Method for expressing protein gene in response to expression of miRNA
11111503 · 2021-09-07
Assignee
Inventors
Cpc classification
C12N15/67
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
International classification
C12N5/10
CHEMISTRY; METALLURGY
Abstract
An mRNA forcibly expresses a protein gene in response to a miRNA, and a method for forcibly expressing the same, are provided. An artificial mRNA comprising a sequence encoding a protein gene, a miRNA target sequence linked to the 3′-terminal side of a Poly A sequence, and a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence; and a method for expressing a protein gene in response to the expression of a miRNA, comprising a step of introducing the artificial mRNA into a cell.
Claims
1. An artificial mRNA comprising: a sequence encoding a protein, a miRNA target sequence linked to the 3′-terminal side of a Poly A sequence of at least 50 nucleotides, and a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence.
2. The artificial mRNA according to claim 1, wherein the translational repression sequence comprises a sequence selected from (i) a nucleotide sequence consisting of 20 or more nucleotides that binds the Poly A sequence in an intracellular environment, (ii) a sequence that binds a 5′ UTR in the artificial mRNA in an intracellular environment, and (iii) a sequence consisting of 100 or more nucleotides.
3. A method for expressing a protein in response to the expression of a miRNA, which comprises a step of introducing the artificial mRNA according to claim 1 into a cell.
4. A method for determining a desired cell type from a cell group comprising two or more cells, using the expression of a miRNA as an indicator, the method comprising the following steps: (1) a step of introducing a first artificial mRNA comprising a sequence encoding a first marker, a first miRNA target sequence linked to the 3′-terminal side of a Poly A sequence of at least 50 nucleotides, and a translational repression sequence linked to the 3′-terminal side of the first miRNA target sequence, into the cell group; and (2) a step of determining the cell type, using the translation level of the first marker as an indicator.
5. The method according to claim 4, wherein the translational repression sequence comprises a sequence selected from (i) a nucleotide sequence consisting of 20 or more nucleotides that binds the Poly A sequence in an intracellular environment, (ii) a sequence that binds a 5′ UTR of the first artificial mRNA in an intracellular environment, and (iii) a sequence consisting of 100 or more nucleotides.
6. The method according to claim 5, wherein the desired cell type is a cell type in which the expression level of the first miRNA greater than in other cell types in the cell group, and the step (2) is a step of determining a cell type in which the translation level of the first marker is greater than in other cell types in the cell group.
7. The method according to claim 4, wherein the first miRNA target sequence comprises a target sequence of miR-302a, and the desired cell type is a pluripotent stem cell.
8. The method according to claim 4, which further comprises: (3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA in the cell group.
9. The method according to claim 8, which further comprises: (4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the mi RNA in the cell group.
10. A method for expressing a protein in response to the expression of a miRNA, which comprises a step of introducing the artificial mRNA according to claim 2 into a cell.
11. The method according to claim 5, wherein the first miRNA target sequence comprises a target sequence of miR-302a, and the desired cell type is a pluripotent stem cell.
12. The method according to claim 6, wherein the first miRNA target sequence comprises a target sequence of miR-302a, and the desired cell type is a pluripotent stem cell.
13. The method according to claim 5, which further comprises: (3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA.
14. The method according to claim 6, which further comprises: (3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA in the cell group.
15. The method according to claim 7, which further comprises: (3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA in the cell group.
16. The method according to claim 5, which further comprises: (4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.
17. The method according to claim 6, which further comprises: (4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.
18. The method according to claim 7, which further comprises: (4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.
19. The method according to claim 8, which further comprises: (4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4) a 20-nt complementary sequence to 5′ UTR in the panel (a),
(5) a 40-nt complementary sequence to 5′ UTR in the panel (b),
(6) 20 nt+stem loop in the panel (c),
(7) an additional sequence consisting of approximately 120 nt in the panel (d),
(8) an additional sequence consisting of approximately 500 nt in the panel (e), and
(9) an additional sequence consisting of approximately 1250 nt in the panel (f).
(10)
(11)
(12)
DESCRIPTION OF EMBODIMENTS
(13) Hereinafter, the present invention will be described in detail in the following embodiments. However, the following embodiments are not intended to limit the scope of the present invention.
First Embodiment
(14) According to a first embodiment, the present invention relates to a method for expressing a protein gene in response to the expression of a miRNA, wherein the method comprises a step of introducing an artificial mRNA comprising a sequence encoding a protein gene, a miRNA target sequence linked to the 3′-terminal side of a Poly A sequence, and a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence into cells.
(15) In the present embodiment, as described in detail below, by designing and preparing an artificial mRNA, any given protein can be expressed in response to the expression of any given miRNA, in cells into which the artificial mRNA has been introduced. In the present description, the present artificial mRNA may be referred to as “miRNA-responsive mRNA” or “miRNA-responsive ON-switch mRNA” in some cases.
(16) In the present invention, miRNA is also referred to as microRNA, and it is an RNA 18 to 25 nucleotides in length, which is present in a cell. miRNA means either one strand of a double-stranded RNA generated by cleaving with Dicer, pre-miRNA generated by partially cleaving a pri-mRNA that is a single-stranded RNA transcribed from DNA with an intranuclear enzyme called Drosha. The number of nucleotides constituting a miRNA is, for example, 18 to 25, preferably 20 to 25, and more preferably 21 to 23. A database that stores information of approximately 1,000 miRNAs can be utilized (for example, miRBase). One skilled in the art could extract any given miRNA information from this database, and could readily extract a miRNA that is specifically expressed in cells expressing a protein gene by using the method of the present invention. In addition, the expression of a miRNA means that a miRNA is in a state in which either one strand of the double-stranded RNA cleaved with the aforementioned Dicer interacts with a plurality of predetermined proteins to form an RNA-induced silencing complex (RISK), in cells into which the mRNA according to the present invention is introduced.
(17) In the present invention, for example, a protein gene encoded by the above-described mRNA can be forcibly expressed cell-specifically. The miRNAs specifically expressed in certain cells have been known from the above-described database, publications, etc., and the elucidation thereof is even now continuing. Accordingly, upon designing the above-described mRNA, it is preferable to extract and select a miRNA that is specifically expressed in cells desirably expressing a protein gene.
(18) The cells according to the present embodiment are not particularly limited, as long as they are cells that desirably express a protein gene. Examples of such cells include pluripotent stem cells including induced pluripotent stem (iPS) cells, somatic cells induced to differentiate from the pluripotent stem cells, cells in a process of being induced to differentiate from the pluripotent stem cells, and cancer cells, but are not limited thereto.
