CN106222199A - Change the means of cell fate - Google Patents
Change the means of cell fate Download PDFInfo
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- CN106222199A CN106222199A CN201610616149.1A CN201610616149A CN106222199A CN 106222199 A CN106222199 A CN 106222199A CN 201610616149 A CN201610616149 A CN 201610616149A CN 106222199 A CN106222199 A CN 106222199A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention discloses a kind of method changing cell fate.The method includes making cell process LAN to have the protein selected from one of following amino acid sequences: (a) SEQ ID NO:1, the aminoacid sequence shown in 2；(b) SEQ ID NO:1, a part for the aminoacid sequence shown in 2；C () has at least 80% with aminoacid sequence shown in (a) and (b), the aminoacid sequence of preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence iden.Utilize the method can make cell process LAN Mkk3 or Mkk6 albumen; and then can effectively change cyto-chromatin structure; promote versatility gene Mechanisms of Histone Acetylation Modification, improve the expression of versatility gene such that it is able to effectively change cell fate and especially promote somatic reprogramming.
Description
Technical field
The present invention relates to biological technical field, more specifically, the present invention relates to a kind of change the method for cell fate, one
For regulating the method for cellular histone acetylation degree, one for changing cells pluripotency gene Oct4, Nanog, Sox2
With the method for Rex1 expression and a kind of for changing cell heterochromatin protein HP1a expression or location in cell
Method, and a kind of construct and a kind of reconstitution cell.
Background technology
Along with going deep into of stem-cell research, scientists finds that the mammalian cell of terminal differentiation is also to have totipotency
, adult cell can be reprogrammed, and changes gene expression profile thus changes its destiny, again has versatility or directly
Transdifferentiation becomes another kind of cell.The method of reprogramming of somatic cells mainly has two kinds in early days: nuclear transplantation and cell merge, but this
Two kinds of methods need nonetheless remain for relating to embryo, is extremely limited in terms of ethics.2006, Japan's Yamanaka laboratory
Obtain breakthrough achievement in research in reprogramming field, find by four transcription factor Sox2 of heterogenous expression, Oct4, Klf4
L cell reprogramming can be made to be pluripotent stem cell (iPSC) with c-Myc, open up one newly for stem-cell research
Direction, and therefore obtain Nobel Prize.Between a few years subsequently, the progress in reprogramming of somatic cells field is abnormal swift and violent.Though
So the induced multi-potent stem-cell research initial stage runs into many problems, inserts including external source oncogene, inefficiency, the most autologous
The problems such as immunoreation, but the proposition of various improved method is greatly facilitated mechanism and the applied research of reprogramming technology.Logical
Method of crossing is improved, and reprogramming technology is applied in various species and cell, and is not only l cell.Scientist
Also filter out various reprogramming combinations of factors, not only increase reprogramming efficiency and quality, but also avoid oncogene
Insert.The methods such as scientists there have been developed protein induced, micromolecular compound induction.
The target of reprogramming technology be exactly solve the special pluripotent cell of patient clinically obtain a difficult problem in a large number.Science
The mice of sickle anemia has successfully been treated at laboratory by reprogramming technology and gene repair technology by family.Although this skill
Art is far away from clinic, but scientists the most successfully induces patient's iPS cell of many single gene mutation diseases, uses
Make drug screening and gene repair.Reprogramming and Development And Differentiation are the changes of cell fate.Cell is to the mistake of other cells switch
What journey there occurs, be that scientists is the most inquisitive.
But, how to change cell fate and especially promote somatic reprogramming, still need to be studied further.
Summary of the invention
It is contemplated that solve one of above-mentioned technical problem the most to a certain extent.To this end, one object of the present invention
It is to propose a kind of can effectively change the method that cell fate especially promotes somatic reprogramming.
It should be noted that the term used in this article " cell fate " refers to from a kind of cell type to another kind
The transformation of cell type, including the differentiation of somatic reprogramming, transdifferentiation and stem cell.
According to the first aspect of the invention, the invention provides a kind of method changing cell fate, it includes changing
The expression of predetermined protein in described cell, described predetermined protein have following amino acid sequences at least one: (a)
SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；(b) SEQ ID NO:1 or 2 at least one shown in ammonia
A part for base acid sequence；C () has at least 80% with aminoacid sequence shown in (a) and (b), and preferably at least 90%, more excellent
Choosing at least 95%, the aminoacid sequence of further preferably at least 99% sequence iden.Wherein, the ammonia shown in SEQ ID NO:1
Base acid sequence is the aminoacid sequence of Mkk3 albumen, particularly as follows: MESPAASPPASLPQTKGKSKRKKDLRISCVSKPPVSNPT
PPRNLDSRTFITIGDRNFEVEADDLVTISELGRGAYGVVEKVRHAQSGTIMAVKRIRATVNTQEQKRLLMDLDINMR
TVDCFYTVTFYGALFREGDVWICMELMDTSLDKFYRKVLEKNMKIPEDILGEIAVSIVRALEHLHSKLSVIHRDVKP
SNVLINKEGHVKMCDFGISGYLVDSVAKTMDAGCKPYMAPERINPELNQKGYNVKSDVWSLGITMIEMAILRFPYES
WGTPFQQLKQVVEEPSPQLPADQFSPEFVDFTSQCLRKNPAERMSYLELMEHPFFTLHKTKKTDIAAFVKEILGEDS
(SEQ ID NO:1).Wherein, the aminoacid sequence that aminoacid sequence is Mkk6 albumen shown in SEQ ID NO:2, particularly as follows:
MSQSKGKKRNPGLKIPKEAFEQPQTSSTPPRDLDSKACISIGNQNFEVKADDLEPIVELGRGAYGVVEKMRHVPSGQ
IMAVKRIRATVNSQEQKRLLMDLDVSMRTVDCPFTVTFYGALFREGDVWICMELMDTSLDKFYKQVIDKGQTIPEDI
LGKIAVSIVKALEHLHSKLSVIHRDVKPSNVLINTLGQVKMCDFGISGYLVDSVAKTIDAGCKPYMAPERINPELNQ
KGYSVKSDIWSLGITMIELAILRFPYDSWGTPFQQLKQVVEEPSPQLPADKFSADFVDFTSQCLKKNSKERPTYPEL
MQHPFFTVHESKAADVASFVKLILGD (SEQ ID NO:2) Mkk3 or Mkk6 belongs to the center kinase of MAPK cascade system,
They have double grading, can be activated by special upstream kinases, can activate again special downstream kinase, thus can guarantee that letter
Number conduction accuracy.It is surprisingly found by the inventors that, change cell Mkk3 or Mkk6 expressing quantity, it is possible to effectively change multipotency
Property gene promoter area acetylation of histone, thus affect chromatin open level, change the expression of versatility gene, thus
Can easily and effectively change cell fate and especially affect somatic reprogramming.Additionally, the method is simple, efficiency
Height, favorable repeatability.
