CN102816796A

CN102816796A - Method of inducing pluripotent stem cells throuhg c-Jun N terminal deletion and applications

Info

Publication number: CN102816796A
Application number: CN2012103245764A
Authority: CN
Inventors: 裴端卿; 韩庆凯; 刘晶; 陈捷凯; 韦备; 彭梅秀
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2012-09-04
Filing date: 2012-09-04
Publication date: 2012-12-12
Anticipated expiration: 2032-09-04
Also published as: CN102816796B

Abstract

The invention relates to the stem cell research in the biological technology field, in particular to a method of inducing pluripotent stem cells throuhg the c-Jun N terminal deletion and applications. The technical scheme aims to provide an application of combined utilization of the c-Jun N terminal deletion and pluripotent stem cell inducing factors in a luripotent stem cell inducing process, and the c-Jun N terminal deletion is a series of clipped amino acid sequences or corresponding nucleotide sequences of amino acids from 170 bits to 334 bits of the c-Jun N protein of AP-1 family protein. According to the method and applications, during the process of pluripotent stem cell inducing, N terminal cut truncated type of the c-Jun N protein of the AP-1 family protein is used, an efficient reprogramming induction system is provided, micro molecules or genes of core reprogramming factors are replaced, and a promoting role of revealing core reprogramming factors in the reprogramming is played.

Description

The methods and applications of the N end disappearance inducing pluripotent stem cells of c-Jun

Technical field

The present invention relates to the stem-cell research of biological technical field, be specifically related to the method and the application thereof of AP-1 family protein c-Jun N end disappearance inducing pluripotent stem cells.

Background technology

AP-1 family is the transcription activating protein that one type of cellular response outer signals comprises growth factor, cytokine and born of the same parents' external pressure.Form homology or heterodimer by different subunits on the structure.These subunits comprise Jun, Fos and ATF.A leucine zipper structure is all arranged on each subunit, form and DNA bonded zinc finger print piece in twos.

ES cell and embryonic stem cell be in one in the inner cell mass of blastaea isolated cell with characteristic of external infinite multiplication, self and multidirectional differentiation.No matter in external still internal milieu, the ES cell can both be induced to differentiate into the nearly all cell type of body.In view of ES cells in vitro infinite multiplication and many differentiation potentials, it has broad application prospects in the regenerative medicine field.

2006, Japanese scientist Yamanaka and colleague thereof with four transcription factors (Oct4, KLF4, Sox2, c-Myc) the successful inoblast reprogrammed with mouse is an embryonic stem cell, this technology is called as inducing pluripotent stem cells (iPS) technology.Thereafter, people and other species such as rat, monkey, the inoblast of pig are also by the reprogrammed of success, and this technology is considered to of paramount importance breakthrough in the regenerative medicine.In recent years, the iPS technical development is rapid, at first is that c-Myc is proved to be nonessential in four rate of rotation factors, although after removing c-Myc, it is extremely low that reprogramming efficiency becomes; Secondly, the micromolecular compound of a series of promotion reprogrammed comes to light, like NSC 630176: VPA, TSA, and Sodium propanecarboxylate, dna methylation enzyme inhibitors: 5-AZA; TGF-beta inhibitor: A83-01 and SB431542, ZNFN3A1 suppressor factor: BIX01294, vitamins C (xitix) etc.The iPS process has been quickened in the discovery of these micromolecular compounds greatly, with reprogramming efficiency from originally about 0.01% level brought up near 1%.In addition; Serum-free and chemical ingredients limit the employing of culture condition; The reprogramming efficiency of three factors is brought up to about 10%, also solved the problem of the experimental repeatability difference that serum batch difference causes in the serum culture system simultaneously, for reprogrammed research provides extraordinary platform.Yet, induce the announcement progress of reprogrammed process mechanism still slow to transcription factor.

Seek the small molecules or the gene that can substitute the core reprogrammed factor, will play a role in promoting disclosing the function of the core reprogrammed factor in rearranging.

Summary of the invention

The purpose of this invention is to provide a kind of method and application of truncation type inducing pluripotent stem cells of c-Jun gene of AP-1 family protein.

Technical scheme of the present invention is to provide the N of a kind of c-Jun to hold disappearance and multipotential stem cell inducible factor to unite the application of use in the inducing pluripotent stem cells process, and the N end disappearance of said c-Jun is brachymemma or its corresponding nucleotide sequences of 170 to 274 amino acids of AP-1 family protein c-Jun to terminal 334 amino acids.

Preferably, above-mentioned multipotential stem cell inducible factor is Klf4, Sox2, Oct4.

Preferably, above-mentioned multipotential stem cell inducible factor is Klf4, Sox2, c-Myc.

Preferably, above-mentioned multipotential stem cell inducible factor is Klf4, Oct4, c-Myc.

Preferably, above-mentioned multipotential stem cell inducible factor is Klf4, Sox2.