(19) When cells into which an mRNA is to be introduced are, for example, pluripotent stem cells, an mRNA that responds to miRNA specifically expressed in pluripotent stem cells can be designed. Such a miRNA specifically expressed in pluripotent stem cells is not particularly limited, as long as it is a miRNA that is known to be specifically expressed in pluripotent stem cells according to publications and the like. For example, such miRNA is either one strand of each of hsa-mir-302a, hsa-mir-302b, hsa-mir-302c, hsa-mir-302d, hsa-mir-367, hsa-5201, hsa-mir-92b, hsa-mir-106a, hsa-mir-18b, hsa-mir-20b, hsa-mir-19b-2, hsa-mir-92a-2, hsa-mir-363, hsa-mir-20a, hsa-mir-17, hsa-mir-18a, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-373, hsa-mir-330, hsa-mir-520c, hsa-mir-182, hsa-mir-183, hsa-mir-96, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-141, hsa-mir-200c, hsa-mir-27a, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-374a, hsa-mir-106b, hsa-mir-93, hsa-mir-25, hsa-mir-584, hsa-mir-374b, hsa-mir-21, hsa-mir-212, hsa-mir-371a, hsa-mir-371b, hsa-mir-372, hsa-mir-200b, hsa-mir-200a, and hsa-mir-429. Other than these, examples include miRNAs appropriately selected from the miRNAs described in Tobias S. Greve, et al., Annu. Rev. Cell Dev. Biol. 2013. 29: 213-239. The miRNA is preferably either one strand of has-mir-302a or hsa-mir-302b, and more preferably hsa-miR-302b-3p.
(20) In the present invention, the target sequence of a miRNA specifically expressed in cells means a sequence capable of specifically binding to the miRNA. For instance, the miRNA target sequence is preferably a sequence complementary to the miRNA specifically expressed in cells. Otherwise, as long as the miRNA target sequence is recognizable by the miRNA, it may also have a mismatch with the completely complementary sequence. The mismatch with the sequence completely complementary to the miRNA may be generally a mismatch, which is recognizable by the miRNA in a desired cell, and it is thought that there may be a mismatch of approximately 40% to 50% with regard to the original function of the cell in a living body. Such a mismatch is not particularly limited, and it is, for example, a mismatch of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or a mismatch of 1%, 5%, 10%, 20%, 30% or 40% of the entire recognition sequence. In addition, in particular, as with the miRNA target sequence on the mRNA contained in a cell, the target sequence may comprise a large number of mismatches in a portion other than a seed region, that is, in a region on the 5′-terminal side in the target sequence, which corresponds to approximately 16 nucleotides on the 3′-terminal side of a miRNA. The seed region may not have such mismatches, or may have a mismatch of 1 nucleotide, 2 nucleotide or 3 nucleotides.
(21) In the present invention, the protein gene encoded by the mRNA may be any given protein gene, and the type of the protein gene is not limited. For instance, the protein gene encoded by the mRNA may be a marker protein gene as described in detail below, or may also be an apoptosis-promoting protein gene, an apoptosis-suppressing protein gene, or a cell surface protein gene.
(22) The marker gene is an RNA sequence encoding any given marker protein, which is translated in a cell, functions as a marker, and enables extraction of differentiated cells, and this RNA sequence can also be referred to as a sequence corresponding to a marker protein. The protein, which is translated in a cell and is able to function as a marker, may be, for example, a protein which can be visualized by fluorescence, luminescence or color development, or by supporting such fluorescence, luminescence or color development, and can be quantified, a membrane-localized protein, or a drug resistance protein, and the like, but the type of the protein is not limited thereto.
(23) Examples of the fluorescent protein include: blue fluorescent proteins such as Sirius or EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, or CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g. hmAG1), ZsGreen, EmGFP, EGFP, GFP2, or HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, or mBanana; orange fluorescent proteins such as KusabiraOrange (e.g. hmKO2) or mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, or mStrawberry; and near infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g. hdKeimaRed), mRasberry, mPlum, or iRFP670, but examples of the fluorescent protein are not limited thereto.
(24) An example of the luminescent protein is aequorin, but examples are not limited thereto. In addition, examples of the protein supporting fluorescence, luminescence or color development include enzymes decomposing fluorescent, luminescent or color development precursors, such as luciferase, phosphatase, peroxidase, or β lactamase, but examples of the type of the protein is not limited thereto. In the present invention, when the substance supporting fluorescence, luminescence or color development is used as a marker, extraction of the differentiated cells is carried out by allowing a cell to come into contact with a corresponding precursor, or by introducing such a corresponding precursor into a cell.
(25) The membrane-localized protein is not particularly limited, as long as it is a membrane-localized protein that is not endogenously expressed in pluripotent stem cells. Examples of the membrane-localized protein include P-gp, MRP1, MRP2 (cMOAT), MRP3, MRP4, MRP5, MRP6, MDR2, and MDR3 proteins. In the present invention, since a membrane-localized protein translated from the introduced mRNA is used as an indicator, a membrane-localized protein that is not endogenously expressed in the target differentiated cells is more preferable. Examples of the drug resistance protein include antibiotic resistance proteins such as a kanamycin resistance protein, an ampicillin resistance protein, a puromycin resistance protein, a blasticidin resistance protein, a gentamycin resistance protein, a kanamycin resistance protein, a tetracycline resistance protein, and a chloramphenicol resistance protein, but are not limited thereto.
(26) Specific examples of the apoptosis-promoting protein gene include Bim-EL, Bax, FADD, and Caspase. Specific examples of the apoptosis-suppressing protein gene include Bcl-xL and Bcl-2. A specific example of the cell surface protein gene is a biotin-added peptide, to which an IgG leader sequence and a PDGFR transmembrane domain are added. However, the protein genes encoded by the mRNA are not limited thereto.
(27) The mRNA preferably comprises, in the direction from the 5′-terminus to the 3′-terminus, a Cap structure (7-methylguanosine 5′-phosphate), an open reading frame encoding a desired protein gene, and a poly A sequence, and also comprises, a miRNA target sequence linked to the 3′-terminal side of the Poly A sequence, and a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence.
(28) The structure of 5′ UTR is not particularly limited in terms of the number of nucleotides and the sequence, as long as it comprises a Cap structure but does not comprise a miRNA target sequence at the 5′-terminus thereof. As an example, the structure of 5′ UTR is composed of 20 or more nucleotides, and it is composed of, for example, 40 to 150 nucleotides, and preferably approximately 40 to 100 nucleotides. The 5′ UTR can be a sequence that hardly has the structure of an RNA such as Stem-loop and does not comprise an initiation codon. However, the 5′ UTR is not limited to a specific sequence. The Poly A sequence does not particularly have the upper limit of the length thereof, and it may be a sequence consisting of, for example, 50 to 300 A nucleotides, and preferably 100 to 150 A nucleotides.