According to embodiments of the invention, the method for above-mentioned change cell fate can further include following supplementary technology
At least one feature:
According to embodiments of the invention, described cell is somatic cell or stem cell.Thereby, it is possible to significantly effectively change body
Cell or the cell fate of stem cell.
According to embodiments of the invention, changing the expression of predetermined protein in described cell is by described in process LAN
Predetermined protein in cell realizes, it is surprisingly found by the inventors that, make cell process LAN Mkk3 or Mkk6 albumen, it is possible to effectively
Promote the acetylation of histone of versatility gene promoter area, thus promote that chromatin is open, promote the expression of versatility gene,
It is thus possible to easily and effectively change cell fate especially affect somatic reprogramming.
According to embodiments of the invention, at least one of described cell process LAN Sox, Klf4, Oct4 and c-Myc in advance,
Optional, described cell process LAN Sox, Klf4 and Oct4 simultaneously in advance.Inventor finds, compared with prior art, and mistake in advance
At least one of expression Sox, Klf4, Oct4 and c-Myc, process LAN Mkk3 or Mkk6 on this basis, can remarkably promote many
The acetylation of histone of energy property gene promoter area, thus it is open to greatly promote chromatin, significantly improves versatility gene
Express such that it is able to more efficiently change cell fate and especially promote somatic reprogramming.
According to embodiments of the invention, changing the expression of predetermined protein in described cell is by described cell
Middle introducing has and carries out selected from the nucleic acid molecule of one of following nucleotide sequences: (a) SEQ ID NO:3 or 4 is at least
Nucleotide sequence shown in one of；(b) SEQ ID NO:3 or 4 at least one shown in the part of nucleotide sequence；(c)
At least 80% is had with nucleotide sequence shown in (a) and (b), preferably at least 90%, more preferably at least 95%, further preferably
The nucleotide sequence of at least 99% sequence iden；D () is under high stringency conditions, it is possible to nucleotide sequence shown in (a) or (b)
At least one or the nucleotide sequence of its complementary sequence hybridization.Wherein, nucleotides sequence shown in SEQ ID NO:3 is classified as Mkk3 egg
The sequence of white encoding gene, particularly as follows: atggagtcgcccgccgcgagcccgccggccagcttgcctcagaccaaagga
aaatccaaaaggaaaaaggacttacggatatcctgcgtgtccaagccacctgtgtccaaccccacacccccccggaa
cctggactcccggaccttcatcactatcggagacagaaacttcgaagtggaggctgatgacttggtgaccatctcag
agctgggtcgtggagcctatggggtggtagagaaagtgcggcatgctcagagtggtaccatcatggctgtcaagcgc
atccgggccacagtgaacacacaggagcagaaacgtctgcttatggacctagacatcaacatgcgcacggtcgactg
cttctacactgtcaccttctatggtgccctcttcagagagggggatgtatggatctgcatggagctcatggacactt
ccctggataagttctaccggaaggtgctggagaagaacatgaaaattccggaagacatcctgggggagatcgctgtg
tctatcgtgcgggccctggagcacctgcatagcaagctgtctgtgatccacagagatgtgaagccatccaatgtcct
catcaacaaggaagggcatgtgaagatgtgcgactttggcatcagtggctacctggtggactctgtggcaaagacaa
tggatgctggctgcaagccttacatggcccctgagaggatcaaccctgaactgaatcagaagggctacaatgtcaag
tctgatgtctggagcctcggcatcaccatgatcgagatggccattctgcgattcccttatgagtcttggggcacacc
gttccagcagctgaagcaggtggtggaggagccatccccacagctcccagcggaccagttctcccctgagtttgtgg
acttcactagccagtgcctaaggaagaaccctgcagagcgcatgagctacctggagctgatggaacacccattcttc
accttgcacaaaactaagaagacagacattgctgcctttgtgaaggagatcctgggagaggattcatag(SEQ ID
NO:3) wherein, nucleotides sequence shown in SEQ ID NO:4 is classified as the sequence of the encoding gene of Mkk6 albumen, particularly as follows:
atgtctcagtcgaaaggcaagaagcgaaacccgggccttaagattccaaaagaagcgtttgaacagcctcagaccag
ttccacgccgcctcgggatttagactccaaggcttgcatatctattggaaaccagaactttgaggtgaaggcagatg
acctggagccgatagtggagctgggacgaggtgcgtacggggtggtggagaagatgcgtcacgtgcccagcgggcag
atcatggcagtgaagcggatacgggccacagttaatagccaggaacagaaacggctgctgatggatttggatgtctc
catgaggacggtggactgtccattcaccgtgaccttctacggtgcactcttccgggagggcgacgtgtggatctgca
tggagctcatggatacgtcactagataaattctacaaacaagttattgataaaggccaaacaattccagaggatatc
ttaggaaagatagcagtttctattgtaaaagcgttagaacatttacacagtaagctgtctgttatccatcgagacgt
caagccttctaatgtgctcattaacacactgggccaggtgaagatgtgtgactttggaatcagtggctacctggtcg
actctgttgctaaaacgatcgatgccggttgcaaaccatacatggctcctgaacgaataaatccagagctcaaccag
aaggggtacagtgtgaagtctgacatttggagcctgggcatcaccatgatcgagctggccatccttcggtttcctta
tgattcttggggaacgcccttccagcagctaaagcaggtggtcgaagagccctctcctcagctcccagcagacaagt
tctccgcggactttgttgactttacctcacagtgcttgaagaaaaattccaaagaacggcccacatatccagagctt
atgcaacatccatttttcaccgtacatgaatccaaagcagcagacgtggcatcttttgtaaaactgatacttgggga
Ctaa (SEQ ID NO:4) inventor finds, introduces above-mentioned nucleic acid molecule and can make cell high-efficient ground process LAN in cell
Mkk3 or Mkk6 albumen, and then the raising of acetylation of histone level can be effectively facilitated, thus improve the table of versatility gene
Reach, and then can effectively change cell fate and especially promote somatic reprogramming.
