WO2017094878A1 - Method for identifying intracellular endogenous protein - Google Patents

Abstract

Provided is a method for detecting an intracellular endogenous protein using living cells. A method for identifying an intracellular endogenous protein, said method comprising the steps of: (1) a step for introducing, into a desired cell, intracellular endogenous protein-responsive mRNA that contains an aptamer sequence unique to an intracellular endogenous protein and also contains a marker gene sequence functionally bonded thereto, or a vector that codes said mRNA; and (2) a step for identifying the intracellular endogenous protein on the basis of the amount of translation of the marker gene.

Description

Identification method of endogenous protein

The present invention relates to a method for identifying a cell endogenous protein, a stabilized aptamer that can be used for the method, and a method for identifying a differentiation and reprogramming state of a cell.

An artificial gene expression control system that functions only in target cells can be a useful tool in biotechnology and medical applications. Cell types and states are mainly determined by proteins. Therefore, it is important to develop a mechanism that can detect protein in human cells and control gene expression.

The mRNA switch is a gene switch having an RNA (aptamer) region that binds to a specific protein in the 5′UTR of mRNA. This switch suppresses its own translation by binding a protein to an aptamer on mRNA. In principle, by incorporating an aptamer that binds to any protein, an mRNA switch that detects the desired protein and suppresses translation can be produced. In addition, direct introduction of mRNA into cells has higher introduction efficiency and lower risk of genome damage than conventional gene introduction using plasmids. Accordingly, the mRNA switch is ideal as an artificial gene expression control system that functions only in target cells.

The following two examples are known as gene switches that are produced by incorporating aptamers into mRNA cells and that target proteins expressed in human cells. One is a switch in which a part of ribozyme incorporated into 3′UTR is changed to an aptamer of human U1A protein (see Non-Patent Document 1). However, in this study, U1A was overexpressed by introducing a plasmid, and a response to a protein endogenous to human cells has not been realized. The other is a switch that incorporates an aptamer into an intron to control splicing in the presence of the target protein and to produce different mature mRNAs (see Non-Patent Document 2). This switch can control the expression of fluorescent proteins and prodrug activating enzymes in response to NF-kB and CTNNB1 (β-catenin). However, in this study, the protein that binds to the aptamer needs to be present in the nucleus, so the target protein is limited to the nuclear localization protein. In addition, since splicing control is used, it is difficult to introduce a system other than DNA such as a plasmid or a viral vector.

From the above, it is desired to develop a switch that is not limited to a target protein, can introduce mRNA, and can detect the expression of an underlying protein. Since mRNA controls gene expression by controlling translation, the function of the switch can be realized by direct introduction of mRNA, and mRNA that detects microRNA and controls translation must be developed (Non-patent Document 3). However, the development of an mRNA switch that can detect human endogenous proteins and control translation has not been realized yet.

】 Human endogenous proteins could not be detected with the mRNA ~ switches produced so far. There is a need for a method for detecting human cell endogenous proteins in living cells without destroying the cells.

The present inventors have discovered that a human cell endogenous protein can be specifically and quantitatively detected in a living cell by introducing an mRNA having an aptamer sequence specific to the cell endogenous protein into the cell. Furthermore, the present inventors designed an mRNA having an aptamer sequence for an endogenous protein specific to an induced pluripotent stem (iPS) cell and an aptamer sequence that has stabilized the aptamer sequence, and introduced this into the cell. Thus, it was discovered that the differentiation state and reprogramming state of cells can be detected, and the present invention has been completed.

Therefore, the problems of the present invention can be solved by the following means.
[1] A method for identifying an endogenous protein, comprising the following steps:
(1) a step of introducing into a desired cell a cell endogenous protein-responsive mRNA or aptamer sequence specific to a cell endogenous protein and a marker gene sequence operably linked thereto, or a vector encoding the mRNA; 2) A step of identifying a cellular endogenous protein based on the translation amount of the marker gene.
[2] A cell identification method comprising the following steps:
(1) A step of introducing a cell endogenous protein-responsive mRNA containing an aptamer sequence specific to a cell endogenous protein and a marker gene sequence operably linked thereto or a vector encoding the mRNA into a desired cell;
(2) A step of identifying cells expressing the protein based on the translation amount of the marker gene.
[3] A structure-stabilized LIN28A aptamer obtained by adding one or more of the following modifications based on the RNA secondary structure formed by the natural LIN28A aptamer sequence represented by SEQ ID NO: 1:
(1) AU base pairs constituting a stem including a 5 ′ terminal base and a 3 ′ terminal base are replaced with GC base pairs;
(2) Replace the AU base pair constituting the stem located between the two loops with a GC base pair; or (3) Add 1 to 5 base pairs to the stem located between the two loops.
[4] A structure-stabilized LIN28A aptamer represented by any one of SEQ ID NOs: 2 to 4.
[5] Based on the RNA secondary structure formed by the natural U1A aptamer sequence represented by SEQ ID NO: 6, the structure-stabilized U1A aptamer is modified as follows:
(1) 1 to 15 AU base pairs or GC base pairs are added to a stem structure containing a 5 ′ terminal base and a 3 ′ terminal base, or 1 to 15 AU base pairs are added to a stem existing between two boxes. Alternatively, a GC base pair is added; or a structure-stabilized U1A aptamer obtained by adding the following modifications based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8:
(2) Add 1 to 15 AU base pairs or GC base pairs to the stem containing the 5 ′ terminal base and the 3 ′ terminal base.
[6] A structure-stabilized p50A aptamer based on the RNA secondary structure formed by the p50A aptamer sequence represented by SEQ ID NO: 83 with the following modifications:
1 to 20 AU base pairs or GC base pairs are added to the stem structure containing the 5 ′ terminal base and the 3 ′ terminal base.
[7] A structure-stabilized U1A aptamer represented by any one of SEQ ID NOs: 7, 9, 10, and 84.
[8] An mRNA comprising the structure-stabilized aptamer sequence according to any one of [3] to [7] and a marker gene sequence operably linked thereto.
[9] A method for identifying the differentiation state of a cell, comprising the following steps:
(1) Introduction of an mRNA containing an aptamer sequence specific to a protein expressed by undifferentiated cells and a marker gene sequence operably linked thereto, or a vector encoding the mRNA into a cell group that can contain pluripotent stem cells And (2) identifying the differentiation state of the cell based on the translation amount of the marker gene.
[10] The method according to [9], wherein the aptamer sequence is a natural LIN28A aptamer sequence or the structure-stabilized aptamer sequence according to [3] or [4].

According to the present invention, it is possible to detect an endogenous protein of a living cell, which has been impossible until now, with high sensitivity and quantitatively. In addition, by detecting endogenous proteins in living cells, it can identify induced pluripotent stem (iPS) cells and detect their differentiation and reprogramming states to classify, isolate or selectively eliminate them. Became possible. Furthermore, a stabilized aptamer particularly useful for these detections could be obtained. These stabilized aptamers can be used as protein-specific sequences in the mRNA used in the method described above, and optionally bind to other molecules to strongly bind to the target protein in the cell and function. Can be useful as an anticancer agent or antagonist, or as a sensor outside the cell.

FIG. 1 is a diagram for explaining the mechanism of action of a protein-responsive mRNA switch. A protein-responsive switch has a sequence (aftermer) that binds to a specific protein in the 5 'untranslated region (5'-UTR) of mRNA, and the target protein is linked to the target protein. Has suppressed the translation of That is, translation of the mRNA switch is suppressed in the presence of the trigger protein (upper right in FIG. 1), and normal translation is performed in the absence (lower right in FIG. 1). This molecular device has been artificially created by inserting an aftermer into the 5'-UTR of mRNA. FIG. 2 is a diagram showing an outline of the plasmid used in the experiment. Map information of the plasmids used as empty vectors for the switch plasmid (top) and trigger plasmid (bottom) was delineated using ApE (http://www.biology.utah.edu/jorgensen/wayned/ape/). All plasmids are transcribed from the CMV promoter. In the switch plasmid, an aptamer sequence was inserted into a restriction enzyme site 14 bases downstream from the transcription start point. The transcription product of the trigger plasmid is bicistronically translated tagRFP with the translation of the trigger protein. The trigger protein and tagRFP are translated as two independent proteins by ribosome skipping that occurs at the T2A site. Therefore, the expression level of the trigger protein and the expression level of tagRFP are considered to be the same. FIG. 3 is a secondary structure prediction diagram of the LIN28A aptamer used in the experiment. FIG. 4 is a graph showing that each switch plasmid responds to LIN28A in HEK293 FT cells and is repressed in translation. FIG. 5 is a graph showing that the switch plasmid into which stbC is inserted responds to LIN28A in HeLa cells and translation is suppressed. FIG. 6 is a graph showing that translation is suppressed when the switch plasmid into which stbC is inserted specifically binds to LIN28A. FIG. 7 is a series of diagrams showing that the translation efficiency of the prepared switch changes according to the amount of LIN28A in the cell. FIG. 7A is a graph showing that the translation efficiency of the switch decreases according to the amount of Lin28A expressed depending on the concentration of doxycycline. FIG. 7B shows the results of confirming the expression levels of Lin28A and GAPDH at each doxycycline concentration by Western blotting. FIG. 7C is a graph showing the relationship between doxycycline concentration by quantifying the relative expression level of LIN28A from the band concentration of FIG. 7B. FIG. 7D is a graph showing the relationship between the relative expression level of LIN28A and the fluorescence ratio of each switch. FIG. 8 is a series of diagrams showing that the prepared LIN28A switch responds to LIN28A even during mRNA transfection. FIG. 8A is a dot plot showing a cell population at each LIN28A mRNA introduction amount for a LIN28A switch into which stbC is inserted. FIG. 8B is a dot plot showing that the population is separated according to the amount of LIN28A mRNA introduced in the LIN28A switch with stbC inserted. The dot plot of stbC is an overlay of the dot plots shown in FIG. 8A. FIG. 8C is a graph showing that translation of a LIN28A switch with stbC inserted is suppressed depending on the amount of LIN28A. FIG. 9 is a series of diagrams illustrating a mechanism for separating iPS cells and differentiated cells using the LIN28A switch. FIG. 9A is a diagram schematically illustrating that translation of the LIN28A switch is suppressed in iPS cells. FIG. 9B is a diagram for explaining that cells into which a LIN28A switch has been introduced can be divided into iPS cells and differentiated cells by flow cytometry. FIG. 10 is a series of diagrams showing that the LIN28A switch inserted with stbC undergoes translational suppression in iPS cells. FIG. 10A is a diagram showing that iPS cells and differentiated cells can be distinguished on the dot plot plane of flow cytometry using the LIN28A switch. FIG. 10B is a histogram showing that the LIN28A switch inserted with stbC undergoes translational suppression in iPS cells rather than in differentiated cells. FIG. 10C is a graph showing how much translation of the mRNA into which each aptamer is inserted is suppressed in iPS cells compared to differentiated cells. FIG. 11 is a series of diagrams showing that the translation efficiency of the LIN28A switch changes with the progress of differentiation induction. FIG. 11A is a graph comparing the translation efficiency of iPS cells and cells of each differentiation induction day. FIG. 11B is a dot plot showing the result of flow cytometry of each cell and its overlay. FIG. 12 is a secondary structure prediction diagram of the U1A aptamer used in the experiment. FIG. 13 is a graph showing that each switch plasmid responds to U1A in HEK293 FT cells and is translationally suppressed. FIG. 14 is a series of diagrams showing that the produced U1A switch undergoes translational suppression in response to U1A inherent in human cells. FIG. 14A is a fluorescent photograph of the U1A switch and its mutants introduced into cells. FIG. 14B is a histogram showing the amount of fluorescence of the U1A switch and its mutants. FIG. 14C is a graph showing the fluorescence ratio of each U1A switch. FIG. 15 is a graph showing the relative translation efficiency at the time of shRNA introduction in the U1A switch. FIG. 16 is a graph showing the expression level of endogenous miRNA upon introduction of the LIN28A switch. FIG. 17 is a predicted secondary structure diagram of the p50 aptamer used in the experiment. FIG. 18 is a graph showing that each switch plasmid responds to p50 in HEK293 FT cells and is repressed in translation.