(29) The number of miRNA target sequences linked to the 3′-terminal side of the Poly A sequence is preferably 1. This is because, if such a sequence remains on the 3′-terminal side of Poly A after the cleavage of the target sequence by the miRNA, it is hardly recognized as Poly A and the expression of a gene does not start (expression suppression cannot be released) in some cases. However, when the expression suppression can be released after the cleavage of the target sequence by the miRNA, the poly A sequence may comprise a plurality of miRNA target sequences. The phrase “a miRNA target sequence linked to the 3′-terminal side of a Poly A sequence” means that the poly A sequence may be directly linked to the miRNA target sequence, or that the poly A sequence and the miRNA target sequence may comprise a sequence that does not affect their functions, for example, a sequence consisting of approximately 1 to 5 nucleotides, between them.
(30) A translational repression sequence is linked to the 3′-terminal side of the miRNA target sequence. The phrase “a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence” means not only a case in which the miRNA target sequence is directly linked to the translational repression sequence, but also a case in which another sequence may be present between them. For example, the miRNA target sequence and the translational repression sequence may comprise an adapter sequence consisting of 20 to 100 nucleotides between them. Moreover, when two or more miRNA target sequences are contained, “the 3′-terminal side of the miRNA target sequence” means the 3′-terminal side of a miRNA target sequence that is located at the most 3′-terminal side.
(31) The translational repression sequence may be a sequence capable of preventing the action associated with the translation of the Poly A sequence, and the translational repression sequence preferably comprises a nucleotide sequence selected from (i) a nucleotide sequence consisting of 20 or more nucleotides that specifically recognizes the Poly A sequence, (ii) a sequence specifically recognizing 5′ UTR, and (iii) a sequence consisting of 100 or more nucleotides.
(32) An example of (i) the nucleotide sequence consisting of 20 or more nucleotides that specifically recognizes the Poly A sequence is a Poly U sequence completely complementary to the Poly A sequence. The Poly U sequence is, for example, a Poly U sequence consisting of 20 or more, 40 or more, 60 or more, or 80 or more uridine nucleotides, but the examples of the Poly U sequence are not limited thereto. Moreover, if the sequence (i) can specifically recognize the Poly A sequence, it may have a mismatch. Furthermore, if the sequence (i) comprises a nucleotide sequence consisting of 20 or more nucleotides, which specifically recognizes the Poly A sequence, it may further comprise any given sequence on the 3′-terminal side and/or 5′-terminal side thereof. In addition, as long as the sequence (i) can specifically recognize the Poly A sequence and can repress the translation thereof, it may not necessarily be a sequence consisting of 20 or more nucleotides, but may be a sequence consisting of 5 or more, 10 or more, or 15 or more nucleotides.
(33) (ii) The sequence specifically recognizing 5′ UTR is preferably a sequence completely complementary to 5′ UTR. However, if this sequence can specifically recognize 5′ UTR, it may have a mismatch with such a completely complementary sequence. Moreover, if the sequence (ii) comprises a sequence specifically recognizing 5′ UTR, it may further comprise any given sequence on the 3′-terminal side and/or 5′-terminal side thereof.
(34) (iii) The sequence consisting of 100 or more nucleotides may be a long sequence capable of translational repression, and the types of the nucleotides and the sequence of the nucleotides are not particularly limited. The sequence may consist of 100 or more nucleotides, and may preferably consist of 300 or more nucleotides, 500 or more nucleotides, 1000 or more nucleotides, or 1500 or more nucleotides.
(35) The nucleotides constituting the miRNA-responsive mRNA preferably comprise modified nucleotides such as pseudo uridine and 5-methylcytidine, instead of ordinary uridine and cytidine. This is because of reduction in cytotoxicity. Such modified nucleotides can be positioned independently, as a whole or as a part of the mRNA, in both cases of uridine and cytidine. In the case of being contained as a part, the nucleotides can be positioned randomly at any given ratio.
(36) The miRNA-responsive mRNA having such structural characteristics can hide the poly A sequence from the translation system in cells and can inhibit the translation thereof. It is thought that, in cells, an mRNA is recognized by the CAP structure existing at the 5′-terminus and the poly A sequence existing at the 3′-terminus, and that activation of translation takes place. It is said that the above-designed artificial miRNA-responsive mRNA is temporarily inactivated.
(37) If the miRNA-responsive mRNA is sequenced as described above, it can be synthesized by one skilled in the art according to any method that is already known in the genetic engineering field. In particular, the miRNA-responsive mRNA can be obtained by an in vitro synthesis method using, as a template, template DNA comprising a promoter sequence. One advantage of the present invention is that an mRNA can be obtained as designed according to a simple method.
(38) In the method for expressing a protein gene according to the present invention, only one type of miRNA-responsive mRNA may be introduced into cells, or two or more, for example, three, four, five, six, seven, or eight or more miRNA-responsive mRNAs may be used. The types, numbers, and structures of individual miRNA-responsive mRNAs to be introduced may be designed, as appropriate, by one skilled in the art depending on the purpose of expressing the protein gene. For example, a plurality of miRNA-responsive mRNAs each having a different miRNA target site and an identical protein gene can be designed. Otherwise, a plurality of miRNA-responsive mRNAs each having a different miRNA target site and a different protein gene can also be designed. The translational repression sequences may be identical to or different from one another, as long as they have necessary translation repression function in each miRNA-responsive mRNA.
(39) In the step of introducing a miRNA-responsive mRNA into a cell, one or more miRNA-responsive mRNAs are directly introduced into a cell by using a lipofection method, a liposome method, an electroporation method, a calcium phosphate co-precipitation method, a DEAE dextran method, a microinjection method, a gene gun method, etc. The miRNA-responsive mRNA can also be introduced into a cell in the form of DNA, using a vector or the like. Also in such a case, the same method as that described above can be used. In the case of introduction of two or more different miRNA-responsive mRNAs, a plurality of mRNAs can be co-introduced into a cell, or can also be introduced therein separately. At this time, the amount of miRNA-responsive mRNA introduced is different, depending on the type of cell, into which the mRNA is introduced, the type of the introduced mRNA, a method of introducing the mRNA, and the types of introduction reagents. In order to achieve a desired translation level, one skilled in the art can appropriately select these conditions.