Another technical scheme of the present invention is the method that the N end disappearance inducing pluripotent stem cells of a kind of c-Jun is provided:

A, 170 to 274 amino acids that will contain donor c-Jun Argine Monohydrochloride sequence are cloned on the expression vector to the corresponding nucleotide sequence of terminal 334 amino acids;

B, use step a gained expression vector and multipotential stem cell inducible factor infected donors somatocyte to make its reprogrammed be multipotential stem cell.

Preferably, said step a is specially: will contain the 254-334 of donor c-Jun Argine Monohydrochloride sequence, and 274-334, the corresponding nucleotide sequence of 170-334 amino acids fragment is cloned on the expression vector.

Preferably, the multipotential stem cell inducible factor of aforesaid method is Klf4, Sox2, Oct4.

Preferably, the multipotential stem cell inducible factor of aforesaid method is Klf4, Sox2.

Preferably, the donor somatocyte of aforesaid method is a l cell.

The present invention has used the proteic N end of the c-Jun of AP-1 family protein truncation type in the process of inducing pluripotent stem cells; Provide a kind of high efficiency reprogrammed to induce system; Substitute the small molecules or the gene of the core reprogrammed factor, will play a role in promoting disclosing the function of the core reprogrammed factor in rearranging.The proteic N end of c-Jun truncation type comprises a series of brachymemmas such as 170 amino acids to 334 amino acids, especially comprises 170 to 274 a series of truncation types to terminal 334 amino acids.170-334 as shown in Figure 2,250-334,274-334 fragment have promoter action to OKS inductive reprogrammed, 170-334 wherein, the 250-334 fragment then has obvious facilitation to OKS inductive reprogrammed.

Description of drawings

Fig. 1 is the structure iron of mouse c-Jun albumen total length and different truncation types.Protein sequence total length and structural domain information according to the last mouse c-Jun of NCBI design different primers, adopt the method for conventional molecular cloning, and the polypeptide of sequence fragment that clones different aminoacids in the diagram is used for experiment.1-334 polypeptide fragment called after c-Jun-FL wherein; 1-256 polypeptide fragment called after c-Jun (bZIP), 170-334 fragment called after c-Jun-DN.Clone's fragment is cloned on the retroviral vector pMXS with suitable restriction enzyme, carries out the segmental expression of purpose.

Fig. 2 is under OKS three factor reprogrammed models, the reprogramming efficiency comparison diagram that c-Jun albumen total length or different truncation type are compared with zero load.With OKS and unloaded virus infection OG2 embryo fibroblast is contrast, c-Jun full length gene or different truncation type virus infection OG2 embryo fibroblast shown in reaching with OKS respectively.Infect the back and in containing the mES substratum of serum, carry out inducing culture, under fluorescent microscope, calculate GFP male fluorescence clone number at suitable fate.The reprogrammed clone number that each fragment obtains is cloned number divided by the reprogrammed under the no cargo conditions, obtain the influence of total length or each truncated segment reprogramming efficiency.

Fig. 3 is that c-Jun is (ZIP) with the influence figure of c-Jun-DN to three factor reprogramming efficiencies.Three factor S KO with shown in behind the virus infection OG2 MEC, cultured continuously in serum or serum free culture system iSF1 is changed substratum every day.At suitable fate, calculate green fluorescence clone number through fluorescent microscope, be reference with three factor infected group fluorescence clone number, calculate the relative reprogramming efficiency of each experimental group.

Fig. 4 is the replacement design sketch of c-JunDN to the different reprogrammed factors.The MEC of virus infection OG2 shown in using respectively infects back cultured continuously in serum free culture system iSF1, changes substratum every day.At suitable fate, calculate green fluorescence clone number through fluorescent microscope.Analyze c-JunDN to Sox2, the metalepsy of Klf4 and Oct4.K：Klf4；S：Sox2，M：c-Myc，O：Oct4。

Fig. 5 be c-JunDN substitute Oct4 with klf4 and Sox2 with l cell reprogrammed design sketch.The MEC of virus infection OG2 shown in using respectively infects back cultured continuously in serum free culture system iSF1, changes substratum every day.At suitable fate, calculate green fluorescence clone number through fluorescent microscope.K：Klf4；S：Sox2。

Fig. 6 is that the SKMJunDN of picking induces reprogrammed clone figure.Said KSMJunDN is the cell that KSM and c-Jun-DN infect.Be cloned in microscopically by SKMJunDN inductive reprogrammed and present fine and close spheroid, edge gloss, the form homogeneous has typical stem cell form; Demonstrate very strong green fluorescence under the fluorescent microscope, show that its endogenous versatility gene Oct4 activates.AP dyeing is positive, and shows the clonal expression SEAP.

Fig. 7 is that SKMJunDN induces reprogrammed clone Oct4 and Nanog promoter region CpG island methylation state figure.Induce the reprogrammed cloned culture to extract genomic dna the SKMJunDN of picking, adopt PCR, clone gene group Oct4 is separated with Nanog promoter region purpose fragment with Auele Specific Primer.Adopt the methylation state on sulphite process analysis purposes zone C pG island.The result shows that the clone Oct4 of picking and the promoter region of Nanog all present the demethylation state, and be similar with embryonic stem cell.Black circle expression CpG island is a methylation state, and white circle expression CpG island is the demethylation state.