Hereinafter, the present invention will be described in detail with reference to embodiments. The following embodiments do not limit the present invention.

[First Embodiment: Method for Detecting Cellular Endogenous Protein]
According to the first embodiment, the present invention relates to a method for identifying a cellular endogenous protein. The detection method includes the following steps.
(1) a step of introducing into a desired cell a cell endogenous protein-responsive mRNA or aptamer sequence specific to a cell endogenous protein and a marker gene sequence operably linked thereto, or a vector encoding the mRNA; 2) A step of identifying a cellular endogenous protein based on the translation amount of the marker gene.

Here, the desired cell is not particularly limited, and the cell may be arbitrary. For example, it may be a cell contained in a cell group collected from a multicellular species, or may be a cell contained in a cell group obtained by culturing isolated cells. The cells are particularly cells collected from mammals (eg, humans, mice, monkeys, pigs, rats, etc.) or cells obtained by culturing cells isolated from mammals or mammalian cell lines. It may be. Examples of somatic cells include keratinized epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface), exocrine glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, , Adrenal medullary cells), metabolism / storage cells (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells of the inner chain (eg, blood vessels) Endothelial cells), ciliated cells with transport ability (eg, airway epithelial cells), cells for extracellular matrix secretion (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system Cells (eg, T lymphocytes), sensory cells (eg, sputum cells), autonomic nervous system neurons (eg, cholinergic neurons), sensory organs and peripheral neuron support cells (eg, associated cells), central nervous system Neurons and glial cells (eg, astrocytes), pigment cells (eg Retinal pigment epithelial cells), and progenitor cells (tissue progenitor cells), etc. There is no particular limitation on the degree of cell differentiation and the age of the animal from which the cells are collected, and this is the same for both undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells. It can be used as the source of somatic cells in the invention. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells. In the present invention, the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human. The cells may be cells obtained by artificial manipulation after collecting the somatic cells.

In the present invention, “identifying a cell endogenous protein” refers to detecting, identifying, classifying, and selecting a cell endogenous protein using a cell alive. Alternatively, the present invention can also be referred to as a method of identifying a cell, and in this case, it can be referred to as a method of identifying a target cell using a specific cell endogenous protein as an index. For cell identification, after identification, the cell type that expresses the protein is identified, classified, isolated, removed, determined to be alive or dead, and a specific biological signal of the cell type is detected. Alternatively, it may include quantification and fractionation based on a specific physical or chemical signal of the cell type.

In the step (1) of this embodiment, intracellular endogenous protein-responsive mRNA is used. In the present specification, intracellular endogenous protein-responsive mRNA is sometimes referred to as protein-responsive mRNA or mRNA switch. The protein-responsive mRNA comprises (i) an aptamer sequence specific for a cellular endogenous protein and (ii) a marker gene sequence operably linked thereto. .

(i) Aptamer Sequence Specific to Cellular Endogenous Protein The aptamer sequence specific to the cell endogenous protein may be any RNA aptamer sequence that specifically binds to the endogenous protein in the desired cell. Therefore, it may be a natural aptamer sequence already known in literature or the like, an artificial aptamer sequence obtained by modifying it, or other artificially produced aptamer sequences. The natural aptamer sequence can be appropriately selected from, for example, a database available on the website: (Apta-Index (trademark): http://www.aptagen.com/aptamer-index/aptamer-list.aspx). Artificial aptamer sequences can be obtained by a technique for selecting RNA that binds to a target protein from an RNA population containing random sequences (reference document [3]). This method is known as an evolutionary engineering method known as in vitro selection method or SELEX method. Thus, aptamers that bind to the desired protein can be obtained from artificial RNA sequences. By incorporating these aptamers into mRNA, an mRNA switch that responds to a desired protein can be created. Specific aptamers and stabilized aptamers suitable for the method of the present invention will be described in detail in the second and third embodiments.

(ii) Marker gene sequence operably linked thereto The “marker gene” of (ii) used in the present invention is any marker gene that is translated in a cell and functions as a marker and enables identification of the cell. It is a gene that encodes a protein. Examples of proteins that can be translated into cells and function as markers include, for example, visualization, quantification by assisting fluorescence, luminescence, coloration, or fluorescence, luminescence, or coloration of fluorescence, fluorescent proteins, etc. Including, but not limited to, a gene that encodes a protein that can be activated, a membrane protein, or a gene that encodes a protein that kills a cell when expressed, such as an apoptosis-inducing gene or a suicide gene. In combination with an apoptosis-inducing gene, an apoptosis-inhibiting gene can also be used as a marker gene. In the present specification, a protein translated from mRNA containing a nucleic acid corresponding to the coding region of the marker gene is referred to as a marker protein.

In the present invention, fluorescent proteins include blue fluorescent proteins such as Sirius, BFP, and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (for example, hmAG1 ), Green fluorescent proteins such as ZsGreen, EmGFP, EGFP, GFP2, HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana; KusabiraOrange (eg, hmKO2), mOrange Orange fluorescent proteins such as: TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry and other red fluorescent proteins; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (eg, hdKeimaRed), mRasberry, Examples include, but are not limited to, near infrared fluorescent proteins such as mPlum.

In the present invention, the photoprotein can be exemplified by aequorin, but is not limited thereto. Examples of proteins that assist fluorescence, luminescence, or coloration include, but are not limited to, enzymes that degrade fluorescence, luminescence, or color precursors such as luciferase, phosphatase, peroxidase, and β-lactamase. Here, in the present invention, when a substance that assists fluorescence, luminescence, or color is used as a marker gene, in selection of a cell that contains or does not contain a specific protein, the corresponding precursor is brought into contact with the cell. Alternatively, it can be done by introducing the corresponding precursor into the cell.

In the present invention, the apoptosis-inducing gene means a gene encoding a protein having apoptosis-inducing activity for cells. For example, IκB, Smac / DIABLO, ICE, HtrA2 / OMI, AIF, endonuclease G, Bax, Bak, Noxa, Hrk (harakiri), Mtd, Bim, Bad, Bid, PUMA, activated caspase-3, Fas, Tk, etc. For example, but not limited to. In the present invention, Bim is preferably used as an apoptosis-inducing gene. .

In the present invention, the suicide gene means a gene whose expression in a cell is lethal to the cell. In the present invention, the suicide gene may be one that causes cell death by itself (eg, diphtheria A toxin) or the expression of this gene sensitizes the cell to a specific drug (eg, Or sensitize cells to antiviral compounds by expression of the herpes simplex thymidine kinase gene. Examples of suicide genes include diphtheria A toxin, herpes simplex thymidine kinase gene (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine Genes encoding kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), etc. However, it is not limited to these. In the present invention, HSV-TK is preferably used as a suicide gene.

In the present invention, the marker gene may include a gene encoding a localization signal. Examples of the localization signal include a nuclear localization signal, a cell membrane localization signal, a mitochondrial localization signal, a protein secretion signal, and the like. Specifically, a classical nuclear translocation sequence (NLS), M9 Examples include, but are not limited to, sequences, mitochondrial target sequences (MTS), and endoplasmic reticulum translocation sequences. Such a localization signal is obtained when, for example, imaging cytometry described later, a cell that contains or does not contain a specific protein is detected, and further, a step of separating and sorting such cells is performed on the image. Particularly advantageous.