According to the second aspect of the invention, the invention provides a kind of construct, including the first nucleic acid molecules, described first
Nucleic acid molecule encoding has the protein selected from one of following amino acid sequences: (a) SEQ ID NO:1 or 2 at least one institute
The aminoacid sequence shown；(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；(c) (a) with
B shown in (), aminoacid sequence has at least 80%, and preferably at least 90%, more preferably at least 95%, the most at least
The aminoacid sequence of 99% sequence iden.It is surprisingly found by the inventors that, the construct utilizing the present invention can be effectively by upper
The related gene stating coding Mkk3 or Mkk6 albumen introduces recipient cell, and makes recipient cell success process LAN Mkk3 or Mkk6 egg
In vain, and then the acetylation of histone of versatility gene promoter area can be effectively facilitated, improve the expression of versatility gene, thus
Can effectively change cell fate and especially promote somatic reprogramming.
According to embodiments of the invention, above-mentioned construct can further include following additional technical feature at least it
One:
According to embodiments of the invention, described first nucleic acid molecules is to have the nucleoside selected from one of following nucleotide sequences
Acid molecule: (a) SEQ ID NO:3 or 4 at least one shown in nucleotide sequence；(b) SEQ ID NO:3 or 4 at least it
A part for nucleotide sequence shown in one；C () has at least 80%, preferably at least with nucleotide sequence shown in (a) and (b)
90%, more preferably at least 95%, the nucleotide sequence of further preferably at least 99% sequence iden；(d) under high stringency conditions,
Can with nucleotide sequence shown in (a) or (b) at least one or the nucleotide sequence of its complementary sequence hybridization.Invention Crinis Carbonisatus
Existing, utilize this construct effectively the related gene of above-mentioned coding Mkk3 or Mkk6 albumen can be introduced recipient cell, and make
Recipient cell process LAN Mkk3 efficiently or Mkk6 albumen, it is possible to effectively facilitate the histone acetyl of versatility gene promoter area
Change, improve the expression of versatility gene, and then can effectively change cell fate and especially promote somatic reprogramming.
According to embodiments of the invention, described construct is selected from plasmid, phage, artificial chromosome, cosmid and virus
The form of at least one.The form of construct is not particularly limited.Optionally, construct is the form of plasmid.Plasmid conduct
Heredity carrier, has simple to operate, can carry the character of larger piece section, it is simple to operates and processes.The form of plasmid is not subject to
Limit especially, both can be circular plasmids, it is also possible to be linear plasmid, and can be i.e. strand, it is also possible to be double-strand.Ability
Field technique personnel can select as required.
According to embodiments of the invention, described construct is for converting process LAN Sox, Klf4, Oct4 and c-Myc's in advance
The cell of at least one, optionally, described cell simultaneously process LAN Sox, Klf4 and Oct4 in advance.Inventor finds, with existing
Technology is compared, in advance at least one of process LAN Sox, Klf4, Oct4 and c-Myc, on this basis process LAN Mkk3 or Mkk6,
The acetylation of histone of versatility gene promoter area can be remarkably promoted, thus it is open to greatly promote chromatin, significantly improves
The expression of versatility gene such that it is able to more efficiently change cell fate and especially promote somatic reprogramming.
According to the third aspect of the invention we, the invention provides a kind of side for regulating cellular histone acetylation degree
Method, it is characterised in that the method includes changing the expression of predetermined protein in described cell, described predetermined protein has
Following amino acid sequences at least one: (a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；(b)SEQ
ID NO:1 or 2 at least one shown in the part of aminoacid sequence；C () (a) has with aminoacid sequence shown in (b)
At least 80%, preferably at least 90%, more preferably at least 95%, the aminoacid sequence of further preferably at least 99% sequence iden
Row, optionally, described regulation cellular histone acetylation degree is for improving cellular histone acetylation degree.According to the present invention's
Embodiment, improves cellular histone acetylation degree and refers to improve the acetylation of histone degree of versatility gene promoter area,
Thus improve the expression of versatility gene, and then can effectively change cell fate and especially promote somatic reprogramming.
According to the fourth aspect of the invention, the invention provides a kind of for change cells pluripotency gene Oct4,
The method of the expressions such as Nanog, Sox2 and Rex1.The method includes the expression water changing predetermined protein in described cell
Flat, described predetermined protein have following amino acid sequences at least one: (a) SEQ ID NO:1 or 2 at least one shown in
Aminoacid sequence；(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；(c) and (a) and
B shown in (), aminoacid sequence has at least 80%, and preferably at least 90%, more preferably at least 95%, the most at least
The aminoacid sequence of 99% sequence iden, optionally, described change cells pluripotency gene Oct4, Nanog, Sox2 and Rex1
Expression is the expression improving cells pluripotency gene Oct4, Nanog, Sox2 and Rex1.Wherein it is desired to explanation
It is that the kind of versatility gene is unrestricted, as long as the expression of versatility gene can be changed i.e. by the method for the present invention
Can, such as Oct4, Nanog and Rex1 gene.