Fig. 8 is that SKMJunDN induces reprogrammed clonal expression Sox2, Nanog, Rex1, versatility molecular marked compound figure such as SSEA-1.The clone of picking is planted on the feeder layer cells, cultivated two days with embryonic stem cell.Adopt the method for immunofluorescence, use Sox2 respectively, Nanog, Rex1, SSEA-1 antibody and corresponding fluorescence two anti-these multi-functional expression of gene situations that detect.

Fig. 9 is that SKMJunDN induces the reprogrammed clone to have correct karyogram.After the clone who chooses handled with NST-757, adopt ordinary method to carry out karyotyping, observe mitotic figure.

Figure 10 is that SKMJunDN induces reprogrammed clone injection blastaea can form chimeric mouse figure.The SKMJunDN of picking clone digestion is expelled to (10-20 cell/blastaea) in the blastaea for after unicellular, again blastaea is transplanted in the female mouse of the false pregnancy uterus of ICR strain (10-15 blastaea/female mouse) continued of sewing up a wound raising.Wait to be born and whether chimericly judge through inspection iris and hair color behind the mouse.The cell preparation of using OG2 to originate goes out iPSCs and the embryoplastic chimeric mouse of ICR strain can present black iris and variegated hair.

Figure 11 is a c-Jun expression contents comparison diagram in embryonic stem cell and the embryo fibroblast.Adopt conventional molecular biology method to extract total RNA of embryonic stem cell and embryo fibroblast respectively, adopt quantitative fluorescent PCR to detect after the total RNA of 2ug is reversed to cDNA.Compare c-jun expression of gene amount in embryonic stem cell and the embryo fibroblast.

Figure 12 is embryonic stem cell c-Jun expression amount variation diagram in the embryoid body atomization.Embryonic stem cell is broken up to embryoid body according to ordinary method, and the differentiation culture thing of collecting different number of days respectively extracts total RNA, the total RNA of 2ug is reversed to adopts behind the cDNA quantitative fluorescent PCR to detect.Relatively break up c-jun expression of gene quantitative changeization in the different number of days process to embryoid body.

Figure 13 is embryonic stem cell c-Jun expression amount variation diagram in atomization.Embryonic stem cell is cultivated under the condition that removes leukocyte inhibitory factor (LIF), and cell will progressively break up.The culture of collecting different number of days extracts total RNA, the total RNA of 2ug is reversed to adopts behind the cDNA quantitative fluorescent PCR to detect.Relatively break up c-jun expression of gene quantitative changeization in the different number of days process.

Figure 14 be c-Jun, c-Jun (bZIP) with c-JunDN to differentiation of stem cells impact effect figure.Making up c-Jun, c-Jun respectively (bZIP) with the strain of c-JunDN stably express stem cell, and carries out continuous passage and cultivates under the culture condition of embryonic stem cell.

Figure 15 is that c-Jun (bZIP) keeps design sketch with c-JunDN to the stem cell versatility.Making up c-Jun respectively (bZIP) with the stably express stem cell strain of c-JunDN, cultivates in the embryonic stem cell substratum after removing LIF.Observe the versatility state of stem cell, and detect versatility molecular marked compound alkaline phosphatase activity with AP dyeing.C-Jun (bZIP) expresses all and can postpone differentiation in the versatility that does not have to keep under the state of LIF stem cell with crossing of c-JunDN.Black is alkaline phosphatase staining among the figure.

Embodiment

By specifying technology contents of the present invention, structural attitude, realized purpose and effect, give explanation below in conjunction with embodiment and conjunction with figs. are detailed.

Practice of the present invention will be used the conventional art of molecular biology, microbiology, cytobiology, immunology and recombinant DNA, and it belongs to the art technology scope.Referring to for example: the molecular cloning experiment guide, the 3rd edition (2002), and Sambrook, people such as Fritsch and Maniatis write; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (people such as F.M.Ausubel writes (1987)); Book series METHODS IN ENZYMOLOGY (Academic Press; Inc.): PCR 2:A PRACTICAL APPROACH (1995), M.J.MacPherson, people such as B.D.Hames and G.R.Taylor write; ANTIBODIES (1988); People such as Harlow and Lane writes, A LABORATORY MANUAL and ANIMAL CELL CULTURE (1987), and people such as R.I.Freshney write; HANDBOOK OFSTEM CELLS, volume 2, people such as W.FrenchAnderson write.

Unless otherwise indicated, used term all has the conventional implication of understanding of those skilled in the art among this paper, for the ease of understanding the present invention, some terms that use among this paper has been carried out following definitions.

" embryonic stem cell " as herein described is such cell.It derives from mouse blastaea inner cell mass separation and Culture, can and have multinomial differentiation potential and be divided into any cell type of three germinal layers at external infinite multiplication, self.Have special molecular marked compound, can form teratoma, be expelled to 3.5 days blastaeas and place the female mouse of replace-conceive uterus class can form chimeric mouse again.