In the present invention, an aptamer sequence and a marker gene sequence are operatively linked to each other within the 5′UTR, 3′UTR, and / or the open reading frame (including the start codon) encoding the marker gene. It means that an aptamer sequence is provided in the open reading frame. The protein-responsive mRNA preferably comprises a Cap structure (7 methylguanosine 5 ′ phosphate), an open reading frame encoding a marker gene, and a poly A tail in the 5 ′ to 3 ′ direction from the 5 ′ end. An aptamer sequence within the 5'UTR, 3'UTR, and / or in the open reading frame. The position of the aptamer sequence in mRNA may be 5′UTR, 3′UTR, or may be within the open reading frame (3 ′ side of the start codon).

Preferably, in the protein-responsive mRNA, the nucleic acid sequences (i) and (ii) are linked in this order in the 5 ′ to 3 ′ direction. At this time, the number of bases and the kind of base between the cap structure and the aptamer sequence may be arbitrary as long as they do not constitute a stem structure or a three-dimensional structure. For example, the number of bases between the cap structure and the aptamer sequence can be designed to be 0 to 50 bases, preferably 0 to 20 bases. The number of bases and the type of base between the aptamer sequence and the start codon may be arbitrary as long as they do not constitute a stem structure or a three-dimensional structure, and the number of bases between the aptamer sequence and the start codon is 0-50. The base can be designed so that the base is preferably 10 to 30 bases.

In the present invention, it is preferable that an AUG serving as a start codon does not exist in the aptamer sequence. For example, if the aptamer sequence is present in the 5 'UTR and AUG is included in the aptamer sequence, it should be designed to be in-frame in relation to the marker gene linked to the 3' side. Is preferred. Alternatively, if the aptamer sequence contains an AUG, it can be converted to another triplet sequence if it is not important for target binding. When AUG is present in the stem structure and the base is substituted, the corresponding base is also substituted so that a base pair is formed after the substitution. In addition, in order to minimize the influence of AUG in the aptamer sequence, the location of the aptamer sequence in the 5 ′ UTR can be changed as appropriate. For example, the number of bases between the cap structure and the AUG sequence in the aptamer sequence is 0 to 60 bases, for example, 0 to 15 bases, 10 to 20 bases, 20 to 30 bases, 30 to 40 bases, 40 to 50 bases , Can be designed in an arrangement of 50-60 bases. However, it is desirable that the aptamer insertion position is close to the cap structure.

Such mRNA can be prepared by genetic engineering if the nucleic acid sequence is designed. For example, it can be prepared by a PCR method based on a predetermined primer and template DNA, but is not limited to a specific adjustment method. Also, a vector encoding such mRNA can be appropriately prepared using a commercially available DNA vector system once a predetermined mRNA sequence is designed and determined.

Next, each step of the present invention will be described. Step (1) is a step of introducing a protein-responsive mRNA or a vector encoding the mRNA into a desired cell (hereinafter sometimes referred to as an introduction step).

The protein-responsive mRNA can be introduced using a lipofection method, a liposome method, an electroporation method, a calcium phosphate coprecipitation method, a DEAE dextran method, a microinjection method, a gene gun method, or the like. Optionally, two, three or four or more protein-responsive mRNAs having different aptamer sequences and marker gene sequences can be co-introduced. Furthermore, at this time, in some cases, control mRNA can be co-introduced into the target cells. Control mRNA refers to mRNA that does not have an aptamer sequence and encodes a marker gene different from the marker gene encoded by protein-responsive mRNA. The activity ratio of marker proteins expressed from two or more co-introduced mRNAs is constant within the cell population. In addition, the introduction amount at this time varies depending on the cell group to be introduced, the mRNA to be introduced, the introduction method and the kind of the introduction reagent, and those skilled in the art can appropriately select these in order to obtain a desired translation amount. The amount of control mRNA introduced can also be appropriately selected by those skilled in the art to obtain a desired translation amount.

The introduction of protein-responsive mRNA can also be carried out by introducing a vector encoding the mRNA. Introduction with a more stable DNA vector is particularly useful when the introduced cell is not used for subsequent transplantation, for example, when used for screening or testing. Introduction of a vector into a cell can be performed by the same means as introduction of mRNA. The same method can be used when introducing a vector encoding two or more kinds of protein-responsive mRNAs having different aptamer sequences and marker gene sequences, or when introducing a vector encoding a control mRNA.

When protein-responsive mRNA is introduced into a cell, if there is a protein specific to the aptamer in the cell, the amount of translation of the marker gene encoded by the protein-responsive mRNA is controlled by binding the protein to the aptamer. For example, the translation amount is suppressed. The amount of translation is controlled quantitatively according to the amount of protein present in the cell. On the other hand, when the protein does not exist in the cell, the translation amount of the marker gene encoded by the protein-responsive mRNA is not suppressed. Therefore, the translation amount of the marker gene differs between a cell in which the protein is present or expressed and a cell in which the protein is not present or not expressed. On the other hand, the control mRNA expresses the marker protein regardless of the presence of the protein. This is because even when the control mRNA is introduced into the cell, the aptamer sequence does not exist in the control mRNA, so that the translation is not controlled according to the presence and / or amount of the predetermined protein.

The subsequent step (2) is a step of identifying a cellular endogenous protein based on the translation amount of the marker gene (hereinafter sometimes referred to as an identification step). In the identification step, the cellular endogenous protein is identified based on the translation amount of the marker gene as described above. That is, when a cell endogenous protein specific to the aptamer sequence provided in the protein-responsive mRNA is present in the cell, the cell endogenous protein is identified based on the phenomenon that the translation amount of the marker gene is suppressed. In the “discriminating” mode, the presence or absence of suppression of marker gene translation is detected, the presence or absence of a predetermined cellular endogenous protein is determined, and the endogenous protein is quantified by measuring the translational suppression efficiency of the marker gene In addition, in the case where the marker gene is a drug resistance gene or the like, it is possible to confirm the presence of the endogenous protein based on the viability of the cell after culturing in the presence of the drug. Is not limited. Other identification modes include control of target cell fate by a marker gene by an apoptosis-inducing gene, cell differentiation by a differentiation-inducing gene or a reprogramming-inducing gene, and detection of a reprogramming cell.

Specifically, the identification step can be performed by detecting a signal from the marker protein using a predetermined detection device. Examples of the detection device include, but are not limited to, a flow cytometer, an imaging cytometer, a fluorescence microscope, a light emission microscope, and a CCD camera. As such a detection apparatus, those suitable for those skilled in the art can be used depending on the marker protein and the detection mode. For example, when the marker protein is a fluorescent protein or a luminescent protein, the marker protein can be quantified using a detection device such as a flow cytometer, an imaging cytometer, a fluorescence microscope, or a CCD camera. In the case of a protein that assists in luminescence or coloration, a marker protein quantification method using a detection device such as a luminescence microscope, a CCD camera, or a luminometer is possible. When the marker protein is a membrane-localized protein, A marker protein quantification method using a detection reagent specific to a cell surface protein such as an antibody and the above-described detection apparatus is possible. In addition, it is an identification method such as a magnetic cell separator (MACS) that does not go through the quantification process of the marker protein, and can be performed by substantially isolating the cells. In this case, a discrimination method by isolating a living cell based on expression of a marker gene by drug administration is possible. .

An example of a preferable identification (detection) method when the marker protein is a fluorescent protein is flow cytometry. Flow cytometry can provide the intensity of light emitted by fluorescent proteins and luminescent enzymes, which are marker proteins translated in individual cells, as identification information, especially the fluorescence intensity ratio measured with a flow cytometer. Thus, identification information can be obtained. For example, when a fluorescent protein is used, cells with different amounts of translation of the marker gene are observed as separate bands on a flow cytometry dot plot. In other words, since cells are separated based on the amount of endogenous protein detected on a flow cytometry dot plot, it is also possible to physically separate these cells after detection. It may be an aspect. Alternatively, it is also effective to obtain identification information by measuring the fluorescence intensity ratio by cell image analysis using an imaging cytometer or the like.

The method for measuring the translation amount of protein-responsive mRNA is not limited to the specific method described above, and can be carried out by any other method. For example, the amount of translation can be measured by a method such as quantification of the protein expression level by Western blot, and the present invention also constitutes the case where measurement is performed using such a method.

The cells after measuring the amount of translation are separated, sorted, and sorted based on the presence or absence of a protein that specifically binds to the aptamer or the difference in the expression level, and further artificial processing or transplantation into the cell. Can be used for applications. Such separation, separation, and sorting are described as one aspect of the identification. In particular, in the step (1), when protein-responsive mRNA is directly introduced into a cell, the mRNA can be metabolized in the cell with a half-life of about 10 hours to 1 day, which is advantageous from the viewpoint of safety. It is. According to the method for identifying a cellular endogenous protein and the method for identifying a cell according to this embodiment, the expression of a specific protein in the cell can be identified alive using a living cell, and This is very useful in that cells expressing a specific protein can be selectively isolated while alive. .

As a further application mode, the protein-responsive mRNA according to the present embodiment and the microRNA-responsive mRNA are used in combination to detect and / or identify cells expressing a specific protein and a specific microRNA. A method is mentioned. The microRNA-responsive mRNA is an mRNA having a microRNA response element instead of the aptamer described above. Such a microRNA response element and a method for selecting a living cell using the same are described in detail in International Publication WO2015 / 105172 by the present inventors.

The following can be expected in the future depending on the protein-responsive mRNA (also referred to as mRNA switch) used in the present embodiment and its production principle. An advantage of an mRNA switch is that a protein expressed as an output can function as an input for another mRNA switch. Therefore, by combining a plurality of mRNA switches, it is possible to construct an artificial genetic circuit based on translation control and having a low risk of genome damage. In addition, by using such an artificial gene circuit, safe and highly efficient cell reprogramming (including initialization and differentiation) can be expected. Furthermore, by designing a circuit that takes a specific gene expression pattern, it can also be applied to maintain the cell state. For example, to prevent spontaneous differentiation of cultured iPS cells. Can be used.