(40) The action of the miRNA-responsive mRNA that has been introduced into a cell will be described. When a miRNA-responsive mRNA is introduced into a certain cell and a miRNA specifically binding to a miRNA target site is present in the cell, the miRNA binds to the miRNA-responsive mRNA and the mRNA is cleaved at a site between the Poly A sequence and the miRNA target site. Thereby, the miRNA-responsive mRNA, which has been temporarily inactivated and the translation of which has been repressed, may be translated, and the expression of a protein gene is initiated and is promoted. When the protein is, for example, a quantitatively measurable marker protein, it can be clearly confirmed that the expression level of the protein shows a correlation with a miRNA in a cell. In addition, such a protein to be expressed may cause cell death or may exhibit predetermined function to cells, depending on the properties of the protein. When a miRNA-responsive mRNA prepared by establishing a plurality of miRNA target sites each binding to different miRNAs in a single mRNA is introduced into a cell, if even one miRNA binding to any of the plurality of miRNA target sites has been expressed in the cell, the translation repression state can be released, and it becomes possible to be translated. On the other hand, when a miRNA specifically binding to such a miRNA target site is not present in the cell, the miRNA-responsive mRNA is not influenced by the miRNA, and thus, the mRNA still remains in the translation repression state. As a result, a protein encoded by the miRNA-responsive mRNA is not expressed, substantially no action takes place, and thus, the miRNA-responsive mRNA is decomposed.
(41) Moreover, when a miRNA-responsive mRNA is introduced into a cell population, in which cells having different properties are present, a protein is expressed only in cells, in which a miRNA specifically binding to a miRNA target site is present, according to the same action as that described above. As a result, cells expressing a predetermined miRNA can be determined based on the expression of the protein, and can be then separated and characterized. The determination of cells using such a miRNA-responsive mRNA will be explained in a second embodiment.
Second Embodiment
(42) According to a second embodiment, the present invention relates to a method for determining a desired cell type from a cell group comprising two or more cells, using the expression of a miRNA as an indicator,
(43) the method comprising the following steps: (1) a step of introducing a first artificial mRNA comprising a sequence encoding a first marker gene, a first miRNA target sequence linked to the 3′-terminal side of a Poly A sequence, and a translational repression sequence linked to the 3′-terminal side of the first miRNA target sequence, into the cell group; and (2) a step of determining the cell type, using the translation level of the first marker gene as an indicator.
(44) In the determination method according to the present embodiment, the cell group used as a target is a cell group comprising two or more cells. This cell group may be either a cell group collected from species of multicellular organisms, or a cell group obtained by culturing isolated cells. The cell group is particularly a cell group comprising two or more somatic cells collected from mammals (e.g., a human, a mouse, a monkey, a swine, a rat, etc.), or a cell group obtained by culturing cells isolated from mammals or mammalian cell lines. Examples of the somatic cells include keratinizing epithelial cells (e.g. keratinized epidermal cells), mucosal epithelial cells (e.g. epithelial cells on a tongue surface layer), exocrine epithelial cells (e.g. mammary gland cells), hormone secreting cells (e.g. adrenomedullary cells), cells for metabolism/storage (e.g. liver cells), inner luminal epithelial cells constituting a boundary surface (e.g. type I alveolar cells), inner luminal epithelial cells in the inner chain tube (e.g. vascular endothelial cells), cells having cilia with transport ability (e.g. respiratory tract epithelial cells), cells for extracellular matrix secretion (e.g. fibroblasts), contractile cells (e.g. smooth muscle cells), cells of blood and immune system (e.g. T lymphocytes), sensory cells (e.g. rod cells), autonomic nervous system neurons (e.g. cholinergic neurons), cells supporting sensory organs and peripheral neurons (e.g. satellite cells), nerve cells and glial cells in the central nervous system (e.g. astroglial cells), pigment cells (e.g. retinal pigment epithelial cells), and the progenitor cells thereof (tissue progenitor cells). The degree of differentiation of cells, the age of an animal from which cells are collected, etc. are not particularly limited. Both undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as an origin of somatic cells in the present invention. Herein, examples of the undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, or dental pulp stem cells. In the present invention, the type of mammal as the source, from which somatic cells are collected, is not particularly limited, and it is preferably a human. In addition, a preferred cell group is a cell group, in which artificial operations are performed on prophase somatic cells after collection of the cells, and it may be a cell group that includes undesired cells. Thus, the cell group is, for example, a cell group comprising iPS cells prepared from the somatic cells, or a cell group obtained after differentiation of pluripotent stem cells such as ES cells or iPS cells, which may include differentiated cells other than desired cells. In the present embodiment, the cell group as a determination target is preferably in a survival state. In the present invention, the expression “cells in a survival state” is used to mean cells in a state in which they maintain metabolic capacity. The present invention is advantageous in that, after cells have been subjected to the method of the present invention and the determination method has then been terminated, the cells remain alive without losing their original properties, and can be used in the subsequent intended use, in particular, while maintaining division capacity.
(45) In the method of the present invention, the “desired cell type” to be determined means a group of cells, which is classified from other cell types, using the expression of a miRNA as an indicator. In particular, the desired cell type means a certain group of cells having common properties, in terms of miRNA activity, which will be described in detail later. In the present invention, such a certain group of cells, which is classified from other cell types, using miRNA as an indicator, is also referred to as “homologous cells.” The desired cell type determined by the method of the present invention may be one, or two or more, for example, three, four, five, six, seven, or eight or more. Theoretically, the determine-possible cell type is not limited, and according to the present invention, 100 or more cells can be determined simultaneously.
(46) In the present invention, the expression “to determine a desired cell type” is used to mean that the detectable signal information of desired specific one or more cell types, which are different from other cell types, is presented from a cell group comprising two or more cells, and in particular, that visually recognizable information is presented. It is to be noted that the visually recognizable information is not limited to emission of visual signals directly from cells, but it also includes information obtained by converting signals emitted from cells to visually recognizable information, using numerical values, charts, images, etc. Thus, the visually recognizable information means information visually recognizable by one skilled in the art. In the present description, the term “determine” may include the meanings that, after completion of the determination, the desired cell type is recognized, the desired cell type is distinguished, the desired cell type is identified, the desired cell type is classified, the desired cell type is isolated, undesired cell types are removed, the life or death of the desired cell type is determined, specific biological signals are detected or quantified in the desired cell type, and the desired cell type is fractionated based on specific physical or chemical signals. In the present invention, determination whereby a cell group, which has been unknown to comprise two or more types of cells, is determined to comprise different cell types by the method of the present invention, is also considered to be one aspect of determination of a desired cell type.
(47) The first artificial mRNA used in the step (1) according to the present embodiment is the miRNA-responsive ON-switch mRNA described in the first embodiment, in which the protein is a marker protein. In the present embodiment, any given mRNA that functions in response to a miRNA for the forced expression of the protein gene is referred to as a first artificial mRNA.