R1 cell as herein described derives from 3.5 days isolated embryonic stem cell lines of blastaea of 129 strain mouse; Be laboratory embryonic stem cell line commonly used; Essential characteristic with embryonic stem cell, can the serum culture condition under, no feeder layer cells upload be commissioned to train foster.

" induction type multipotent stem cells (iPS cell) " as herein described is such cell; Its source is that somatocyte forms through inducing reprogramming of somatic cells to change; It is under the embryonic stem cell culture condition; Express, be transplanted to that subcutaneous to form the aspects such as teratoma that comprise 3 germinal layer histocyte structures closely similar with mouse embryo stem cell in cellular form, growth characteristics, surface marker with embryonic stem cell, and also almost completely similar at aspects such as dna methylation mode, gene expression profile, chromatin state, formation chimeric animals with mouse embryo stem cell.

Used term " somatocyte " is the notion for " sexual cell " and " embryonic stem cell " among this paper; It is no longer to have a versatility by what " embryonic stem cell " differentiation produced; But has the cell of a certain concrete function; It is no longer to possess versatility by what " embryonic stem cell " differentiation or inner cell mass continue to grow produced; As the cell that generally has concrete function, one of which from the etap be arranged in blastula stage (being specially after fertilization 3.5 days) mouse afterwards the tire mouse or become mouse to draw materials, generally avoid getting sexual cell and source (like stem spermatogonium, the sexual fold stem cell etc.) thereof that possibly have versatility when drawing materials.

Somatocyte used among this paper preferably derives from Mammals, more preferably derives from people, monkey, dog, cat, rat or mouse, most preferably, derives from mouse.Somatocyte among this paper can be the somatocyte of any kind in the body, is preferably inoblast.

Term as herein described " induces " (also claiming " inducing reprogrammed ") to be meant somatocyte is dedifferented the process into multipotent stem cells.Preferably, import somatocyte and can the inductor cell be dedifferentiated into and be multipotent stem cells (Takahashi K, Yamanaka S.Cell.2006 through keeping the required versatility factor cDNA of stem cell versatility; 126:663 – 676; Wernig M, Meissner A, Foreman R waits the people, Nature.2007; 448:318-324; Yu J, Vodyanik MA, people Science.2007 such as Smuga-Otto K; 318:1917-1920).

Term as herein described " reprogrammed ": refer to the cell that has broken up is dedifferented, be returned to the state of multipotential cell again.Reprogrammed among the present invention refers to becoming the contrary state that is divided into embryonic stem cell of somatocyte.

It can be multiple technologies well known to those skilled in the art that said stem cell versatility factor cDNA is imported somatic method, comprises the various methods that DNA changed over to cell such as virus infection, liposome transfection, transposon-mediated insertion expression, membrane-spanning protein, drug-induced, electroporation, particle bombardment.Preferably, use the virus vector that comprises cDNA to carry out transfection, said virus vector comprises several diseases poisonous carriers such as lentiviral vectors, retroviral vector.Retroviral vector (for example pMX carrier) preferably.

" virus infection " described in this paper promptly refers to and above-mentioned stem cell versatility factor cDNA imported somatic method." Sox2/KLF4/Oct4/c-Myc " among this paper promptly refers to above-mentioned stem cell versatility factor cDNA or has the virus vector of these cDNA; Stem cell versatility factor cDNA can be imported in the somatocyte by " virus infection "; It can translate the synthetic gene product in expression systems such as somatocyte.

What OKSM represented among this paper is the cell of Oct4, Klf4, Sox2, c-Myc virus infection; What " KSM " represented is the cell of Klf4, Sox2, c-Myc virus infection; What " OSM " represented is the cell of Oct4, Sox2, c-Myc virus infection.What " 4F " represented is Oct4, Klf4, Sox2, four factors of c-Myc, and what " 3F " represented is Oct4, Klf4, three factors of Sox2.

The cultural method of cell is conventional cell culture processes and condition among this paper; Comprise some suitable each concrete clones, but do not influence the modification of cell essential property, the cultural method of various cell types and culture condition; Can be referring to people such as W.French Anderson; HANDBOOK OF STEM CELLS, volume 2, or application number is China's mandate invention of 200910038883.4.

" serum free medium SF1 " as herein described is that the iPS-SF1 substratum in the invention is authorized by China of 200910038883.4 for application number.The invention is not restricted to use the substratum identical with efficient culture medium, other contain basic medium and serum or can both add the c-Jun truncation type for the substratum of serum additive and induce the induction type multipotent stem cells.

The method of reporter gene has been adopted in the discriminating of iPS cell among this paper; Described reporter gene is meant and can induces the stage of changing similar embryonic stem cell into through adding by indicator cells; Comprise and utilize transgenic or homologous recombination means to add the sequence that a segment table reaches GFP (like the GFP among this paper) or resistance; This section sequence is in the control of promotor of some genes of embryonic stem cell specifically expressing; So can when cell arrives the embryonic stem cell state, activate the expression of this section GFP or resistant gene, thereby make this cell have the properties and characteristics (as sending green glow) that some can be to be detected and be different from other not cells of reprogrammed to this state.Art technology reporter gene commonly used comprises green fluorescent protein, and resistant gene is ampicillin resistance gene etc. for example.The reporter gene that those skilled in the art can be suitable for various embodiments according to the culture condition and the purposes selection of cell.People such as reference example such as Young II Yeom; Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells; Development 122,000-000 (1996), Printed in Great Britain; The company of Biologists Limited 1996, the 881-894 page or leaf; People such as Shin-yaHatano, Pluripotential competence of cells associated with Nanog activation, Mechanisms of Development 122 (2005), 67-79.