Furthermore, since the translation efficiency of the invented switch correlates with the amount of intracellular protein expression, it is considered that the amount of protein in living cells can be quantified using a protein-responsive mRNA switch. This method does not require disruption of cells as in Western blotting, and it is not necessary to prepare a fusion protein with a detection tag, so it can be used to observe the amount of protein in living cells in a more natural state. . In addition, by increasing the transfection efficiency, the protein expression level at the level of one cell can be expected. Furthermore, by combining with FACS, it becomes possible to isolate cells expressing a specific amount of protein. .

[Second embodiment: Structure-stabilized LIN28A aptamer]
According to the second embodiment, the present invention is a structure-stabilized LIN28A aptamer. The LIN28 aptamer is a stabilized LIN28A aptamer obtained by adding one or more of the following modifications based on the RNA secondary structure formed by the natural LIN28A aptamer sequence represented by SEQ ID NO: 1.
(1) AU base pairs constituting a stem including a 5 ′ terminal base and a 3 ′ terminal base are replaced with GC base pairs;
(2) The AU base pair constituting the stem located between the two loops is replaced with a GC base pair; or (3) The non-Watson-Crick base pair constituting the stem located between the two loops is replaced with the GC base pair. Replace with.

The natural LIN28A aptamer represented by SEQ ID NO: 1 has been reported to have the secondary structure represented by Original in FIG. 3 (Nam, Y., Chen, C., Gregory, R. I., Chou, J. J. & Sliz, P. Molecular basis for interaction of let-7 microRNAs with Lin28. Cell 147, 1080-91 (2011), on the other hand, by modifying (1), the 5 'end The stem including the base and the 3 ′ terminal base can be further stabilized, and it is not necessary to replace all AU base pairs present in the natural LIN28A aptamer with GC base pairs, and at least one AU base pair can be replaced with GC. Stabilization can be achieved by substituting base pairs, but from the standpoint of further stabilization, preferably more, more preferably all AU base pairs are replaced with GC base pairs.

Next, it is located between two loops by modifying (2) the secondary structure represented by Original in FIG. 3 or the stabilized LIN28A aptamer modified in (1). The stem can be further stabilized. Again, it is not necessary to replace all AU base pairs with GC base pairs, but preferably more, more preferably all AU base pairs are replaced with GC base pairs.

Further, the modification (3) is performed on the secondary structure represented by Original in FIG. 3, the stabilized LIN28A aptamer modified (1), or the stabilized LIN28A aptamer modified (2). Thus, the stem located between the two loops can be further stabilized. .

In making these modifications, the base sequence important for binding to LIN28A (except the base at the root of the stem, which forms the AU base pair or GC base pair) is changed, or the loop It is not preferable to change the structure. This is because, in the natural type LIN28A aptamer, the loop structure is known as a structure that specifically recognizes the LIN28A protein. In general, in the LIN28A aptamer, base sequences important for binding to LIN28A are GNGAY (N is an arbitrary base, Y is C or U) and GGAG. In the specific LIN28A aptamer shown below, GGGAU and GGAG (shown underlined in the table).

Further optional modifications include (4) adding 1 to 15 base pairs to the stem containing the 5 'terminal base and the 3' terminal base.

As specific embodiments, the stabilized LIN28A aptamer stbA (SEQ ID NO: 2, stbA in FIG. 3) modified by (1) above, and stabilized by modifying (1) and (2) above. There are stbB (SEQ ID NO: 3, stbB in FIG. 3) and stbC (SEQ ID NO: 4, stbC in FIG. 3) which are LIN28A aptamers. Table 1 below shows the sequences of natural and stabilized three aptamers. Bases modified from the original aptamer are shown in lower case.

The aptamers represented by SEQ ID NOs: 1 to 4 can be used as aptamer sequences for protein-responsive mRNA according to the first embodiment. The design and production of a protein-responsive mRNA having an aptamer sequence is described in detail in the first embodiment. And, the translational repression efficiency of the LIN28A-responsive mRNA used with these aptamer sequences is, in descending order, the LIN28A-responsive mRNA having the stbC sequence, the LIN28A-responsive mRNA having the stbB sequence, the LIN28A-responsive mRNA having the stbA sequence, It is a LIN28A-responsive mRNA having a natural LIN28A aptamer sequence.

The stabilized LIN28A aptamer according to the second embodiment is useful for detecting specific expression of LIN28A protein, and particularly useful for detecting pluripotent stem cells such as iPS cells and ES cells. In addition, LIN28A-responsive mRNA (also referred to as LIN28A switch) can be expected to be used as a stem cell sorting technique. Since LIN28A is a protein present in the cytoplasm, as long as RNA enters the cytoplasm, there is an advantage that intracellular proteins can be easily detected with an mRNA switch such as LIN28A-responsive mRNA. By changing the protein expressed by the switch from a reporter protein to an apoptosis-inducing protein, cell death is induced specifically in non-stem cells. As described above, the use of a protein-responsive mRNA switch has a low risk of genome damage, and can be expected to be applied to the field of regenerative medicine as a safe stem cell / differentiated cell selection technique. Therefore, development of a new nucleic acid drug with few side effects can be expected. .

[Third Embodiment: Modified U1A Aptamer]
According to the third embodiment, the present invention is a structure-stabilized U1A aptamer. The U1A aptamer is a structure-stabilized U1A aptamer based on the RNA secondary structure formed by the natural U1A aptamer sequence represented by SEQ ID NO: 6, with the following modifications.
(1) Add 1 to 15 AU base pairs or GC base pairs to a stem structure containing a 5 ′ terminal base and a 3 ′ terminal base. Alternatively, 1 to 15 AU base pairs or GC base pairs are added to the stem between the two boxes. By the above modification, the aptamer is designed to form a secondary structure necessary for binding to the target.
Alternatively, it is a structure-stabilized U1A aptamer based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8, with the following modifications.
(2) Add 1 to 15 AU base pairs or GC base pairs to the stem containing the 5 ′ terminal base and the 3 ′ terminal base.

It has been reported that the natural U1A aptamer represented by SEQ ID NO: 6 has a secondary structure represented by the lower U1utr in FIG. 12 (reference document [4]). On the other hand, the stem existing between Box1 and Box2 can be stabilized by modifying (1). The 1 to 15 base pairs to be added may be AU base pairs, GC base pairs, or combinations thereof, but preferably more GC base pairs are added, More preferably, all added base pairs are GC base pairs.

As a specific embodiment, there is s U1utr_stb (SEQ ID NO: 7, U1utr_stb in FIG. 12), which is a stabilized U1A aptamer modified in (1) above. This sequence and the sequence of natural U1utr are shown in Table 2 below.

The U1A aptamer known in the literature represented by SEQ ID NO: 8 has been reported to have a secondary structure represented by U1LSL in FIG. 12 (reference document [5]). On the other hand, the stem containing the 5 ′ terminal base and the 3 ′ terminal base can be stabilized by modifying (2). The 1 to 15 base pairs to be added may be AU base pairs, GC base pairs, or combinations thereof, but preferably more GC base pairs are added, More preferably, all added base pairs are GC base pairs.

As specific embodiments, U1LSL + 10 bp (SEQ ID NO: 9, U1LSL + 10 bp in FIG. 12) and U1LSL + 15 bp (SEQ ID NO: 10, FIG. 12), which are the stabilized U1A aptamers modified in (2) above. U1LSL + 15bp). These sequences and the sequence of natural U1LSL are shown in Table 2 below. .

 

The aptamers and stabilized aptamers represented by SEQ ID NOs: 5 to 10 can be used as aptamer sequences of protein-responsive mRNA according to the first embodiment. The design and production of a protein-responsive mRNA having an aptamer sequence is described in detail in the first embodiment. .

The stabilized U1A aptamer according to the third embodiment may be useful for detecting specific expression of U1A protein. U1A protein has been reported to be highly expressed in certain types of cancer cells, and can be used to detect and separate cancer cells by applying the method for detecting cellular endogenous proteins based on the first embodiment. There is expected.

[Fourth Embodiment: Modified p50 aptamer]
The present invention is a structure-stabilized p50 aptamer according to the fourth embodiment. The p50 aptamer is an artificially obtained aptamer represented by SEQ ID NO: 83, and based on the RNA secondary structure formed by the artificial p50 aptamer reported by the literature [6], the following modifications:
(1) A structure-stabilized p50 aptamer obtained by adding 1 to 20 AU base pairs or GC base pairs to a stem structure containing a 5 ′ terminal base and a 3 ′ terminal base.

It has been reported that the p50 aptamer represented by SEQ ID NO: 83 has the secondary structure shown in the left diagram of FIG. 17 (reference document [6]). On the other hand, the stem can be stabilized by modifying (1). The 1 to 20 base pairs to be added may be AU base pairs, GC base pairs, or a combination thereof, but preferably more GC base pairs are added, More preferably, all added base pairs are GC base pairs. The added base pair is preferably 5 to 20 base pairs, more preferably 10 to 20 base pairs.

As a specific embodiment, there is p50A-stb (SEQ ID NO: 84, right diagram p50A-stb in FIG. 17) which is a stabilized p50 aptamer modified in (1) above. This sequence and the sequence of natural U1utr are shown in Table 3 below.

 

The aptamer and the stabilized aptamer represented by SEQ ID NO: 84 can be used as the aptamer sequence in the protein-responsive mRNA described in the first embodiment. The design and production of a protein-responsive mRNA having an aptamer sequence is described in detail in the first embodiment.

The stabilized p50 aptamer according to the fourth embodiment may be useful for detecting specific expression of cancer-associated protein p50. p50 has been reported to be highly expressed in certain types of cancer cells, and can be used for detection and sorting of cancer cells by applying the method for detecting cellular endogenous proteins based on the first embodiment. Be expected.