According to the fifth aspect of the invention, the invention provides one for changing cell heterochromatin protein HP1a carefully
Expression or the method for location in born of the same parents.According to embodiments of the invention, the method includes changing predetermined protein in described cell
Expression, described predetermined protein have following amino acid sequences at least one: (a) SEQ ID NO:1 or 2 is at least
Aminoacid sequence shown in one of；(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；(c)
At least 80% is had with aminoacid sequence shown in (a) or (b), preferably at least 90%, more preferably at least 95%, further preferably
The aminoacid sequence of at least 99% sequence iden.The heterochromatin protein HP1a transcriptional control for gene, the silence of telomere
It is closely related with capping etc..According to embodiments of the invention, cell process LAN Mkk3 or Mkk6 albumen, it is possible to effectively change cell
Heterochromatin protein HP1a expression in cell and location, thus affect the transcriptional control of multipotency gene, improve versatility
The expression of gene, and then can effectively change cell fate and especially promote somatic reprogramming.
According to the sixth aspect of the invention, the invention provides a kind of side for changing cellular histone H1 mobility
Method, the method includes changing the expression of predetermined protein in described cell, and described predetermined protein has following aminoacid
Sequence at least one: (a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；(b) SEQ ID NO:1 or 2
At least one shown in the part of aminoacid sequence；C () has at least 80% with aminoacid sequence shown in (a) or (b),
Preferably at least 90%, more preferably at least 95%, the aminoacid sequence of further preferably at least 99% sequence iden, optionally,
Described change cellular histone H1 mobility is the mobility improving cellular histone H1.H1 then DNA with between nucleosome is combined.
It is surprisingly found by the inventors that, improve the mobility of cellular histone H1, be conducive to opening chromatinic structure, promote multipotency gene
Expression, and then can effectively change cell fate and especially promote somatic reprogramming.
According to embodiments of the invention, said method can further include at least one following additional technical feature:
According to embodiments of the invention, at least one of above-mentioned cell process LAN Sox, Klf4, Oct4 and c-Myc in advance,
Optionally, described cell simultaneously process LAN Sox, Klf4 and Oct4 in advance.Compared with prior art, in advance process LAN Sox,
At least one of Klf4, Oct4 and c-Myc, on this basis process LAN Mkk3 or Mkk6, can remarkably promote versatility gene
The acetylation of histone of promoter region, significantly changes cell heterochromatin protein HP1a expression or location in cell, significantly
Change cellular histone H1 mobility, thus it is open to greatly promote chromatin such that it is able to more efficiently change cell fate
Especially promote somatic reprogramming.
According to embodiments of the invention, changing the expression of predetermined protein in described cell is by described in process LAN
Predetermined protein in cell realizes, process LAN Mkk3 or Mkk6, it is possible to effectively changes cell fate and especially promotes body
The reprogramming of cell.
According to embodiments of the invention, process LAN realizes by introducing construct in described cell.Construct is made
For heredity carrier, the nucleic acid molecules of coding Mkk3 or Mkk6 is imported host cell, thus realizes process LAN in host cell
Mkk3 or Mkk6, thus change cell fate, promote somatic reprogramming.
According to the seventh aspect of the invention, present invention also offers a kind of reconstitution cell, compared with wild-type cell, described
In reconstitution cell, the expression of predetermined protein is changed, described predetermined protein have following amino acid sequences at least it
One: (a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；(b) SEQ ID NO:1 or 2 at least one institute
A part for the aminoacid sequence shown；C () (a) has at least 80% with aminoacid sequence shown in (b), and preferably at least 90%,
More preferably at least 95%, the aminoacid sequence of further preferably at least 99% sequence iden.The cell of the present invention can cross table
Reaching Mkk3 or Mkk6 albumen, reprogramming efficiency is high such that it is able to act effectively as the model of research cell fate change.
According to embodiments of the invention, above-mentioned reconstitution cell can further include following additional technical feature at least it
One:
According to embodiments of the invention, above-mentioned reconstitution cell process LAN Sox, Klf4, Oct4 and c-Myc's the most in advance
At least one, optionally, described cell simultaneously process LAN Sox, Klf4 and Oct4 in advance.Compared with prior art,
At least one of process LAN Sox, Klf4, Oct4 and c-Myc, on this basis process LAN Mkk3 or Mkk6, reconstitution cell in advance
Reprogramming efficiency significantly improve, it is possible to act effectively as research cell fate change model.
According to embodiments of the invention, in reconstitution cell, the expression of predetermined protein is changed is by process LAN institute
State what predetermined albumen realized.Process LAN Mkk3 or Mkk6, the reprogramming efficiency of reconstitution cell significantly improves, it is possible to acts effectively as and grinds
Study carefully the model of cell fate change
According to embodiments of the invention, above-mentioned process LAN realizes by introducing construct in described cell.Build
The nucleic acid molecules of coding Mkk3 or Mkk6, as heredity carrier, is imported host cell, thus realizes crossing table at host cell by body
Reach Mkk3 or Mkk6, form above-mentioned reconstitution cell.The acetylation of histone degree of reconstitution cell versatility gene promoter area is high,
Cell heterochromatin protein HP1a expression or location in cell are changed, and cellular histone H1 mobility is high, so that
The expression of the multipotency gene of reconstitution cell is high, and reconstitution cell reprogramming efficiency significantly improves.
It should be noted that inventor finds, it is extremely inefficient that independent process LAN Mkk3 or Mkk6 makes that cell reprograms,
Only process LAN Mkk3 or Mkk6 on the basis of at least one of Sox, Klf4, Oct4 and c-Myc at process LAN in advance,
Can make cell that above-mentioned change occurs.The term " high stringency conditions " used in this article refers to form specific hybrid and non-shape
Become the condition of non-specific hybrids.Typical high stringency conditions such as can be enumerated at potassium concn about 25mmol/L～about
In 50mmol/L and magnesium density about 1.0mmol/L～about 5.0mmol/L, carry out the condition hybridized.Condition as the present invention
Example, can enumerate at Tris-HCl (pH8.6), the MgCl of KCl and 1.5mmol/L of 25mmol/L2Under hybridize
Condition, but be not limited to this.Additionally, high stringency conditions is documented in Molecular Cloning 3rd (J.Sambrook et
Al., Cold Spring Harbor Lab.Press, 2001) in.The document is by referring to being incorporated to this specification.This area skill
Art personnel can be readily selected above-mentioned condition by changing hybridization, the salinity etc. of hybridization reaction solution.