(48) Upon designing the first artificial mRNA, the marker gene is expressed, and then, a miRNA, which is specifically expressed in desired target cells to be determined, is extracted, and then, a miRNA target site suitable for this miRNA can be selected. Also, a marker gene suitable for the aspect of desired determination can be selected. The type of such a marker gene can be selected from those described in detail in the first embodiment. A preferred marker gene may be a fluorescent protein gene capable of quantitative determination, but it is not limited to a specific marker gene. In the present step, only one first artificial mRNA can be used, or two or more first artificial mRNAs can also be used. In the latter case, a plurality of first artificial mRNAs may be different from one another in terms of the miRNA target site and the marker protein. Otherwise, in some cases, the first artificial mRNAs may be identical to one another in terms of a certain condition. Such first artificial mRNA can be designed, as appropriate, by one skilled in the art, depending on the aspect of determination.
(49) The first artificial mRNA, which has been designed and then prepared according to a genetic engineering means, can be introduced into a cell group by the method described in the first embodiment. At this time, the first artificial mRNA and an mRNA used as a reference (hereinafter also referred to as a “reference mRNA”) can be introduced into such a cell group. The reference mRNA is an mRNA, which can be used to increase separation ability (fold change) in the determination method of the present embodiment, and which behaves specifically or non-specifically to the expression of a miRNA, in a way different from the behavior of the miRNA-responsive ON-switch mRNA.
(50) The reference mRNA may be, for example, a second artificial mRNA comprising a second marker gene, which is operably linked to a second miRNA target sequence. The second artificial mRNA is an artificial mRNA, which reduces the translation level of the second marker gene in response to the expression level of the miRNA, and which is the miRNA-responsive OFF-switch mRNA disclosed in Patent Literature 1 and the like. Regarding this second artificial mRNA, the second miRNA target sequence is a sequence specifically recognized by the same miRNA as that of the first miRNA target sequence. Thus, although the first miRNA target sequence is not necessarily identical to the second miRNA target sequence, they can respond to the same miRNA. Moreover, the second marker gene is different from the aforementioned first marker gene, and signals expressed from these marker genes are separable and distinguishable.
(51) The reference mRNA may also be a third artificial mRNA that does not comprise the target sequence of a miRNA but comprises a third marker gene. The third artificial mRNA is an artificial mRNA, which translates the third marker gene without being influenced by the expression level of the miRNA, and which is the control mRNA disclosed in Patent Literature 1 and the like. Regarding this third artificial mRNA, the third marker gene is different from the first marker gene. In addition, the third marker gene is also different from the second marker gene.
(52) The determination method according to the present embodiment can be carried out only using the first artificial mRNA, or the first and second artificial mRNAs can also be used in combination. All of the first, second, and third artificial mRNAs can also be used. Further, it is also possible to use a plurality of each of the first, second, and third artificial mRNAs.
(53) In the step (1), in the case of using two or more first artificial mRNAs, and/or in the case of using a first artificial mRNA and a reference mRNA, a plurality of mRNAs are preferably co-introduced into a cell group. This is because the ratio of the activities of marker proteins expressed from the thus co-introduced two or more mRNAs is constant in a cell population. At this time, the amount of the mRNA introduced is different, depending on the type of cell group, into which the mRNA is introduced, the type of the introduced mRNA, a method of introducing the mRNA, and the types of introduction reagents. In order to achieve a desired translation level, one skilled in the art can appropriately select these conditions. Also, with regard to the amount of the reference mRNA introduced, in order to achieve a desired translation level, one skilled in the art can appropriately select these conditions.
(54) When the first artificial mRNA is introduced into a cell, the translation of the marker gene encoded by the first artificial mRNA is initiated, if a certain miRNA is present as RISK in the cell. The regulation of the translation level is quantitatively carried out depending on miRNA activity. In contrast, if the certain miRNA is not present in the cell, or if the certain miRNA is not present as RISK, the marker gene encoded by the first artificial mRNA is not translated. Accordingly, the translation level of the marker gene is different between a cell in which the certain miRNA is present as RISK and a cell in which it is not present. It is to be noted that, in the present description, the case where the certain miRNA is present as RISK is also referred to as a “case where miRNA activity is present.” On the other hand, the third artificial mRNA, that is, a control mRNA expresses a marker protein, regardless of miRNA activity. This is because, even if such a control mRNA is introduced into a cell, translation is not regulated depending on the expression level of a miRNA, since the miRNA target sequence is not present therein. In the case of the second artificial mRNA, that is, in the case of a miRNA-responsive OFF-switch mRNA, when the certain miRNA is present as RISK in the cell, the translation of a marker gene is suppressed.
(55) Subsequently, a step of determining a cell is carried out, using the translation level of the marker gene in the step (2) as an indicator. In this step, a cell is determined based on the aforementioned translation level of the marker gene. That is, this step can be a step of determining a desired cell type that is a cell in which the expression level of the miRNA used as an indicator is low and the translation level of the marker gene is low, and/or a step of determining a desired cell type that is a cell in which the expression level of the miRNA used as an indicator is high and the translation level of the marker gene is high. Such a cell in which the expression level of the miRNA used as an indicator is low, or a cell in which the expression level of the miRNA used as an indicator is high, can be determined by obtaining the ratio of the translation levels of the marker genes among cells belonging to a cell group comprising two or more cells.
(56) Specifically, the determination step can be carried out by detecting signals from a marker protein, employing a predetermined detection apparatus. Examples of the detection apparatus include a flow cytometer, an imaging cytometer, a fluorescence microscope, a luminescence microscope, and a CCD camera, but examples are not limited thereto. As such a detection apparatus, one skilled in the art can use a suitable apparatus, depending on a marker protein and the mode of determination. For instance, when the marker protein is a fluorescent protein or a luminescent protein, it is possible to quantify the marker protein using a detection apparatus such as a flow cytometer, an imaging cytometer, a fluorescence microscope or a CCD camera. When the marker protein is a protein supporting fluorescence, luminescence or color development, a method of quantifying the marker protein using a detection apparatus such as a luminescence microscope, a CCD camera or a luminometer can be applied. When the marker protein is a membrane localization protein, a method of quantifying the marker protein using a detection reagent specific to a cell surface protein, such as an antibody, and the aforementioned detection apparatus, can be applied, and also, a method of isolating cells without performing the process of quantifying the marker protein, such as a magnetic cell separation device (MACS), can be applied. When the marker protein is a drug resistance gene, a method, which comprises detecting the expression of the marker gene by administration of a drug and then isolating living cells, can be applied.
(57) An example of a preferred detection method, which is applied when the marker protein is a fluorescent protein, is flow cytometry. In the flow cytometry, the intensity of light emitted from a fluorescent protein, luciferase, that is a marker protein translated in each cell, can be provided as information for determination.