The method of detection cell versatility as herein described is well known to those skilled in the art; Referring to for example Yamanaka; S.Strategies and new developments in the generation of patient-specific pluripotent stem cells.Cell Stem Cell 1,39-49 (2007) etc.Said method comprises that the methylation state of the expression of identifying the versatility molecule marker, cell detects, formation, the teratomatous formation of embryo's corpusculum EB and use through the inductive multipotent stem cells and form chimeric mouse or the like.

" chimeric mouse " as herein described is " chimeric mouse " technology implementation of knowing by one of ordinary skill in the art.Be meant with embryonic stem cell or through the iPS injection cell that this paper technology obtains to go in the mouse blastaea; Make it mix with embryonic cell in the blastaea of being injected into mouse; Intrauterine in the female mouse of replace-conceive grows jointly, and each about the whole body of mouse birth back organized promptly and be made up of two kinds of embryonic cell mixed together, like mosaic picture mosaic appearance; Such mouse is called as chimeric mouse, and (Evans M J waits the people; The ability of EK cell to form chimeras after selection of clones in G418 and some observation on the intergration of retroviral vector proviral DNA into EK cells [M]; Cold Spring Harbor Symposia on Quantitative Biology; 1985; Xian MW, Wu BY, Hu XL, Shang KG, Wu HL, 1996.Construction of chimeric mice of ES cells by microinjection method.Hereditas 18 (1): 7-10).Use iPS can form chimeric mouse be check iPS whether with embryonic stem cell have similarity the most directly and the evidence of most critical.

" basic medium " as herein described is the cultivation article that contain cell growth desired nutritional materials such as carbohydrate, amino acid, inorganic salt, VITAMINs, lipid of artificial preparation.Basic medium according to the invention comprises Dulbecco ' s Modified Eagle ' s Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME); F-10; F-12, RPMI 1640, Glasgow ' s Minimal Essential Medium (GMEM); α Minimal Essential Medium (α MEM); Iscove ' s Modified Dulbecco ' s Medium and M199 etc., but be not limited only to above-mentioned basic medium, wherein the base of optimum selection substratum is high sugared DMEM basic medium.

The aminoacid sequence of c-Jun total length described herein (SEQ ID NO:1): (1-334)

1 mtakmettfyddalnasflqsesgaygysnpkilkqsmtlnladpvgslkphlraknsdl

61 ltspdvgllkl aspelerlii qssnghittt ptptqflcpk nvtdeqegfa egfvralae

121 lhsqntlpsv tsaaqpvsga gmvapavasv agagggggys aslhseppvy anlsnfnpga

181 lssgggapsygaaglafpsqpqqqqqppqpphhlpqqipvqhprlqalkeepqtvpempg

241 etpplspidmesqerikaerkrmrnriaaskcrkrkleriarleekvktlkaqnselast

301 anmlreqvaq lkqkvmnhvn sgcqlmltqq lqtf

C-JunDN truncation type described herein (170-334) sequence (SEQ ID NO:2):

yanlsnfnpgalssgggapsygaaglafpsqpqqqqqppqpphhlpqqipvqhprlqalkeepqtvpempgetpplspidmesqerikaerkrmrnriaaskcrkrkleriarleekvktlkaqnselastanmlreqvaqlkqkvmnhvnsgcqlmltqqlqtf

C-Jun-Bzip described herein (1-256) sequence (SEQ ID NO:3):

1 mtakmettfyddalnasflqsesgaygysnpkilkqsmtlnladpvgslkphlraknsdl

61 tspdvgllklaspelerliiqssnghitttptptqflcpknvtdeqegfaegfvralae

121 lhsqntlpsvtsaaqpvsgagmvapavasvagagggggysaslhseppvyanlsnfnpga

181 lssgggapsygaaglafpsqpqqqqqppqpphhlpqqipvqhprlqalkeepqtvpempg

241 etpplspidmesqeri

The cDNA sequence of c-Jun total length described herein (SEQ ID NO:4):