It addition, described in description: " homogeneity " is known in the art, refers between two or more nucleotide sequence
The relation determined after gene comparision；In the art, " homogeneity " also refers to the sequence degree of correlation between sequence of nucleic acid molecules,
Determined by the Mismatching degree of these sequences；" homogeneity " can be easily computed according to known method, these methods
Including, but not limited to be described in Computer Molecular Biology, Lesk edits, and Oxford University publishes, New York
1993；Biocomputing:Infornatics and Genome Projects, Smith edits, and Academic publishes, New York
1988；Computer Analysis of Sequence Data, Part I, Griffin and Griffin edits, Humana
Publish, New Jersey, 1994；Sequence Analysis in Molecular Biology, von Heinje, Academic go out
Version 1987；Sequence Analysis Primer, Gribskov and Devereux edit, and Stockton publishes, New York 1991；
With Carillo and Lipman, SIAM J, applied mathematics, 48:1073,1988.).
Finally, it should be noted that the present invention's changes the method that cell fate especially promotes somatic reprogramming
It is that present inventor just completes through arduous creative work and continuous Optimization Work.
Accompanying drawing explanation
Fig. 1 is the result schematic diagram that Mkk3 or Mkk6 albumen according to embodiments of the present invention promotes reprogramming experiment；
Fig. 2 is the cells pluripotency gene promoter during Mkk6 albumen according to embodiments of the present invention improves reprogramming
The experimental result schematic diagram of district's acetylation of histone level；
Fig. 3 is that Mkk6 albumen according to embodiments of the present invention improves Oct4, Nanog, Sox2 and Rex1 versatility gene
The experimental result picture of expression；
Fig. 4 is that Mkk6 albumen according to embodiments of the present invention changes heterochromatin protein HP1a expression and the experiment of location
Result figure；And
Fig. 5 is the experimental result picture that Mkk6 albumen according to embodiments of the present invention improves histone h1 mobility.
Detailed description of the invention
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete technology or bar in embodiment
Part, according to the technology described by the document in this area or condition, (such as writing with reference to J. Pehanorm Brooker etc., yellow training hall etc. is translated
" Molecular Cloning: A Laboratory guide ", the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Unreceipted production firm person, be can by city available from conventional products, such as can purchase from Illumina company.
Below in an example, retrovirus package carrier pMXs is selected.
" cell " specifically described herein is any cell that can obtain, and below in an example, selects mice embryonic
Fibroblast (mouse embryo fibroblast cell, MEF cell).
Can be transfected or infection cell by any means known in the art, below in an example, select to reverse
Record virus infected cell.
The impact that cell fate is changed by various methods known in the art detection protein can be used.In following reality
Executing in example, Chromatin immunoprecipitation assay can be used to study Mkk3 or Mkk6 and c-Myc be attached to the shadow of Oct4 promoter level
Ring；Fluorescent quantitative PCR technique can be used to the impact studying Mkk3 or Mkk6 to versatility gene expression doses such as Oct4.
Embodiment 1:Mkk3 or the clone of Mkk6 gene
With the cDNA of mouse embryo fibroblast (MEF) cell as template, utilize primer amplification Mkk3 or Mkk6 shown in table 1
Segment, then reclaims with agarose gel electrophoresis.By inserting PCR sheet after the multiple clone site PmeI enzyme action of pMXs carrier
Section, connects, and converts, chooses bacterium, and order-checking finally obtains the retrovirus packaging plasmid of Mkk3 or Mkk6.
The primer of Mkk3 or Mkk6 protein fragments cloned by table 1
Embodiment 2: process LAN Mkk3 or Mkk6 albumen promote reprogramming experiment
Pack out reprogramming factor S ox2 with single preferendum (Plat-E) Retronituse encapsulated cell line, Klf4, Oct4 and
The retrovirus of c-Myc and Mkk3 or Mkk6.
Detailed process is as follows: Plat-E cell is inoculated in culture dish 12 hours or overnight can transfect, and sees before transfection
Examining cell density, cell density can transfect at 70%-90%.During transfection, first DNA and OPTI-MEM is added, mixing, mix mutually
Closing and add PEI in liquid, the most reverse mixing, mixed liquor stands 13-15 minute, is finally joined by mixed liquor and change liquid
In Plat-E cell, mixing, afterwards cell is put into incubator and cultivates.After 8-10 hour, change MEF to Plat-E cell and cultivate
Base.By syringe collecting virus, with the filter of 0.45 micron by virus filtration to centrifuge tube, and add in centrifuge tube in right amount
Fresh MEF culture medium (general 12 milliliters of the cell culture that diameter is 10 centimetres), add polybrene (polybrene) mixing,
Infect MEF (4 DEG C can also be saved in by collecting virus, standby) first.Again after 24 hours, collect virus by same method,
Superinfection MEF.Every subinfection 24 hours.
Mouse embryo fibroblast (MEF) cell after superinfection is used mESC inducing culture instead and is cultivated, wherein,
The basal medium of mESC inducing culture is DMEM in high glucose (wherein the content of glucose sugar is 4500mg/L), and mESC induction
Containing 15 volume %FBS in culture medium, 1mM Sodium Pyruvate, the nonessential aminoacid (NEAA) of 100 times of dilutions, 100 times of dilutions
The l-glutamine (200mM, purchased from GlutaMax) of stable state, the dual anti-of 100 times of dilutions (wherein contain 10000units/ml
Penicillin and 10000ug/ml streptomycin) and the murine interleukin inhibitive factor (Lif) of 1000units/ml restructuring.Afterwards, often
It changes fresh mESC inducing culture, until occurring after 12-15 days that the cell experiment of colony morphology terminates.