(58) In a first aspect of the second embodiment of the present invention, separation and extraction of a predetermined cell group from a cell population, in which undifferentiated cells after completion of the induction of differentiation from pluripotent stem cells are present, will be explained. At this time, the first artificial mRNA can be designed, so that it has a miRNA target sequence that is specifically expressed in pluripotent stem cells. When the first artificial mRNA is introduced into the cell population, the translational repression sequence is cleaved by the expression of the miRNA in the pluripotent stem cells. Then, the first marker gene is expressed and is to be determined. On the other hand, since such a miRNA specifically expressed in pluripotent stem cells is not present as RISK in differentiated cells, the translational repression sequence is not cleaved, and thus, the translation of the first marker gene does not take place. In short, the translation of the marker gene is carried out only in pluripotent stem cells. Accordingly, in one embodiment of the present invention, by extracting cells in which the marker gene has been translated, it becomes possible to selectively extract only pluripotent stem cells from a cell population, in which undifferentiated cells are present after completion of the induction of differentiation from the pluripotent stem cells.
(59) In the step of extracting cells in which the marker gene has been translated, wherein the step may be optionally carried out, the above-described marker gene is translated, and cells, in which the expression of the marker protein has been confirmed, are extracted as pluripotent stem cells. Specifically, the extraction step can be carried out by detecting signals from the marker protein, using a predetermined detection apparatus. Detection of signals from the marker protein may be either digitalization and quantification of the signals or detection of only the presence or absence of the signals. Examples of the detection apparatus include a flow cytometer, an imaging cytometer, a fluorescence microscope, a luminescence microscope, and a CCD camera, but examples are not limited thereto. As such a detection apparatus, one skilled in the art can use a suitable apparatus, depending on the type of a marker protein. For instance, when the marker protein is a fluorescent protein or a luminescent protein, it is possible to confirm the presence or absence of the expression of the marker protein and/or to quantify the marker protein, using a detection apparatus such as a flow cytometer, an imaging cytometer, a fluorescence microscope or a CCD camera. When the marker protein is a protein supporting fluorescence, luminescence or color development, a method of confirming the presence or absence of the expression of the marker protein and/or quantifying the marker protein, using a detection apparatus such as a luminescence microscope, a CCD camera or a luminometer, can be applied. When the marker protein is a membrane localization protein, a method of confirming the presence or absence of the expression of the marker protein and/or quantifying the marker protein, using a detection reagent specific to a cell surface protein, such as an antibody, and the aforementioned detection apparatus, can be applied, and also, a method of isolating cells without performing the process of quantifying the marker protein, such as a magnetic cell separation device (MACS), can be applied. When the marker protein is a drug resistance protein, a method, which comprises detecting the expression of the marker protein by administration of a drug and then isolating living cells, can be applied.
(60) According to a second aspect of the second embodiment, in separation and extraction of a predetermined cell group from a cell population, in which undifferentiated cells are present after completion of the induction of differentiation from pluripotent stem cells, the first artificial mRNA and the second artificial mRNA can be introduced into the cell population. The first and second artificial mRNAs can be designed, so that they have a miRNA target sequence that is specifically expressed in pluripotent stem cells. The first artificial mRNA that has been introduced into the cell population shows the same behavior as that in the previous first aspect. On the other hand, when the second artificial mRNA is introduced into the cell population, the expression of the second marker gene is suppressed by the expression of the miRNA in the pluripotent stem cells, and in differentiated cells, the second marker gene is expressed. Accordingly, cells expressing the first marker gene can be separated as pluripotent stem cells, whereas cells expressing the second marker gene can be separated as undifferentiated cells.
EXAMPLES
(61) Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are not intended to limit the scope of the present invention.
(62) Experiments
(63) 1. Production of PCR Product Having Sequence of EGFP
(64) The sequence of EGFP was obtained by subjecting a plasmid containing a cloned EGFP to PCR, using two synthetic oligo DNAs, namely, TAPEGFPIVTfwd and TAP IVTrev, and then decomposing the plasmid with DpnI. The sequence of 5′ UTR was produced by hybridizing synthetic oligo DNAs, that is, IVT 5prime UTR and Rev5UTR, and then elongating the resultant. The sequence of 3′UTR was produced by hybridizing two synthetic oligo DNAs, that is, IVT 3prime UTR and Rev3UTR2T20, and then elongating the resultant. The thus produced EGFPORF, 5′ UTR, and 3′UTR were purified, and they were then linked to one another by being subjected to PCR using TAP T7 G3C fwd and Rev3UTR2, which had been mixed and phosphorylated. The PCR product was cloned into a pUC19 vector, thereby producing EGFP comprising UTR (pUC19-EGFPfull). Hereafter, the sequence of EGFP was obtained by performing PCR on this plasmid. The following Table 1 shows the sequences of synthetic oligo DNAs used.
(65) TABLE-US-00001 TABLE 1 Name of Sequence Oligo DNA Sequence (5′.fwdarw.3′) ID No. TAP_IVTrev GCCCCGCAGAAGGTCTAGACTATCACTCGAGATGCATATGAG 1 ATC TAPEGFP_ CACCGGTCGCCACCATGGGATCCGTGAGCAAGGGC 2 IVTfwd IVT_5prime_ CAGTGAATTGTAATACGACTCACTATAGGGCGAATTAAGAGA 3 UTR GAAAAGAAGAGTAAGAAGAAATATAAGACACCGGTCGCCACC ATG Rev5UTR CATGGTGGCGACCGGTGTCTTATATTTCTTCTTACTC 4 IVT_3prime_ TCTAGACCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTC 5 UTR TCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAG TAGG Rev3UTR2T TTTTTTTTTTTTTTTTTTTTCCTACTCAGGCTTTATTCAAAGACC 6 20 AAG TAP_T7_G3C_ CAGTGAATTGTAATACGACTCACTATAGGGC 7 fwd_primer Rev3UTR2 CCTACTCAGGCTTTATTCAAAGACCAAG 8
(66) The produced pUC19-EGFPfull was used as a template, and PCR was then carried out using the primers shown in the following Table 2, each having TAP T7 G3C fwd and the complementary sequence to a miRNA. Poly A and the complementary sequence to a miRNA were added, and thereafter, the plasmid was digested with Dpn I.