ATGACTGCAAAGATGGAAACGACCTTCTACGACGATGCCCTCAACGCCTCGTTCCTCCAGTCCGAGAGCGGTGCCTACGGCTACAGTAACCCTAAGATCCTAAAACAGAGCATGACCTTGAACCTGGCCGACCCGGTGGGCAGTCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTTCTCACGTCGCCCGACGTCGGGCTGCTCAAGCTGGCGTCGCCGGAGCTGGAGCGCCTGATCATCCAGTCCAGCAATGGGCACATCACCACTACACCGACCCCCACCCAGTTCTTGTGCCCCAAGAACGTGACCGACGAGCAGGAGGGCTTCGCCGAGGGCTTCGTGCGCGCCCTGGCTGAACTGCATAGCCAGAACACGCTTCCCAGTGTCACCTCCGCGGCACAGCCGGTCAGCGGGGCGGGCATGGTGGCTCCCGCGGTGGCCTCAGTAGCAGGCGCTGGCGGCGGTGGTGGCTACAGCGCCAGCCTGCACAGTGAGCCTCCGGTCTACGCCAACCTCAGCAACTTCAACCCGGGTGCGCTGAGCAGCGGCGGTGGGGCGCCCTCCTATGGCGCGGCCGGGCTGGCCTTTCCCTCGCAGCCGCAGCAGCAGCAGCAGCCGCCTCAGCCGCCGCACCACTTGCCCCAACAGATCCCGGTGCAGCACCCGCGGCTGCAAGCCCTGAAGGAAGAGCCGCAGACCGTGCCGGAGATGCCGGGAGAGACGCCGCCCCTGTCCCCTATCGACATGGAGTCTCAGGAGCGGATCAAGGCAGAGAGGAAGCGCATGAGGAACCGCATTGCCGCCTCCAAGTGCCGGAAAAGGAAGCTGGAGCGGATCGCTCGGCTAGAGGAAAAAGTGAAAACCTTGAAAGCGCAAAACTCCGAGCTGGCATCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTTAAGCAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCAGTTGCAAACGTTTTGA

The structure of experimental example 1:c-Jun and different absence type thereof, c-Jun brachymemma promote SKO inductive reprogramming efficiency.

Adopt conventional molecular cloning method will contain c-Jun albumen total length (324 amino acid) respectively, and 1-256,75-324; 170-334; 254-334,274-334,6 kinds of segmental nucleotide sequences of different aminoacids such as 170-334 are cloned on the pMXs retrovirus expression vector.Final cloned sequence is as shown in Figure 1.

After l cell digestion, in 12 orifice plates, infect with the expression vector that makes up among OKS or OKSM and the embodiment 1 respectively with every hole 20,000 cell seedings.Infect the back with general reprogrammed culture medium culturing, change substratum every day, and under fluorescent microscope, calculate fluorescence clone number at suitable fate.

As shown in Figure 2, compare with zero load, C-Jun albumen total length, the 1-256 fragment, the 75-324 fragment all induces reprogrammed that restraining effect is arranged to OKS, and wherein C-Jun albumen total length suppresses the most remarkable.And 170-334, the 250-334 fragment then has obvious facilitation to OKS inductive reprogrammed, and wherein 170-334 promoter actions are especially remarkable.170-282 and 274-334 fragment are influential but not obvious to OKS inductive reprogrammed.In ensuing embodiment, we are with c-Jun total length called after c-Jun FL, with c-JUN 170-334 fragment called after c-jun-DN.

Experimental example 2:c-Jun-DN promotes SKO inductive reprogramming efficiency, and can substitute reprogrammed factor Oct or Sox2.

C-JunDN infects the MEF cell with three factor S KO or different combinations of factors respectively, and in serum or serum-free inducing culture system, cultivates, and changes substratum every day and calculates reprogramming efficiency at suitable fate.Observe c-JunDN to the influence of reprogramming efficiency and the substitution effect of counterweight programmed factors.

" serum free medium SF1 " as herein described is that the iPS-SF1 substratum in the invention is authorized by China of 200910038883.4 for application number.The mouse embryo stem cell cell culture medium that blood serum medium as herein described is well known to those skilled in the art, staple are that basic medium adds foetal calf serum, and add leukocyte inhibitory factor LIF." blood serum medium " of present embodiment is that the mES substratum in the invention is authorized by China of 200910038883.4 for application number.

As shown in Figure 3, at serum-free or have under the culture system of serum, c-Jun-DN all can improve SKO inductive reprogramming efficiency.

As shown in Figure 4, under serum free medium iPS-SF1 condition, c-JunDN can realize effective reprogrammed under the condition that does not have Sox2 or Oct4.

As shown in Figure 5, under serum free medium iPS-SF1 condition, c-JunDN can with Sox2, Klf4 is together with the l cell reprogrammed.

As shown in Figure 6, c-Jun-DN substitutes the KSMJunDN clonal expression green fluorescence that Oct4 produces, and has typical stem cell form, and it is positive that AP dyeing also becomes.Said KSMJunDN is the cell that KSM and c-Jun-DN infect.

As shown in Figure 7, KSMJunDN clone Oct4 and Nanog promoter region that c-Jun-DN substitutes the Oct4 generation are the demethylation state, and be similar with embryonic stem cell.

As shown in Figure 8, c-Jun-DN substitutes the KSMJunDN clonal expression Sox2 that Oct4 produces, Nanog, Rex1, versatility molecular marked compounds such as SSEA-1.

As shown in Figure 9, the KSMJunDN clone that c-Jun-DN substitutes the Oct4 generation has correct caryogram.

Shown in figure 10, the KSMJunDN clone that c-Jun substitutes the Oct4 generation can produce allophenic mice.