(endogenous Oct4 starts to use OG2-MEF cell (from Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health)
On son, coupling has GFP albumen), measure the efficiency of the multipotency differentiation of the inducing cell obtained in above-mentioned experiment.When iPS clone has
During GFP fluorescence, show that endogenous Oct4 promoter is expressed, be the mark successfully reprogrammed.Utilize that to count GFP under microscope glimmering
The clone that light is positive, is the induced efficiency of iPS.At four factor S KOM (reprogramming factor S ox2, Klf4, Oct4 and c-Myc)
On the basis of individually process LAN Mkk3 or Mkk6 albumen, Mkk3 or Mkk6 albumen promotes the result of reprogramming experiment as shown in Figure 1.Its
In, Flag group is to have infected the comparison MEF cell of empty carrier, Mkk3 or Mkk6 group is to have infected the MEF of Mkk3 or Mkk6 virus
Cell, then statistics GFP positive colony quantity.
Fig. 1 result shows: the GFP positive colony number of Mkk3 or Mkk6 group is about 2.5 times of Flag group, show when four because of
On the basis of sub-SKOM, infect Mkk3 or Mkk6 and can be greatly increased GFP positive colony quantity, show the base in four factor S KOM
On plinth, process LAN Mkk3 or Mkk6 can significantly improve the reprogramming efficiency of reconstitution cell.
Embodiment 3:Mkk3 or the Mkk6 impact on versatility gene promoter area acetylation of histone level
Analyze Mkk3 or Mkk6 on when affecting of versatility gene promoter area Acetylation Level, first packaging Mkk3 or
The retrovirus of Mkk6, infecting mouse embryo fibroblast (MEF) cell, wherein it is desired to explanation, any MEF is all can
The recipient cell that virus infects, and process LAN SKO (reprogramming factor S ox2, Klf4, Oct4) the most in advance.Collect as comparison
The MEF cell having infected empty carrier and express Mkk3 or Mkk6 virus MEF cell, fix with the formaldehyde of final concentration 1%, then
Terminate with the glycine of final concentration 0.125M.After PBS washes twice, with appropriate PBS re-suspended cell, divide by often pipe 10,000,000 cell
Dress.It is centrifuged and removes supernatant.Cell precipitation is placed on ice, adds 1 milliliter of lysis buffer A, wherein, the composition of lysis buffer A
As follows: 50mM HEPES-KOH pH7.3；140mM NaCl；1mM EDTA pH8.0；10%glycerol；0.5%NP-40；
0.25%Triton-100；Protease inhibitor cocktail, after cracking 10 minutes on ice, 1400g 4 DEG C centrifugal 5
Minute.Adding 200 Al lysis buffer B after removing supernatant, wherein, the composition of lysis buffer B is as follows: 1%SDS；50mM
Tris-HCl pH8.0；10mM EDTA；Protease inhibitor cocktail, on ice cracking 10 minutes.Ultrasonic, by base
Because group is broken into the sample about segment 400-500bp.
Take protein A and protein G magnetic bead (Life technologies, the 26162) mixing of proper volume, take
Go out after preserving liquid, resuspended with 200 microlitre PBST, add acetylation of histone antibody and each 1 μ g of IgG such as H3K9Ac or H4K16Ac,
4 DEG C of rotations combine 3 hours.Magnetic bead is washed with PBST.Above-mentioned ultrasonic sample IP (immunoprecipitation) buffer is diluted 10 times, takes 10
J diluent does Input comparison, takes 400 j diluent and joins magnetic bead, and 4 DEG C rotate combination overnight.Within second day, use less salt
Cleanout fluid, high salt cleanout fluid, LiCl cleanout fluid and TE solution wash magnetic bead.It is eventually adding the Chelex-100 solution and 1 of 200 microlitres
Micro L protease, 56 DEG C of 1100g react half an hour, and boiling water bath 10 minutes, centrifuging and taking supernatant, supernatant can make quantitative fluorescent PCR
(QPCR) template.Finally combine promoter region DNA feelings with the histone of quantitative fluorescent PCR (QPCR) method detection acetylation modification
Condition.
Result shows, Mkk3 or Mkk6 albumen can significantly improve versatility gene promoter area acetylation of histone journey
Degree.
Wherein, result such as Fig. 2 of the experiment of Mkk6 albumen raising versatility gene promoter area acetylation of histone degree
Shown in.Fig. 2 result shows, in Flag group, i.e. in the MEF cell having infected empty vector control, acetylizad histone combines
The promoter region sequence arrived is less, and in Mkk6 experimental group, the promoter region sequence that acetylizad histone is attached to is more.
It can thus be seen that Mkk6 albumen improves versatility gene promoter area acetylation of histone degree.
The impact of the versatility gene expression doses such as Oct4 is tested by embodiment 4:Mkk3 or Mkk6
Collect and infected empty vector control and the MEF cell of Mkk3 or Mkk6 virus, be SKOM+Flag group and SKOM respectively
+ Mkk3 or Mkk6 group, add Trizol reagent cell lysis, six 1 milliliter of the every holes of orifice plate.Careful piping and druming, until cell quilt used
Cracking, is sucked in EP pipe, is placed in-80 DEG C of preservations.When RNA extracts, it is put thawed on ice from-80 DEG C of taking-ups.Add 200 micro-
Rise chloroform, reverse mixing, centrifugal 15 minutes of 13000g 4 DEG C.Take supernatant, supernatant is transferred in new EP pipe, add
400 microliters isopropanol, reverse mixing.Room temperature stands 10 minutes, and 13000g4 DEG C is centrifuged 15 minutes, removes supernatant, stay precipitation.Precipitation
Wash once with 70% ethanol.Air is dried 10-20 minute, adds appropriate RNAse-Free water, and 55 DEG C dissolve 20 minutes, measure
Concentration.It is template that reverse transcription system takes the RNA of appropriate quality, carries out reverse transcription with Oligo-dT for primer.Reverse transcription is obtained
CDNA sample with distilled water dilute 50-200 times.The upstream and downstream primer of QPCR is diluted to together, final concentration of 2.5 μMs, its
In, primer sequence is as follows:
Forward primer: CTGTAAGGACAGGCCGAGAG (SEQ ID NO:9)
Downstream primer: CAGGAGGCCTTCATTTTCAA (SEQ ID NO:10)
According to QPCR test kit (Bio-rad, SsoAdvancedTM Universal Green Supermix-
Explanation operation 172-5272).