[Fifth Embodiment: Method for Identifying Cell Differentiation (Reprogramming) State]
According to the fifth embodiment, the present invention is a method for identifying a differentiation state of a cell. The method includes the following steps.
(1) Introduction of an mRNA containing an aptamer sequence specific to a protein expressed by undifferentiated cells and a marker gene sequence operably linked thereto, or a vector encoding the mRNA into a cell group that can contain pluripotent stem cells And (2) identifying the differentiation state of the cell based on the translation amount of the marker gene.

In this embodiment, “identification of the differentiation state of a cell” refers to identifying that a cell is differentiated or undifferentiated, its change, degree, and the like. For example, in one embodiment (a), cells that have been contacted with the reprogramming factor are cultured for a predetermined method and for a period of time, and then reprogrammed to become induced pluripotent stem (iPS) cells. Detecting, detecting, recognizing, identifying, and separating the state of initialization such as whether or not reprogramming is not successful and the target iPS cell is not included Shall. It is known from previous studies that iPS cells express LIN28A (Tano, K. et al. A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using acientcientHighly Culture System. PLoS One 9 e110496 (2014).) Among cells that have been contacted with reprogramming factors, cells that express a sufficient amount of LIN28A (cells with endogenous LIN28A) are in the desired reprogramming state. It can be said that it is an iPS cell of interest. In another embodiment (b), pluripotent stem cells remaining without differentiation are detected in a group of cells that have undergone a predetermined differentiation operation for pluripotent stem cells including iPS cells. Detecting and recognizing, or selectively separating and sorting the cells. When iPS cells and other pluripotent stem cells are differentiated into desired cells and used clinically, the most hindrance is the cells that have not differentiated and have tumorigenic potential (PLoS One . 2012; 7 (5): e37342. Doi: 10.1371 / journal.pone.0037342. Epub 2012 May 17.). Therefore, a technique for selectively removing cells that retain pluripotency from the differentiated cell group has been strongly desired. In the present invention, an aptamer sequence specific to a protein expressed by undifferentiated cells, typically a LIN28A-responsive mRNA that specifically detects LIN28A, is used to detect cells that retain pluripotency, Can be detected and recognized, and in preferred embodiments can be separated and selectively removed. At this time, it has been confirmed that the LIN28A switch can identify undifferentiated cells without significant side effects. However, the differentiation state of cells is not limited to a specific mode, and undifferentiated cells are detected, identified, separated, and removed from a group of cells that may be a mixture of cells having different differentiation states. Make it possible.

The “cell group that can contain pluripotent stem cells” used in step (1) of the present embodiment is, for example, cells that have been brought into contact with an reprogramming factor and cultured after a predetermined method and period, It may be a cell that is programmed to be predicted to be in the state of an induced pluripotent stem (iPS) cell. Alternatively, it is a cell group obtained by performing a predetermined differentiation operation on pluripotent stem cells, and refers to a cell group in which undifferentiated cells can remain. However, the cell group may not contain undifferentiated cells. Alternatively, it may be a cell group that may contain any other pluripotent stem cells, and the purpose of detection and the origin of the cell group are not particularly limited. *

Next, cells brought into contact with the reprogramming factor, which is a cell group that can contain pluripotent stem cells preferably used in the embodiment (a), will be described. In this embodiment, the cell brought into contact with the reprogramming factor typically refers to a cell that has been artificially manipulated for the purpose of preparing iPS cells. iPS cells can be generated by introducing specific reprogramming factors into somatic cells in the form of DNA, RNA, or protein. Proliferation with almost the same characteristics as ES cells, such as differentiation pluripotency and self-renewal. An artificial stem cell derived from a somatic cell having the ability (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666). The reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-cording RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be constituted by a low molecular compound. Examples of genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination. As combinations of reprogramming factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010/056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010/111409, WO 2010/111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. 2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467 -2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008 ), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-13 5, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. ( 2009), Proc Natl Acad Sci U S A. 106: 8912-8917, Kim89JB, 17et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5 : 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. ( 2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 、 1293, M344, siRNA and shRNA against HDAC (eg Nucleic acid expression inhibitors such as HDAC1DACsiRNA Smartpool (registered trademark) (Millipore), HuSH 29mershRNA Constructs against HDAC1 (OriGene), etc.], MEK inhibitors (eg, PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitors (eg, Bio and CHIR99021), DNA methyltransferase inhibitors (eg, 5-azacytidine), histone methyltransferase inhibitors (eg, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2, SetDBl And nucleic acid expression inhibitors such as siRNA and shRNA against G9a), L-channel calcium agonist (eg Bayk8644), butyric acid, TGFβ inhibitor or ALK5 inhibitor (eg LY3649) 47, SB431542, 616453 and A-83-01), p53 inhibitors (eg siRNA and shRNA against p53), ARID3A inhibitors (eg siRNA and shRNA against ARID3A), miR-291-3p, miR-294, miR- MiRNA such as 295 and mir-302, Wnt Signaling (eg soluble Wnt3a), neuropeptide Y, prostaglandins (eg prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISL, PITX2 , DMRTBl and other factors used for the purpose of increasing the establishment efficiency are also included.In this specification, the factors used for the purpose of improving the establishment efficiency are also distinguished from the initialization factor. Shall not.

In the case of a protein form, the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, HIV-derived TAT and polyarginine), or microinjection.

On the other hand, in the case of DNA, it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection. Virus vectors include retrovirus vectors, lentivirus vectors (cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054), and the like. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed. For example, selectable marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can be included. In addition, the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.

In the case of RNA, it may be introduced into somatic cells by techniques such as lipofection and microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine® (TriLink® Biotechnologies) is used. Yes (Warren L, (2010) Cell Stem Cell. 7: 618-630).

As a culture solution for inducing iPS cells, for example, DMEM, DMEM / F12 or DME culture solution containing 10-15% FBS (in addition to these culture solutions, LIF, penicillin / streptomycin, puromycin, L- Glutamine, non-essential amino acids, β-mercaptoethanol, etc. may be included as appropriate.) Or a commercially available culture medium (for example, a culture medium for mouse ES cell culture (TX-WES culture medium, Thrombo X), primate ES Medium for cell culture (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology, Essential 8, Life Technologies, StemFit, Ajinomoto).

As an example of the culture method, for example, a somatic cell is brought into contact with a reprogramming factor on a DMEM or DMEM / F12 medium containing 10% FBS at 37 ° C. in the presence of 5% CO ₂ for about 4 to 7 days. Then, re-spread the cells on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact between the somatic cells and the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.

Alternatively, 10% FBS-containing DMEM medium (including LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) in the presence of 5% CO ₂ at 37 ° C. Can be suitably included with puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) and can form ES-like colonies after about 25 to about 30 days or more . Desirably, instead of feeder cells, somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349), Matrigel (BD) and iMatrix511 (Nippi)).

In addition to this, a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106: 15720-15725). Furthermore, in order to increase the establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845). .

During the culture, the culture medium is exchanged with a fresh culture medium once a day from the second day onward. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish. As used herein, the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell. Say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines. Specifically, somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells. When iPS cells are used as a material for transplantation cells, it is desirable to use somatic cells having the same or substantially the same HLA genotype of the transplant destination individual from the viewpoint of preventing rejection. Here, “substantially the same” means that the HLA genotype matches the transplanted cells to such an extent that an immune response can be suppressed by an immunosuppressive agent. For example, HLA-A, HLA-B And somatic cells having an HLA type in which 3 loci of HLA-DR or 4 loci plus HLA-C are matched.

Next, a cell group obtained by performing an operation for predetermined differentiation on the pluripotent stem cell, which is a cell group that can contain the pluripotent stem cell preferably used in the embodiment (b), will be described. In the present invention, a pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into many cells existing in a living body and also has proliferative ability, and can be arbitrarily induced into a desired differentiated cell. Cells are included. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic Examples include germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts and bone marrow stem cell-derived pluripotent cells (Muse cells). A preferred pluripotent stem cell is an iPS cell, more preferably a human iPS cell, from the viewpoint that it can be obtained without destroying an embryo, an egg or the like in the production process. Other pluripotent stem cells other than the above iPS cells will be described below.

(A) Embryonic stem cells Embryonic stem cells (ES cells) are established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice, and have the ability to proliferate through self-replication. It has stem cells.

ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod 55: 254-259; JA JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution to which substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (basic fibroblast growth factor (bFGF)) are added. It can be carried out. For methods of establishing and maintaining human and monkey ES cells, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl Acad. Sci. USA, ； 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485. *

For example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF is used as the culture medium for ES cell production. Human ES cells can be maintained in a humid atmosphere at 37 ° C., 5% CO ₂ (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932). ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl ₂ and 20% KSR. Can be used.

ES cells can be generally selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index. In particular, in the selection of human ES cells, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).

Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute of Regenerative Medicine (Kyoto, Japan), Kyoto University Is possible.

(B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is capable of self-replication in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and by repeating subculture under the same culture conditions as ES cells, Stem cells can be obtained (Masatake Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).

(D) A cloned embryo-derived ES cell obtained by nuclear transfer A cloned embryo-derived ES cell (nt ES cell) obtained by nuclear transfer is a cloned embryo-derived ES cell produced by nuclear transfer technology, It has almost the same properties as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72 : 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell. For the production of nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology is used (Kiyaka Wakayama et al. ( 2008), Experimental Medicine, Vol.26, No.5 (extra number), 47-52). Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.

(E) Multilineage-differentiating Stress Enduring cells
Multilineage-differentiating Stress Enduring cells (Muse cells) are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time. Preferably, it is a pluripotent cell obtained by trypsin treatment for 8 hours or 16 hours and then suspension culture, and is positive for SSEA-3 and CD105.