(67) TABLE-US-00002 TABLE 2 Name of Oligo Sequence DNA Sequence (5′.fwdarw.3′) ID No. 3UTR-109A- tagcttatcagactgatgttgaattttttttttttttttttttttt 9 Tg21 tttttttttttttttttttttttttttttttttttttttttttttt (Complementary ttttttttttttttttttttttttttttttttttttttttcctactc sequence to miR- aggctttattc 21-5p) 3UTR-109A- ACTTAAACGTGGATGTACTTGCTttttttttttttttttttttttt 10 Tg302 ttttttttttttttttttttttttttttttttttttttttttttttt (Complementary tttttttttttttttttttttttttttttttttttttttcctactcag sequence to miR- gctttattc 302a-5p) 3UTR-109A-N gttgcgattatgaacctattagattttttttttttttttttttttttt 11 tttttttttttttttttttttttttttttttttttttttttttttttt ttttttttttttttttttttttttttttttttttttcctactcaggctt tattc
2. Production of DNA Template, with 3′-Terminus of Which Additional Sequence is Fused
(68) In order to search for a sequence that inhibits translation when added to poly A sequence or later, the following sequences were added. The EGFP having a miRNA-responsive sequence produced in the above 1 was used as a template, and in order to finally unify the 3′-terminus to an M13 sequence or an Rn2 sequence having no complementary sequences on the plasmid, the synthetic oligo DNAs shown in the following Table 3 were used as adapters.
(69) TABLE-US-00003 TABLE 3 Sequence Name of Oligo DNA Sequence (5′.fwdarw.3′) ID No. Tg21-M13NRev tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgc 12 (Synthetic oligo DNA caagcttgcatgcctgcaggtcgactctagaggatccccgggtaccGGTCTCTt adding M13 sequence to agcttatcagactgatgttgaa complementary sequence to miR-21-5p, used for addition of poly U) N-Mi3NRev (Synthetic tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgc 13 oligo DNA adding M13 caagcttgcatgcctgcaggtcgactctagaggatccccgggtaccGGTCTCTg sequence to control ttgcgattatgaacctattaga sequence, used for addition of poly U) Tg21-M13Rev (Synthetic cacacaggaaacagctatgaccatgtagcttatcagactgatgttgaattt 14 oligo DNA adding M13 sequence to complementary sequence to miR-21-5p) N-Mi3Rev (Synthetic cacacaggaaacagctatgaccatggttgcgattatgaacctattagattt 15 oligo DNA adding M13 sequence to control sequence) Tg302-M13Rev cacacaggaaacagctatgaccatgACTTAAACGTGGATGTACTT 16 (Synthetic oligo DNA GCTttt adding M13 sequence to complementary sequence to miR-302a-5p) Tg21-Rn2-Rev (Synthetic GTTACATTGTGCCACGGAGTCGATCtagcttatcagactgatgtt 17 oligo DNA adding Rn2 gaattt sequence to complementary sequence to miR-21-5p) Tg302-Rn2-Rev GTTACATTGTGCCACGGAGTCGATCACTTAAACGTG 18 (Synthetic oligo DNA GATGTACTTGCTttt adding Rn2 sequence to complementary sequence to miR-302a-5p)
(70) Using the M13 sequence or the Rn2 sequence as a joint (adapter sequence), the PCR product having the sequence of EGFP produced in the above 1, the above-described synthetic oligo DNAs, and synthetic oligo DNAs having various sequences were mixed, and the obtained mixture was then subjected to PCR to produce templates for transcription having various 3′-terminus additional sequences. Hereafter, the synthetic oligo DNAs each having a 3′-terminus additional sequence used in the production of templates and the final products (RNAs) are described in terms of only the portions downstream of the poly A sequences. The following Table 4 specifically shows synthetic oligo DNAs having poly U with a length of 80. Other than those, templates having poly U with a length of 20, 40 and 60 were also produced. The dotted line portion indicates an adapter. In the case of using M13, the sequence becomes CAUGGUCAUAGCUGUUUCCUG; in the case of using M13RV, it becomes CCGCUCACAAUUCCACA; and in the case of using Rn2, it becomes GTTACATTGTGCCACGGAGTCGATC. In order to prevent generation of non-specific by-products upon production of such templates, a sequence consisting of GAAUUCUCGCAGCCCGAAGA that is not complementary to the sequences of other regions was added to the 3′-terminus.
(71) Templates, to which a poly U sequence was added, are shown in the following Table 4.
(72) TABLE-US-00004 TABLE 4 Name of Oligo Sequence DNA/RNA Sequence (5′ .fwdarw. 3′) ID No. YF549-M13- TCTTCGGGCTGCGAGAATTCaaaaaaaaaaaaaaaaaaaatgtggaatt 19 T20N20 gtgagcgg YF550-M13- TCTTCGGGCTGCGAGAATTCaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 20 T40N20 aaaaaaaaaaatgtggaattgtgagegg YF551-M13- TCTTCGGGCTGCGAGAATTCaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 21 T60N20 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaatgtggaattgtgagegg YF552-M13- TCTTCGGGCTGCGAGAATTCaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 22 T80N20 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aatgtggaattgtgagegg Tg21-U80 UUCAACAUCAGUCUGAUAAGCUAAGAGACCGGUACCCGGGGAUCCUCUA 23 GAGUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUAGCUGU
(73) Templates, to which a long chain RNA (approximately 500 or 1200 nt) was added, are shown in the following table, in terms of only the sequence downstream of each poly A sequence. The region of a pGEM-T easy vector was amplified using the following primers and was then bound according to fusion PCR. The dotted line portion in the RNA sequence shown in Table 5 indicates an adapter sequence.
(74) TABLE-US-00005 TABLE 5 Name of Oligo Sequence DNA/Name of RNA Sequence (5′ .fwdarw. 3′) ID No. M13Rev-Fwd catggtcatagctgtttcctgtgtg 24 M13Rev-1000-Rev GCATTGGTAACTGTCAGACCAAGTTTACTC 25 M13Rev-500-Rev GGAGCCTATGGAAAAACGCCAGCaacg 26 Additional
(75) Additional sequences, to which the complementary sequence to 5′ UTR was added, are shown in the following Table 6. In the RNA sequences shown in Table 6, the dotted line portion indicates an adapter sequence, the bold indicates a complementary sequence to 5′ UTR, the underlined portion indicates a stem loop, and the 3′-terminus is a sequence that is not complementary to the sequences of other regions.