Test result indicate that, Mkk3 or Mkk6 can significantly improve the table of Oct4, Nanog, Sox2 and Rex1 versatility gene
Reach level.
Wherein, Mkk6 improves result such as Fig. 3 institute of expression of Oct4, Nanog, Sox2 and Rex1 versatility gene
Show.Fig. 3 result shows, versatility gene relative expression's discharge curve is SKOM+Mkk6, SKOM+flag, SKO+ the most successively
In Mkk6, SKO+flag cell, the expression of multipotency gene, therefore deduces that, Mkk6 can significantly improve Oct4, Nanog, Sox2
Expression with Rex1 versatility gene.
The impact of heterochromatin protein HP1a is tested by embodiment 5:Mkk6
At analysis Mkk3 or Mkk6 on when affecting of heterochromatin protein HP1a, first pack the reverse transcription of Mkk3 or Mkk6
Virus, infects MEF (in the present embodiment, MEF cell does not has any gene of process LAN, is the primary MEF of wild type).Cultivate 3 days
After, wash one time with PBS, fix half an hour by 4% paraformaldehyde room temperature.Draw paraformaldehyde after Gu Ding, change PBS.Use sweet ammonia
Acid solution is washed one time, then washes 3 times with PBS, then with the penetrating closing of mixed solution 1 hour of Triton X 100 and BSA.PBS
One anti-(HP1a antibody) is hatched at least 1 hour after washing 3 times；Hatch fluorescence two after washing 5 times by washing liquid to resist.Finally contaminate cell with DAPI
Core, PBS washes one time, is enclosed on microscope slide by slide by mountant, then sees with laser confocal microscope (Zeiss 710)
Examine and take pictures.
Result shows, Mkk3 or Mkk6 albumen increases the expression in nucleus of the MEF cell HP1 α albumen and gathering.
Wherein, Mkk6 albumen increases result such as Fig. 4 institute of the expression in nucleus of the MEF cell HP1 α albumen and gathering
Show.Fig. 4 result shows, in the MEF cell having infected empty vector control, HP1a expression in nucleus is strong, forms one
Individual gathering；And in the MEF that Mkk6 infects, although in nucleus, also can form the gathering of some HP1a, but integral level comparison
It is remarkably decreased according to group.These results show that Mkk6 can affect the expression of HP1a albumen and the location in cell.
The impact of histone h1 mobility is tested by embodiment 6:Mkk3 or Mkk6
MEF cell is seeded in the glass bottom capsule with coated 35 millimeters of poly-D-lysine.Then fluorescent fusion is used
Protein G FP-Histone1 infects, and the virus then packed with Mkk3 or Mkk6 infects MEF, and (in the present embodiment, MEF cell does not has
Have any gene of process LAN, be the primary MEF of wild type), carry out FRAP detection after 3 days.
Select the cell that fluorescence intensity (referring to H1-GFP) is moderate, first take pictures as comparison, more selected region to be bleached:
The circle of one a diameter of 25 pixel, with this border circular areas of laser intensity transmission of one times or several times of normal laser irradiation value,
Bleaching green fluorescence.Then taking the image after a series of photobleaching (the most every 0.5 second), after general 2 minutes, fluorescence is strong
Degree tends towards stability, and stops taking pictures, and preserves image.
Result shows, Mkk3 or Mkk6 albumen can dramatically increase the mobility of heterochromatic H1.
Wherein, Mkk6 albumen in MEF cell the mobility of histone h1 affect experimental result as shown in Figure 5.Fig. 5 ties
Fruit display, Mkk6 can dramatically increase the mobility of heterochromatic H1, shows that Mkk6 albumen can open chromatinic structure.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although above it has been shown and described that embodiments of the invention, it is to be understood that above-described embodiment is example
Property, it is impossible to be interpreted as limitation of the present invention, those of ordinary skill in the art is without departing from the principle of the present invention and objective
In the case of above-described embodiment can be changed within the scope of the invention, revise, replace and modification.
Claims (20)
1. the method changing cell fate, it includes changing the expression of predetermined protein in described cell, described pre-
Determine protein have following amino acid sequences at least one:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () has at least 80% with aminoacid sequence shown in (a) and (b), and preferably at least 90%, more preferably at least 95%, enter
The aminoacid sequence of one step preferably at least 99% sequence iden.
Method the most according to claim 1, it is characterised in that described cell is somatic cell or stem cell.
3. according to the method described in claim 1, it is characterised in that the expression of predetermined protein in the described cell of described change
It is to be realized by the predetermined protein in cell described in process LAN.
Method the most according to claim 1, it is characterised in that farther include, described cell process LAN Sox in advance,
At least one of Klf4, Oct4 and c-Myc,
Optional, described cell process LAN Sox, Klf4 and Oct4 simultaneously in advance.
Method the most according to claim 1, it is characterised in that the expression water of predetermined protein in the described cell of described change
Flat is to be had by introducing in described cell to carry out selected from the nucleic acid molecule of one of following nucleotide sequences:
(a) SEQ ID NO:3 or 4 at least one shown in nucleotide sequence；
(b) SEQ ID NO:3 or 4 at least one shown in the part of nucleotide sequence；
C () has at least 80% with nucleotide sequence shown in (a) and (b), and preferably at least 90%, more preferably at least 95%, enter
The nucleotide sequence of one step preferably at least 99% sequence iden；
D () is under high stringency conditions, it is possible to nucleotide sequence shown in (a) or (b) at least one or its complementary sequence hybridization
Nucleotide sequence.
6. a construct, it is characterised in that include that the first nucleic acid molecules, described first nucleic acid molecule encoding have selected from following
The protein of one of aminoacid sequence:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () (a) has at least 80% with aminoacid sequence shown in (b), and preferably at least 90%, more preferably at least 95%, enter one
The aminoacid sequence of step preferably at least 99% sequence iden.