A method for inducing differentiation of these pluripotent stem cells into predetermined differentiated cells is known, and those skilled in the art can implement a specific differentiation induction method according to the target cell. As an example, cardiomyocytes can be produced from pluripotent stem cells as a method for inducing differentiation into cardiomyocytes, for example, by a method reported by Laflamme MA et al. (Laflamme MA & Murry CE, Nature 2011, Review). Although not particularly specified, for example, a method of producing cardiomyocytes by forming a cell mass (embryoid body) by suspension culture of induced pluripotent stem cells, myocardium in the presence of a substance that suppresses BMP signaling A method for producing cells (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), and producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signaling pathway And a method for isolating Flk / KDR positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporin A (WO2009 / 118928). As another example, as a method of inducing differentiation into tissue cells such as endothelial cells, hepatocytes or insulin-producing cells, for example, Kajiwara M, et al, Proc Natl Acad Sci U S A. 109: 12538-12543, 2012, Kunisada Y, et al, Stem Cell Res. 8: 274-284, 2012, Nakagawa, M, et al, Sci Rep 4, 3594, 2014, etc., to produce tissue cells from pluripotent stem cells Can do. As yet another example, as a method for inducing differentiation into nerve cells, for example, nerve cells can be produced from pluripotent stem cells by the methods disclosed in WO2011 / 019092 and WO2011 / 158960.

Step (1) is a step of introducing a vector encoding an mRNA having an aptamer sequence specific to a protein expressed by an undifferentiated cell into a cell group that can contain pluripotent stem cells such as the cell group exemplified above. is there. The step of introducing mRNA or vector into the cell may be the same as the method described in step (1) of the first embodiment. This step may be performed, for example, at any time during the initialization step or during the differentiation step for the embodiments (a) and (b). Differentiated state can be obtained. Embodiment (a) can be performed in particular at the time when iPS-like colonies are formed and iPS cells are predicted to be generated, that is, when it is predicted that they have been reprogrammed. This is because the iPS cell culture medium contains a mixture of reprogrammed iPS cells and cells that have not been initialized at this point, and has an advantage of identifying the reprogrammed iPS cells. The embodiment (b) can be carried out at a time when it is predicted that a predetermined differentiation-induced cell, for example, a cardiomyocyte or a nerve cell is obtained.

Step (2) is a step of identifying the differentiation state of the cell based on the translation amount of the marker gene. The identification step can be performed by a method substantially similar to that described in the first embodiment. For example, by measuring the translational suppression efficiency of a marker gene, the expression level of a protein expressed by an undifferentiated cell, typically, LIN28A in the cell can be quantified. Thus, information about whether the cell is a reprogrammed iPS cell or another pluripotent stem cell (undifferentiated cell) can be obtained. IPS cells reprogrammed to a desired state and undesired undifferentiated cells remaining in differentiated cells can be selectively separated after identification or simultaneously with identification. These separations can be typically performed by using fluorescent proteins as marker genes and separating cells in a predetermined translation state by flow cytometry and imaging cytometry. Alternatively, when a drug resistance protein or a lethal gene is used as a marker protein, selective separation can be performed simultaneously with identification.

Conventionally, pluripotent stem cells such as iPS cells and ES cells can be selected according to the shape of the formed colonies. In addition, cell surface proteins expressed when somatic cells are reprogrammed (for example, SSEA-1, SSEA-3, SSEA-4, TRA-2-54, TRA-1-60 and TRA-1- 80) was selected as an indicator. According to the method for detecting a differentiation state and a reprogramming state of a cell according to the fifth embodiment of the present invention, mRNA comprising an aptamer sequence specific to a protein expressed by an undifferentiated cell, particularly, a LIN28A-responsive mRNA is used. It is possible to detect and differentiate iPS cells or undifferentiated cells selectively by detecting the differentiation state and reprogramming state of living cells using the expression of undifferentiated cells, especially LIN28A as an index. Become. Compared with the conventional method, there is an advantage that the gene expression state in the cell can be detected while living, not the cell surface protein, and the information inside the cell can be visualized while living.

Hereinafter, the present invention will be described in more detail using examples. The following examples do not limit the invention.

[Plasmid construction]
First, pTAPmyc-T2A-tagRFP and pAptamerCassette-EGFP were prepared as empty vectors for the trigger plasmid and the switch plasmid, respectively.
In preparation of pTAPmyc-T2A-tagRFP, specifically, first, pIRES2-DsRed Express (Clontech) was cleaved with BamHI and NheI, and then double-stranded DNA annealed with synthetic oligos SKW003 and SKW004 was inserted, and pTAP- IRES2-DsRed Ex was produced. Further, this plasmid and pTAP-myc were cleaved with BamHI and XbaI and inserted into pTAP-IRES2-DsRed Ex to prepare pTAPmyc-IRES2-DsRed Ex. Next, the target portion was PCR amplified using pTAP-BS15-T2A-tagRFP as a template and synthetic oligos KWC0093 and KWC0094 as a primer set. This PCR product and pTAPmyc-IRES2-DsRed Ex were cleaved with BglII and NotI, and the digested PCR product was inserted to obtain pTAPmyc-T2A-tagRFP.

PAptamerCassette-EGFP was prepared by cutting pBoxCDGC-kMet-EGFP (reference document [1]) with NheI and AgeI, and then inserting double-stranded DNA annealed with synthetic oligos SKC0041 and SKC0042. In preparation of a LIN28A expression plasmid (pLIN28Amyc-T2A-tagRFP), first, the target portion was amplified using pTrg5H-hLin28 as a template and synthetic oligos SKC0052 and KWC0053 as a primer set. This PCR product and pTAPmyc-T2A-tagRFP were cleaved with SalI and BamHI and then ligated to obtain pLIN28Amyc-T2A-tagRFP.

In preparation of the U1A expression plasmid (pu1Amyc-T2A-tagRFP), first, the target portion was amplified using pTrg5H-U1A as a template and synthetic oligos SKC0052 and KWC0053 as a primer set. This PCR product and pTAPmyc-T2A-tagRFP were cleaved with SalI and BamHI and then ligated to obtain pU1Amyc-T2A-tagRFP.
The sequences of the synthetic oligo DNA used so far are shown in Table 4 below.

In preparation of each switch plasmid, first, pAptamerCassette-EGFP was cut with AgeI and BamHI. Next, double-stranded DNAs obtained by annealing synthetic oligos with the combinations shown in Table 5 below were prepared and inserted into digested pAptamerCassette-EGFP to obtain each LIN28A switch plasmid.

 

Each U1A switch plasmid was prepared in the same manner as the LIN28A switch using oligos in the combinations shown in Table 6.

All plasmids for transfection into cultured cells were purified in large quantities using JETSTAR 2.0 (VERITAS).

[Build IVT template]
The 5′UTR and 3′UTR sequences for protein coding region (ORF), trigger mRNA and reference mRNA preparation were PCR amplified using appropriate primers from plasmid or oligo DNA. The plasmids used as templates and the primers used, and the oligo DNAs used as templates and the primers used are shown in Tables 7, 8, and 9, respectively.

 

 

 

In order to prepare the IVT template for synthesizing the trigger mRNA and the reference mRNA, the 5 ′ UTR fragment, 3 ′ UTR fragment, and ORF were ligated by PCR amplification using the T7FwdA and Rev120A primer sets shown in Table 10.

In order to prepare an IVT template for switch mRNA synthesis, 3 ′ UTR fragment, spacer sequence and ORF were ligated by PCR amplification using 5 ′ UTR primer and Rev120A primer set. Table 5 shows 5 ′ UTR primers and spacer sequences used for the preparation of each switch mRNA.

 

The PCR product was purified using MinElute PCR purification kit (QIAGEN) according to the manufacturer's instructions. When the PCR product was amplified from the plasmid before purification, it was digested with Dpn I (TOYOBO) at 37 ° C for 30 minutes.

[Synthesis and purification of mRNA]
mRNA synthesis was adjusted using MegaScript T7 kit (Ambion) according to reference [2]. In this reaction, except for switch mRNA, pseudouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate (TriLink BioTechnologies) were used instead of uridine triphosphate and cytidine triphosphate, respectively. did. The guanosine triphosphate used was diluted 5-fold with Anti-Reverse Cap Analog (New England Biolabs). The reaction mixture was reacted at 37 ° C for 6 hours, TURBO DNase (Ambion) was added, and the mixture was further incubated at 37 ° C for 30 minutes. The obtained mRNA was purified by FavorPrep Blood / Cultured Cell total RNA extraction clumn (Favorgen Biotech), and incubated at 37 ° C. for 30 minutes using Antarctic Phosphatase (New England Biolabs). Then, it further refine | purified by RNeasy MinElute Cleanup Kit (QIAGEN).

[Cell culture]
HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) -F12 (Invitrogen) containing 10% Fetal Bovine Serum (FBS). HEK293FT cells were cultured in DMEM (Nacalai Tex) supplemented with 10% FBS, 2 mM L-Glutamine (Invitrogen), 0.1 mM Non-Essential Amino Acids (Invitrogen), and 1 mM Sodium Pyruvate (Sigma). iPS cells were cultured on Laminin-coated 6-well plates using StemFit (Ajinomoto) under feeder-free conditions.

[Plasmid transfection]
The cultured cells were seeded in a 24-well plate, and the purified plasmid was introduced the next day so that the total amount became 500 ng. In the experiment using the mutant, HEK293FT cells were seeded in a 96-well plate, and the purified plasmid was introduced the next day so that the total amount was 125 ng. The introduction was performed using 2 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The used plasmid was mixed in Opti-MEM (Invitrogen) so that the quantitative ratio of the switch plasmid and the trigger plasmid was 1: 4. The medium was changed 4 to 6 hours after transfection.

