | 杭州观梓健康科技有限公司 | It is a kind of to screen the method for participating in multipotential stem cell vitro directed differentiation regulatory factor |
| CN107164527B (en) * | 2017-06-29 | 2018-04-20 | 杭州观梓健康科技有限公司 | It is a kind of to screen the method for participating in multipotential stem cell vitro directed differentiation regulatory factor |
| CN110095610A (en) * | 2018-01-31 | 2019-08-06 | 中国科学院上海生命科学研究院 | A kind of high throughput protein analysis method and its applicable library |
| WO2019149040A1 (en) * | 2018-01-31 | 2019-08-08 | 中国科学院上海生命科学研究院 | Method for high-throughput analyzing proteins and applicable library thereof |
| JP2021524016A (en) * | 2018-01-31 | 2021-09-09 | 中国科学院分子細胞科学卓越創新中心Ｃｅｎｔｅｒ Ｆｏｒ Ｅｘｃｅｌｌｅｎｃｅ Ｉｎ Ｍｏｌｅｃｕｌａｒ Ｃｅｌｌ Ｓｃｉｅｎｃｅ， Ｃｈｉｎｅｓｅ Ａｃａｄｅｍｙ Ｏｆ Ｓｃｉｅｎｃｅｓ | High-throughput analysis method for proteins and its application library |
| CN110157661A (en) * | 2018-02-12 | 2019-08-23 | 中国科学院上海生命科学研究院 | A kind of method for separating of monoploid embryo stem cell |
| CN108504690A (en) * | 2018-03-21 | 2018-09-07 | 陈子江 | A kind of edit methods being directed to male gene |
| CN108504691A (en) * | 2018-03-21 | 2018-09-07 | 陈子江 | A method of being directed to female gene editor |
| CN108504691B (en) * | 2018-03-21 | 2021-09-03 | 陈子江 | Method for editing female gene |
| CN108504690B (en) * | 2018-03-21 | 2021-09-03 | 陈子江 | Editing method for male gene |
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| CN106676075B (en) | 2020-06-26 |
| WO2017080452A1 (en) | 2017-05-18 |
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