Construct the most according to claim 6, it is characterised in that described first nucleic acid molecules is to have selected from following nucleoside
The nucleic acid molecule of one of acid sequence:
(a) SEQ ID NO:3 or 4 at least one shown in nucleotide sequence；
(b) SEQ ID NO:3 or 4 at least one shown in the part of nucleotide sequence；
C () has at least 80% with nucleotide sequence shown in (a) and (b), and preferably at least 90%, more preferably at least 95%, enter
The nucleotide sequence of one step preferably at least 99% sequence iden；
D () is under high stringency conditions, it is possible to nucleotide sequence shown in (a) or (b) at least one or its complementary sequence hybridization
Nucleotide sequence.
Construct the most according to claim 7, it is characterised in that described construct is selected from plasmid, phage, artificially colored
The form of at least one of body, cosmid and virus.
Construct the most according to claim 7, it is characterised in that described construct be used for converting in advance process LAN Sox,
The cell of at least one of Klf4, Oct4 and c-Myc,
Optionally, described cell simultaneously process LAN Sox, Klf4 and Oct4 in advance.
10. one kind for the method that regulates cellular histone acetylation degree, it is characterised in that the method include changing described carefully
The expression of predetermined protein in born of the same parents, described predetermined protein have following amino acid sequences at least one:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () (a) has at least 80% with aminoacid sequence shown in (b), and preferably at least 90%, more preferably at least 95%, enter one
The aminoacid sequence of step preferably at least 99% sequence iden,
Optionally, described regulation cellular histone acetylation degree is for improving cellular histone acetylation degree.
11. 1 kinds are used for the method changing cells pluripotency gene Oct4, Nanog, Sox2 and Rex1 expression, and its feature exists
In, the method includes changing the expression of predetermined protein in described cell, and described predetermined protein has following aminoacid
At least one sequence:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () has at least 80% with aminoacid sequence shown in (a) and (b), and preferably at least 90%, more preferably at least 95%, enter
The aminoacid sequence of one step preferably at least 99% sequence iden,
Optionally, described change cells pluripotency gene Oct4, Nanog, Sox2 and Rex1 expression is for improving cell multipotency
The expression of property gene Oct4, Nanog, Sox2 and Rex1.
12. 1 kinds for changing cell heterochromatin protein HP1a expression or method of location in cell, it is characterised in that
The method includes changing the expression of predetermined protein in described cell, and described predetermined protein has following amino acid sequences
At least one:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () has at least 80% with aminoacid sequence shown in (a) or (b), and preferably at least 90%, more preferably at least 95%, enter
The aminoacid sequence of one step preferably at least 99% sequence iden.
13. 1 kinds for the method changing cellular histone H1 mobility, it is characterised in that the method includes changing described cell
The expression of middle predetermined protein, described predetermined protein have following amino acid sequences at least one:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () has at least 80% with aminoacid sequence shown in (a) or (b), and preferably at least 90%, more preferably at least 95%, enter
The aminoacid sequence of one step preferably at least 99% sequence iden,
Optionally, described change cellular histone H1 mobility is the mobility improving cellular histone H1.
14. according to the method described in any one of claim 10～13, it is characterised in that described cell process LAN Sox in advance,
At least one of Klf4, Oct4 and c-Myc,
Optionally, described cell simultaneously process LAN Sox, Klf4 and Oct4 in advance.
15. according to the method described in any one of claim 10～13, it is characterised in that predetermined egg in the described cell of described change
The expression of white matter is to be realized by the predetermined protein in cell described in process LAN.
16. methods according to claim 15, farther include, and described process LAN is by introducing power in described cell
Profit requires what the construct described in 7～9 any one realized.
17. 1 kinds of reconstitution cells, it is characterised in that compared with wild-type cell, the table of predetermined protein in described reconstitution cell
The level of reaching is changed, described predetermined protein have following amino acid sequences at least one:
(a) SEQ ID NO:1 or 2 at least one shown in aminoacid sequence；
(b) SEQ ID NO:1 or 2 at least one shown in the part of aminoacid sequence；
C () (a) has at least 80% with aminoacid sequence shown in (b), and preferably at least 90%, more preferably at least 95%, enter one
The aminoacid sequence of step preferably at least 99% sequence iden.
18. reconstitution cells according to claim 17, it is characterised in that described reconstitution cell process LAN the most in advance
At least one of Sox, Klf4, Oct4 and c-Myc,
Optionally, described cell simultaneously process LAN Sox, Klf4 and Oct4 in advance.
19. reconstitution cells according to claim 17, it is characterised in that the expression of predetermined protein in described reconstitution cell
Level is changed and is realized by predetermined albumen described in process LAN.
20. methods according to claim 19, farther include, and described process LAN is by introducing power in described cell
Profit requires what the construct described in 7～9 any one realized.
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| CN107286232A (en) * | 2017-05-23 | 2017-10-24 | 中国科学院广州生物医药与健康研究院 | Efficient induction of the POU domains engineered protein to multipotential stem cell |
| CN110029086A (en) * | 2019-03-13 | 2019-07-19 | 中国科学院广州生物医药与健康研究院 | The factor of inducing somatic reprogramming and the method reprogrammed using the factor inducing somatic |
| CN112063656A (en) * | 2020-08-28 | 2020-12-11 | 中国科学院广州生物医药与健康研究院 | Use of Map2k3 or Map2k6 for improving efficiency of inducing adult cells to generate pluripotent stem cells |
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| CN110029086A (en) * | 2019-03-13 | 2019-07-19 | 中国科学院广州生物医药与健康研究院 | The factor of inducing somatic reprogramming and the method reprogrammed using the factor inducing somatic |
| CN112063656A (en) * | 2020-08-28 | 2020-12-11 | 中国科学院广州生物医药与健康研究院 | Use of Map2k3 or Map2k6 for improving efficiency of inducing adult cells to generate pluripotent stem cells |
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