[MRNA transfection]
The cultured cells were seeded in a 24-well plate, and the synthesized mRNA was introduced the next day. The introduction was performed using 1 μL of StmFect (Stemgent) according to the manufacturer's instructions. In experiments using HEK293FT cells, 100 ng of switch mRNA, 0 to 300 ng of trigger mRNA, and 50 ng of reference mRNA were co-introduced. In experiments using iPS cells, 100 ng of switch mRNA and 100 ng of reference mRNA were co-introduced. The medium was changed 4 to 6 hours after transfection. For transfection into iPS cells, the medium was further changed at least 1 hour before introduction.

[Flow cytometry]
The day after transfection, cells were detached from the plate and analyzed by flow cytometry through a mesh. In experiments using 24-well plates, Accuri C6 (BD Biosciences) was used. EGFP and hmAG1 were detected by FL1 (530/30 nm) filters. tagRFP and mKO2 were detected with FL2 (585/40 nm) filters. iRFP670 was detected by FL4 (675 / 12.5 nm) filters filter. In experiments using 96-well plates, BD LSRFORTESSA (BD Bioscuence) was used. EGFP and tagRFP were analyzed by a Blue laser equipped with a FITC filter (530/30 nm) and a Green laser equipped with a PE filter (585/42 nm), respectively. Dead cells and debris were excluded by forward and side light scatter signals. For the calculation of the translation efficiency using a plasmid, among the living cells, those with tagRFP fluorescence values above a certain level were analyzed. For the calculation of translation efficiency using mRNA, living cells with iRFP670 or mKO2 fluorescence values above a certain level were analyzed.

[LIN28A expression induction and transfection by doxycycline] Cell lines that can induce LIN28A expression by adding doxycycline are 10% FBS, 2 mM L-Glutamine (Invitrogen), 0.1 mM Non-Essential Amino Acids (Invitrogen), 1 mM Sodium The cells were cultured in DMEM (Nacalai Tex) supplemented with Pyruvate® (Sigma). Cells were seeded in a 12-well plate and replaced with a medium containing doxycycline (0 ng / mL to 10 ng / mL) before transfection. Transfection was performed the day after seeding, and the purified plasmid was introduced so that the total amount was 400 ng. The introduction was performed using 4 μL of Lipofectamine 2000® (Invitrogen) according to the manufacturer's instructions. The used plasmid was mixed in Opti-MEM (Invitrogen) so that the quantitative ratio of the switch plasmid and the trigger plasmid was 1: 1. The medium was changed 4 hours after transfection.

[Western blotting] Half of the cells to be analyzed by flow cytometry were collected in a microtube and centrifuged at 300 g, 5 min, and 4 ° C. The supernatant was removed, 200 μl PBS was added and resuspended, and then centrifuged at 300 μg, 5 μmin, 4 ° C. This process was performed twice and stored at -80 ° C. To the collected cells, 50 μl of RIPA buffer (25 mM Tris (ph7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) was added on 30 μmin ice to extract the protein. This was centrifuged at 15000 μg for 20 μmin, and the supernatant was collected in another eppen and stored at −20 ° C. For each sample, the protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Scientific). Then, the sample was electrophoresed using Extra <PAGE> One <Precast> Gel <10>-<20% (nacalai> tesque). The marker was prepared by adding 0.5 uL, MagicMark (trademark), XP, Western Protein, Standard, (invitrogen) to 3 uL, Full-Range, Rainbow, Molecular, Weight, Markers, (GE, health care). Each sample was diluted with MilliQ so that the total protein concentration was 5 μg / 15 μL. 6 × SDS buffer 4 μl was added to the prepared sample and boiled at 95 ° C. for 3 μmin. The entire amount was applied to a well and electrophoresed at 20 μmA for 120 μmin. After electrophoresis, the gel was transferred to a membrane by iBlot drive blotting system (Invitrogen) and blocked using Blocking®One® (nacalai®tesque). GAPDH was detected using GAPDH Antibody (FL-335) (Santa Cruz Biotechnology), Goat anti-Rabbit IgG (H + L)-HRP conjugate (bio-rad). LIN28A was detected using Anti-human LIN28A (Goat) (R and D Systems), Rabbit anti-Goat IgG (H + L) Secondary Antibody, and HRP conjugate (Life Technologies). After the antibody treatment, chemiluminescence was performed using ECL®Plus®Western®Blotting®Detection®System® (GE®Healthcare), and observed with LAS-4000® (FUJIFILM).

Example 1
[Modification of natural aptamer]
The aptamer used in the experiment was designed to modify the aptamer known as the LIN28A binding motif (Figure 3 left; Original aptamer) so that structure formation is likely to occur on mRNA. The AU base pair present in the original sequence was gradually changed to a stronger GC base pair (FIG. 3, right; stbA, stbB, stbC). According to the modification, the probability of base pairing increases and the stabilization of the structure is expected. CentroidFold (http://www.ncrna.org/centroidfold) was used to predict the secondary structure of the aptamer. The aptamer containing the original shown in FIG. 3 was inserted into the plasmid of FIG. 2 to obtain a switch plasmid. Aptamer sequences other than the stabilized aptamer sequences shown in Table 1 are shown in Table 12 below. Bases modified from the original aptamer are shown in lower case.

[Confirmation of translation efficiency of the invented switch plasmid]
The prepared switch plasmid was co-introduced into HEK293FT cells together with a trigger plasmid expressing LIN28A. FIG. 4 is a graph comparing the translation efficiency of each switch plasmid. The better the switch, the lower the translation efficiency. Translation efficiency was obtained by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when LIN28A was introduced and the value when it was not introduced. Furthermore, the control switch plasmid (FIG. 4; No aptamer) was used as a reference. Error bars indicate mean ± standard deviation (n = 2, two independent experiments, averaged from 3 samples each time). Stronger translational suppression was observed in the switch with a more stable structure compared to the switch with the original inserted. However, stbD, which is considered to have the most stable structure, did not show strong translational repression. This is because the structure of the terminal loop is changed in stbD, as is apparent in FIG. Therefore, it is considered that an appropriate secondary structure needs to be stably formed in order to suppress translation of a stronger switch.

Next, it was confirmed whether the switch inserted with stbC, in which particularly strong translational suppression was observed, could function in other cell types. The experiment and analysis method are the same as in the case of HEK293FT. FIG. 5 is a graph showing the translation efficiency of the LIN28A switch in HeLa cells. Error bars indicate mean ± standard deviation (n = 3). It was shown that the switch inserted with stbC caused translational suppression in response to LIN28A even in HeLa cells.

FIG. 6 is a graph showing the translation efficiency when the LIN28A switch or a mutant thereof is introduced into HEK293FT cells. The experiment and analysis method are the same as above. Error bars indicate mean ± standard deviation (n = 3). The mutant means a substance in which a base involved in binding to LIN28A existing in the aptamer sequence is substituted. Moreover, deletion means the thing which removed the base involved in the coupling | bonding with LIN28A from an aptamer sequence. Therefore, these two mutants are inhibited from binding to LIN28A, and recovery of translation efficiency is expected. In fact, these two mutants did not show strong translational repression, like the switch with stbC inserted.

[Confirmation of correlation between LIN28A switch translation efficiency and intracellular LIN28A content]
By introducing a switch plasmid into a cell line capable of inducing LIN28A expression by doxycycline addition, the correlation between the translation efficiency of the switch and the amount of intracellular LIN28A was verified. This cell line can regulate the expression level of LIN28A depending on the concentration of doxycycline added. That is, it is expected that the fluorescence ratio of the switch decreases as the doxycycline concentration increases. FIG. 7A is a graph showing the added doxycycline concentration and the translation efficiency of each switch. It was confirmed that the switch inserted with stbC was most sensitive to translational suppression depending on the doxycycline concentration. FIG. 7B shows the result of confirming whether the expression level of LIN28A is increased depending on the doxycycline concentration by Western blotting. As an internal standard, the expression of GAPDH was also detected at the same time. In fact, it was confirmed that the expression level of LIN28A increased according to the doxycycline concentration. FIG. 7C is a graph quantifying the band concentration obtained in FIG. 7B and showing the relationship with doxycycline concentration. It was confirmed that the relative expression level of LIN28A reached the upper limit when the doxycycline concentration was 3 to 4 ng / mL. This concentration corresponds to the concentration at which the fluorescence ratio of the switch with the stbC inserted reaches the lower limit in FIG. 7A. Therefore, it was confirmed that the LIN28A switch is subject to translational suppression depending on the intracellular LIN28A amount. FIG. 7D is a graph showing the relationship between the relative expression level and the fluorescence ratio of each switch in the compartment until the relative expression level of LIN28A reaches the upper limit. It was shown that the fluorescence ratio of each LIN28A switch and the relative expression level of LIN28A are correlated, and that the switch inserted with stbC can detect the amount of LIN28A expressed in cells with high sensitivity.

[Confirmation of translation efficiency in mRNA transfection of LIN28A switch]
It was verified whether the LIN28A switch would function even when protein-responsive mRNA was directly introduced into cultured cells. A switch mRNA, a trigger mRNA expressing LIN28A, and a reference mRNA (iRFP mRNA) for confirming whether a series of mRNAs were introduced into the cells were co-introduced into HEK293FT cells. The amount of trigger mRNA to be introduced was changed in 6 stages of 0 ng (non-introduced), 50 ng, 100 ng, 150 ng, 200 ng and 300 ng. Theoretically, translation efficiency from switch mRNA decreases with the amount of trigger mRNA introduced. FIG. 8A is a plot of the fluorescence of a switch into which stbC has been inserted at the time of introduction of each amount of trigger mRNA by flow cytometry. It was confirmed that the population moved downward (translation amount from the switch mRNA decreased) according to the amount of trigger mRNA introduced.