The above results shows that c-JunDN-DN can effectively substitute Oct4 or Sox2 with the embryo fibroblast reprogrammed.

Embodiment 3:c-Jun-DN can keep the versatility of stem cell.

In order to study the effect of C-Jun gene in the embryonic stem cell versatility is kept; We studied this gene embryonic stem cell with become somatocyte and embryonic stem cell atomization in the expression situation, analyzed the influence that different truncation types is kept and broken up in the embryonic stem cell versatility simultaneously.

Respectively the embryo fibroblast (MEF) of mouse and embryonic stem cell (ES) are extracted RNA, use the expression amount of c-Jun in two kinds of cells of methods analyst of RT-PCR again.

Show that like Figure 11 the expression amount of c-Jun is far above the expression amount in the embryonic stem cell in the MEF cell.Show that the c-Jun factor possibly be unfavorable for keeping of versatility.

Adopt the mode of standard to be divided into embryoid body (EB) in the ES cell, collect sample, extract RNA, the expression amount of c-Jun in two kinds of cells of methods analyst of employing RT-PCR at different fates.

Shown in figure 12, the expression amount of ES cell c-Jun in the EB process of differentiation constantly raises.

The ES cell is not cultivated in having the substratum of LIF, is collected cell sample, extract RNA, the expression amount of c-Jun in two kinds of cells of methods analyst of employing RT-PCR in different number of days.

Shown in figure 13, the ES cytodifferentiation is crossed in the title, and the expression amount of c-Jun constantly raises.

With the c-Jun total length, (bZIP), c-JunDN changes among the R1 c-Jun, adopts the clone of resistance screening ability stably express target gene fragment respectively.

Shown in figure 14, the c-Jun total length cross to be expressed in R1, can cause the differentiation of stem cell, and c-Jun (bZIP), c-JunDN then can not cause differentiation of stem cells.

Shown in figure 15, removing in the ES substratum of LIF, (bZIP), c-JunDN can keep the versatility state of cell to c-Jun, delays differentiation of stem cells.

The above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to be done; Or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.

Sequence table

< 110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health

< 120>methods and applications of the N of c-Jun end disappearance inducing pluripotent stem cells