(76) TABLE-US-00006 TABLE 6 Name of Oligo Sequence DNA/Name of RNA Sequence (5′ .fwdarw. 3′) ID No. M13Rev-5Cmp20N20 TCTTCGGGCTGCGAGAATTCgggcgaattaagagagaaaacacacagg 29 aaacagctatgaccatg M13Rev-5Cmp40N20 TCTTCGGGCTGCGAGAATTCgggcgaattaagagagaaaagaagagta 30 agaagaaatatacacacaggaaacagctatgaccatg M13Rev-5Cmp20SLN20 TCTTCGGGCTGCGAGAATTCCGCGCTGGACtcccGTCCAGCGCGgggc 31 gaattaagagagaaaacacacaggaaacagctatgaccatg Additional sequence
3. Transcription
(77) Transcription was carried out using MEGA shortscript TM T7 Transcription Kit (Thermo Fisher Scientific) in accordance with the instruction manual included therewith. A CAP analog, ARCA, was added to GTP at a ratio of 4:1, and 5-methyl-cytosine and pseudo-uridine were used instead of CTP and UTP, respectively. The transcription scale was set at 10 μL. After completion of the transcription, using DNase I (Thermo Fisher Scientific) and rAPid alkaline phosphatase (Roche), the RNA was subjected to decomposition of the template DNA and dephosphorylation. The reaction was carried out by adding 4 μL of 10×rAPid alkaline phosphatase buffer, 1 μL of rAPid alkaline phosphatase, 0.5 μL of Turbo DNase I, and 40 μL of H.sub.2O to 10 μL of a transcription reaction solution, and then incubating the mixture at 37° C. for 30 minutes. Thereafter, the RNA was purified using RNAeasy MiniElute column (QIAgen) or FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech), and was eluted with H.sub.2O, followed by measuring the concentration, and the resultant was finally adjusted to 100 ng/μL and was then preserved at −20° C.
(78) 4. Introduction into Cells and Quantification of Fluorescence
(79) Cells were seeded on a 24 well plate (HeLa: 0.5×10.sup.5, 293FT: 1×10.sup.5, iPSC (201B7): 1×10.sup.5), and one day later, the medium was replaced with a fresh one before transfection. Transfection of the RNA into the cells was carried out using Stemfect RNA Transfection kit (Stemgent) in accordance with the protocols included therewith. With regard to the amount of the RNA used, 1 μL of Stemfect was used with respect to 100 ng of the mRNA of iRFP670 as an internal control, and with respect to 100 ng of the RNA of an ON switch expressing EGFP. Twelve to twenty-four hours after the introduction, the resulting cells were observed under a fluorescence microscope. Thereafter, the cells were peeled from the plate using Trypsin-EDTA or AccuMax, and then, were fully suspended. The suspension was passed through a mesh to remove large masses. Subsequently, using Accuri C6, the fluorescence of iRFP670 and that of EGFP were quantified, and finally, were quantified by the ratio of EGFP/iRFP670.
(80) The sequence names and sequence numbers of a miRNA-responsive ON-switch mRNA (first artificial mRNA), a miRNA-responsive OFF-switch mRNA (second artificial mRNA), and a control mRNA (third artificial mRNA), which were used in the experiments, are shown in the following Table 7.
(81) TABLE-US-00007 TABLE 7 Name of RNA Sequence ID No miR21-responsive OFF switch EGFP mRNA 35 Control EGFP mRNA 36 Control iRFP670 mRNA 37 EGFP-A109-Tg21-U80 38 EGFP-A109-Tg21-U60 39 EGFP-A109-Tg21-U40 40 EGFP-A109-Tg21-U20 41 EGFP-A109-N-U80 42 EGFP-A109-N-U60 43 EGFP-A109-N-U40 44 EGFP-A109-N-U20 45 EGFP-A109-Tg21-120nt 46 EGFP-A109-Tg21-5UTRcomp20nt 47 EGFP-A109-Tg21-5UTRcomp40nt 48 EGFP-A109-Tg21-5UTRcomp20nt-stemloop 49 EGFP-A109-Tg21-5 00nt 50 EGFP-A109-Tg21-1250nt 51 miR-302a-5p-respnsive ON-switch mRNA 52
Results
1. Change in Expression of EGFP in HeLa Cells or 293FT Cells in Case of Linking Poly U with Complementary Sequence to miR-21
(82) An mRNA, in which poly U had been added to the 3′-terminal side of the poly A of EGFP across the complementary sequence (Tg21) to miR-21 or the sequence (N) obtained by shuffling the complementary sequence to miR-21, was introduced into HeLa cells. Thereafter, the fluorescence of EGFP and the fluorescence of iRFP670 used as a reference were measured with a flow cytometer, and the ratio thereof was then calculated (
(83) 2. Change in Expression Level of EGFP in HeLa Cells in Case of Linking Complementary Sequence to 5′ UTR with Complementary Sequence to miR-21
(84) A pGEM-T easy-derived sequence was added to the 3′-terminal side of the poly A of EGFP across the complementary sequence (Tg21) to miR-21 or the sequence (N) obtained by shuffling the complementary sequence to miR-21. On the other hand, HeLa cells, in which miR-21 was highly expressed, were transfected with an mRNA to which the complementary sequence to 5′ UTR had been added, and with the mRNA of iRFP670. In these cases, the expression levels of EGFP and iRFP670 were two-dimensionally plotted. The results are shown in
(85) 3. Response of miR-21 to Mimic or Inhibitor in HeLa Cells or 293FT Cells
(86) Using HeLa cells in which miR-21 was highly expressed, and 293FT cells in which miR-21 was not expressed, the response of an mRNA comprising a Tg21 sequence was confirmed. At this time, regarding HeLa cells, the case of adding 2 pmol of inhibitor to the cells and the case of not adding such inhibitor to the cells were subjected to experiments. Regarding 293FT cells, the case of adding 2 pmol of mimic to the cells and the case of not adding such mimic to the cells were subjected to experiments. Regarding the HeLa cells, the results were shown by comparing the case of adding the inhibitor with the case of not adding the inhibitor (
(87) 4. Improvement of Separation by Utilizing Reference of ON Switch
(88) A miR-21-responsive iRFP670 ON switch was used as a reference, instead of an iRFP670 mRNA used as a reference of miR-21 OFF switch. It is thought that, since the expression of iRFP670 is suppressed in the absence of miR-21, and thereby the value of EGFP/iRFP670 is increased, the miR-21-expressing cell population is separated from the miR-21-non-expressing cell population. The complementary sequence to 5′ UTR used in the above 2 was added to the mRNA of iRFP670 according to the same method as described above, and thereafter, HeLa cells or 293FT cells were transfected with this mRNA and with the miR-21-responsive OFF switch of EGFP. The results are shown in
(89) 5. Identification of iPS Cells by Using ON Switch, into Which Complementary Sequence to miR-302a-5p has been Inserted
(90) The complementary sequence to miR-21 was replaced with the complementary sequence to miR-302a-5p, so as to produce an mRNA in which the expression of EGFP is increased in response to miR-302a-5p. This mRNA was co-introduced with the mRNA of iRFP670 as a reference into 293FT cells and iPS cells (201B7), and the expression level of fluorescence was then analyzed by flow cytometry. The results are shown in