<160>4

<170>PatentIn version 3.3

<210>1

<211>333

<212>PRT

< 213>mouse c-Jun aminoacid sequence

<400>1

Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala

1 5 10 15

Ser Phe Leu Gln Ser Glu Ser Gly Ala Tyr Gly Tyr Ser Asn Pro Lys

20 25 30

Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser

35 40 45

Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Thr Ser Pro Asp

50 55 60

Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile Ile

65 70 75 80

Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln Phe

85 90 95

Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu Gly

100 105 110

Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro Ser

115 120 125

Val Thr Ser Ala Ala Gln Pro Val Ser Gly Ala Gly Met Val Ala Pro

130 135 140

Ala Val Ala Ser Val Ala Gly Ala Gly Gly Gly Gly Gly Tyr Ser Ala

145 150 155 160

Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe Asn

165 170 175

Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala Ala

180 185 190

Gly Leu Ala Phe Pro Ser Gln Pro Gln Gln Gln Gln Gln Pro Pro Gln

195 200 205

Pro Pro His His Leu Pro Gln Gln Ile Pro Val Gln His Pro Arg Leu

210 215 220

Gln Ala Leu Lys Glu Glu Pro Gln Thr Val Pro Glu Met Pro Gly Glu

225 230 235 240

Thr Pro Pro Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys

245 250 255

Ala Glu Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg

260 265 270

Lys Arg Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr

275 280 285

Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg

290 295 300

Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met Asn His Val Asn Ser

305 310 315 320

Gly Cys Gln Leu Met Leu Thr Gln Gln Leu Gln Thr Phe

325 330

<210>2

<211>165

<212>PRT

< 213>mouse c-Jun truncation type (170-334) amino acids sequence

<400>2

Tyr Ala Asn Leu Ser Asn Phe Asn Pro Gly Ala Leu Ser Ser Gly Gly

1 5 10 15

Gly Ala Pro Ser Tyr Gly Ala Ala Gly Leu Ala Phe Pro Ser Gln Pro

20 25 30

Gln Gln Gln Gln Gln Pro Pro Gln Pro Pro His His Leu Pro Gln Gln

35 40 45

Ile Pro Val Gln His Pro Arg Leu Gln Ala Leu Lys Glu Glu Pro Gln

50 55 60

Thr Val Pro Glu Met Pro Gly Glu Thr Pro Pro Leu Ser Pro Ile Asp

65 70 75 80

Met Glu Ser Gln Glu Arg Ile Lys Ala Glu Arg Lys Arg Met Arg Asn

85 90 95

Arg Ile Ala Ala Ser Lys Cys Arg Lys Arg Lys Leu Glu Arg Ile Ala

100 105 110

Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu

115 120 125

Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln

130 135 140

Lys Val Met Asn His Val Asn Ser Gly Cys Gln Leu Met Leu Thr Gln

145 150 155 160

Gln Leu Gln Thr Phe

165

<210>3

<211>255

<212>PRT

< 213>mouse c-Jun truncation type (1-256) amino acids sequence

<400>3

Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala

1 5 10 15

Ser Phe Leu Gln Ser Glu Ser Gly Ala Tyr Gly Tyr Ser Asn Pro Lys

20 25 30

Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser

35 40 45

Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Thr Ser Pro Asp

50 55 60

Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile Ile

65 70 75 80

Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln Phe

85 90 95

Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu Gly

100 105 110

Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro Ser

115 120 125

Val Thr Ser Ala Ala Gln Pro Val Ser Gly Ala Gly Met Val Ala Pro

130 135 140

Ala Val Ala Ser Val Ala Gly Ala Gly Gly Gly Gly Gly Tyr Ser Ala

145 150 155 160

Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe Asn

165 170 175

Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala Ala

180 185 190

Gly Leu Ala Phe Pro Ser Gln Pro Gln Gln Gln Gln Gln Pro Pro Gln

195 200 205

Pro Pro His His Leu Pro Gln Gln Ile Pro Val Gln His Pro Arg Leu

210 215 220

Gln Ala Leu Lys Glu Glu Pro Gln Thr Val Pro Glu Met Pro Gly Glu

225 230 235 240

Thr Pro Pro Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile

245 250 255

<210>4

<211>1005

<212>DNA

< 213>artificial sequence

<400>4

atgactgcaa agatggaaac gaccttctac gacgatgccc tcaacgcctc gttcctccag

60

tccgagagcg gtgcctacgg ctacagtaac cctaagatcc taaaacagag catgaccttg

120

aacctggccg acccggtggg cagtctgaag ccgcacctcc gcgccaagaa ctcggacctt

180

ctcacgtcgc ccgacgtcgg gctgctcaag ctggcgtcgc cggagctgga gcgcctgatc

240

atccagtcca gcaatgggca catcaccact acaccgaccc ccacccagtt cttgtgcccc

300

aagaacgtga ccgacgagca ggagggcttc gccgagggct tcgtgcgcgc cctggctgaa

360

ctgcatagcc agaacacgct tcccagtgtc acctccgcgg cacagccggt cagcggggcg

420

ggcatggtgg ctcccgcggt ggcctcagta gcaggcgctg gcggcggtgg tggctacagc

480

gccagcctgc acagtgagcc tccggtctac gccaacctca gcaacttcaa cccgggtgcg

540

ctgagcagcg gcggtggggc gccctcctat ggcgcggccg ggctggcctt tccctcgcag

600

ccgcagcagc agcagcagcc gcctcagccg ccgcaccact tgccccaaca gatcccggtg

660

cagcacccgc ggctgcaagc cctgaaggaa gagccgcaga ccgtgccgga gatgccggga

720

gagacgccgc ccctgtcccc tatcgacatg gagtctcagg agcggatcaa ggcagagagg

780

aagcgcatga ggaaccgcat tgccgcctcc aagtgccgga aaaggaagct ggagcggatc

840

gctcggctag aggaaaaagt gaaaaccttg aaagcgcaaa actccgagct ggcatccacg

900

gccaacatgc tcagggaaca ggtggcacag cttaagcaga aagtcatgaa ccacgttaac

960

agtgggtgcc aactcatgct aacgcagcag ttgcaaacgt tttga

1005

Claims

1.c-Jun N end disappearance unite the application of use in the inducing pluripotent stem cells process with the multipotential stem cell inducible factor, the N end disappearance of said c-Jun is 170 to 274 amino acids of AP-1 family protein c-Jun brachymemma or its corresponding nucleotide sequences to terminal 334 amino acids.

2. application according to claim 1 is characterized in that, said multipotential stem cell inducible factor is Klf4, Sox2, Oct4.

3. application according to claim 1 is characterized in that, said multipotential stem cell inducible factor is Klf4, Sox2, c-Myc.

4. application according to claim 1 is characterized in that, said multipotential stem cell inducible factor is Klf4, Oct4, c-Myc.

5. application according to claim 1 is characterized in that, said multipotential stem cell inducible factor is Klf4, Sox2.

6. use the N of the c-Jun of AP-1 family to hold the method that lacks inducing pluripotent stem cells, it is characterized in that,

7. the method for the N end disappearance inducing pluripotent stem cells of the c-Jun of use according to claim 6 AP-1 family; It is characterized in that; Said step a is specially: the 254-334 that will contain donor c-Jun Argine Monohydrochloride sequence; 274-334, the corresponding nucleotide sequence of 170-334 amino acids fragment is cloned on the expression vector.

8. the method for the N end disappearance inducing pluripotent stem cells of the c-Jun of use according to claim 6 AP-1 family is characterized in that said multipotential stem cell inducible factor is Klf4, Sox2, Oct4.

9. the method for the N end disappearance inducing pluripotent stem cells of the c-Jun of use according to claim 6 AP-1 family is characterized in that said multipotential stem cell inducible factor is Klf4, Sox2.

10. the method for the N end disappearance inducing pluripotent stem cells of the c-Jun of use according to claim 6 AP-1 family is characterized in that said donor somatocyte is a l cell.