FIG. 8B is a dot plot in which the switch in which another aptamer is inserted and the measurement result of FIG. 8A are superimposed. Among the switches into which each aptamer was inserted, only in the switch into which stbC was inserted, the movement of the cell population with the amount of trigger mRNA was observed. Therefore, it was suggested that the cell population can be separated depending on the expression level of LIN28A in the cell by introducing the switch into which the stbC is inserted into the cell population. FIG. 8C is a graph showing the translation efficiency of switch mRNA at each trigger mRNA introduction amount. The translation efficiency was determined by first dividing the fluorescence intensity of hmAG1 by the fluorescence intensity of iRFP670, and then dividing the value when the trigger mRNA was introduced and the value when it was not introduced. Of the switches inserted with each aptamer, strong translational suppression was observed in the switch inserted with stbC. Furthermore, a decrease in translation efficiency was confirmed with an increase in the amount of trigger mRNA introduced.

(Example 2)
[Identification of iPS cells by LIN28A switch]
LIN28A is a protein that is highly expressed in stem cells including iPS cells and ES cells. Therefore, iPS cells and differentiated cells can be differentiated from living cells based on the difference in the expression level of LIN28A. This suggests that iPS cells and differentiated cells can be distinguished from each other in the translation efficiency of each cell of the LIN28A switch. In other words, when LIN28A switch is introduced into cells, LIN28A is highly expressed in iPS cells, so that suppression of translation should be observed, and translational suppression should not be observed in differentiated cells (FIG. 9A). At this time, cells not transfected with the switch mRNA can be removed by measuring the expression of the reference mRNA. Furthermore, by performing flow cytometry, the iPS cell population and the differentiated cell population can be separated from the difference in the fluorescence value of the switch mRNA with respect to the fluorescence value of the reference mRNA (FIG. 9B).

FIG. 10A is a dot plot showing the measurement results when each LIN28A switch and reference mRNA (mKO2) are co-introduced. In the control switch, the populations of iPS cells and differentiated cells matched, but it was confirmed that the cell populations were clearly separated in the switch into which stbC was inserted. FIG. 10B is a histogram showing the ratio of two fluorescent signals in the dot plot of FIG. 10A. FIG. 10C is a graph comparing the translation efficiency of the LIN28A switch between iPS cells and differentiated cells. The translation efficiency was obtained by first dividing the fluorescence intensity of hmAG1 by the fluorescence intensity of mKO2, and then dividing the value in iPS cells and the value in differentiated cells. It was observed that the translation efficiency of the LIN28A switch in iPS cells was reduced by more than 50% compared to differentiated cells.

FIG. 11A is a comparison of the translation efficiency of the switches between iPS cells on differentiation induction days 14 and 34 and undifferentiated iPS cells. For the translation efficiency, the fluorescence intensity of EGFP was first divided by the fluorescence intensity of tagRFP, and then the value in iPS cells and the value in differentiated cells were divided. Furthermore, the values normalized with the value of the control (No aptamer) are shown in the graph. It was confirmed that the translation efficiency of iPS cells was reduced compared to differentiated cells. FIG. 11B is a dot plot showing the result of flow cytometry of each cell and its overlay. It was observed that the cell population moved upward (translation amount increased) with differentiation induction.

(Example 3)
[Scalability of human endogenous protein detection using mRNA switch]
In the present invention, a highly sensitive mRNA switch was successfully developed by stabilizing the structure of the aptamer inserted into mRNA. Whether this design principle can be applied to detection of proteins other than LIN28A was verified by creating an mRNA switch that responds to U1A. U1A exists in human cells as a splicing-related protein. FIG. 12 is an aptamer that binds to U1A used in the experiment. U1hp and U1utr are naturally occurring aptamers. The lower secondary structure ((b) in FIG. 12) was reprinted from Reference [4]. U1utr in the upper part of FIG. 12 is a predicted secondary structure using CentroidFold based on the reported sequence. This resulted in the possibility that U1utr was not stable and did not form the reported structure. Therefore, we designed U1utr_stb to form a structure close to that reported by modifying multiple bases. U1LSL is an aptamer used in the reference [5]. By adding a long stem structure to this aptamer, it was made difficult to interact with other regions on the mRNA, and the aptamer structure was protected. Aptamer sequences other than the stabilized aptamer sequences shown in Table 2 are shown in Table 13 below. Bases modified from the original aptamer are shown in lower case.

FIG. 13 is a graph comparing the translation efficiency of each switch plasmid. The prepared switch plasmid was co-introduced into HEK293FT cells together with a trigger plasmid expressing U1A. The translation efficiency was obtained by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when U1A was introduced and the value when not introduced. Furthermore, the control switch plasmid (FIG. 4; No aptamer) was used as a reference. Error bars indicate mean ± standard deviation (n = 3). As a result, stronger translational suppression was observed in U1utr_stb, which is considered to be more stable than U1utr. Furthermore, for U1LSL, stronger translational suppression was observed in aptamers that had a longer stem structure added to stabilize the structure.

Furthermore, U1LSL and aptamer with a stem structure added thereto were subjected to base substitution to produce mutants. These mutants form the same structure as the wild type, but lose the ability to bind U1A. Therefore, recovery of translation efficiency is expected for mRNA with a mutant inserted. FIG. 14A is a fluorescence micrograph when each switch plasmid is introduced into HEK293FT cells together with a control trigger plasmid. Stronger translational suppression was observed in the switch inserted with the wild type (WT) aptamer than in the switch inserted with the mutant (mut) aptamer. FIG. 14B is a histogram obtained by flow cytometric analysis of the cells observed in FIG. 14A. FIG. 14C is a graph showing the fluorescence ratio of the switch inserted with the wild type aptamer and the switch inserted with the mutant aptamer. The fluorescence ratio was determined by first dividing the fluorescence intensity of GFP by the fluorescence intensity of DsRed Ex and then dividing the value of the switch into which the wild type and the mutant were inserted. Error bars indicate mean ± standard error (n = 3 independent experiments). From these results, it was suggested that the produced U1A switch can respond to U1A in cells. In other words, an mRNA switch that can detect a desired protein can be produced by stabilizing the secondary structure of an aptamer that binds to an arbitrary protein.

(Example 4) [Knockdown analysis]
The cultured cells were seeded in a 24-well plate, and the next day, a total amount of 500 ng of plasmid and 5 pmol of shRNA were co-introduced. The introduction was performed using 2 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The used plasmid was mixed in Opti-MEM (Invitrogen) so that the quantitative ratio of the switch plasmid and the reference plasmid (pTAPmyc-T2A-tagRFP) was 1: 4. The medium was changed 4 to 6 hours after transfection. The day after transfection, cells were detached from the plate and analyzed by flow cytometry through a mesh. In experiments using 24-well plates, Accuri C6 (BD Biosciences) was used. EGFP was detected with a FL1 (530/30 nm) filters filter. tagRFP was detected with the FL2 (585/40 nm) filters filter. The shRNA used is as follows.

[Quantitative RT-PCR]
The day after the mRNA transfection, iPS cells were separated from the plate, and the cells were collected. Differentiated cells that were not transfected were collected and used in the same manner. Total RNA recovery, reverse transcription, and quantitative PCR were performed using miRNA Cells-to-CT kit (Applied Biosystems). The miRNA TaqMan probe (Applied Biosystems) was used for the expression level of mature miRNA. The values were normalized using RNU6B. Expression levels were calculated using the ΔΔCt method. The TaqMan probes used are as follows.

[Confirmation that U1A switch responds to endogenous U1A]
FIG. 15 is a graph showing the relative translation efficiency at the time of shRNA introduction in the U1A switch. The prepared switch plasmid was co-introduced into HEK293FT cells together with a reference plasmid expressing TAGRFP and shRNA. The relative translation efficiency was obtained by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when shRNA was not introduced and the value of each sample. Error bars indicate mean ± standard error (n = 3, 3 independent experiments, averaged from 3 samples each time). As a result, an increase in translation was observed when shUA1 was introduced in a switch into which a stable aptamer was inserted. This result means that endogenous U1A can be detected with higher sensitivity in a switch in which an aptamer having a more stable structure is inserted. Therefore, by stabilizing the secondary structure, an mRNA switch having sufficient sensitivity for detecting endogenous protein can be produced.

[Influence of introduction of LIN28A switch on endogenous miRNA expression]
LIN28A is involved in the maturation of a series of miRNAs belonging to the let7 family in nature. Specifically, there is a LIN28A binding site at the loop site in the precursor of let7 miRNA, and binding of LIN28A here inhibits expression of mature miRNA. Therefore, introduction of an mRNA switch capable of binding to LIN28A may affect the process of miRNA expression control. In other words, binding of endogenous LIN28A to the mRNA switch may result in insufficient miRNA maturation suppression and increased let7 family expression. To investigate this, the expression levels of three typical mature let7 families were measured by quantitative RT-PCR.

FIG. 16 is a graph showing the expression level of mature let7 miRNA when the LIN28A switch is introduced. The expression level in differentiated cells was expressed as 1. Error bars indicate mean ± standard deviation (n = 3). As a result, when LIN28A switch was introduced into iPS cells (no aptamer, stbC), no significant change in the expression of mature miRNA was observed. Therefore, the LIN28A switch invented this time can distinguish iPS cells from differentiated cells without significantly affecting the miRNA expression profile.

[Preparation of mRNA switch using artificial aptamer]
The aptamers used in the U1A switch and LIN28A switch were either created using naturally occurring ones as they were, or were created using a partially extracted motif. In principle, even aptamers acquired artificially, a highly sensitive mRNA switch can be produced by utilizing the technique used this time. In order to verify this, an mRNA switch was prepared using an aptamer (p50A: SEQ ID NO: 83) that binds to p50, which is one of cancer-related proteins (FIG. 17, left diagram, reference [6]). By adding a long stem structure to this aptamer, it was made difficult to interact with other regions on the mRNA, so that the aptamer structure was protected (p50A-stb: SEQ ID NO: 84, FIG. 17, right) Figure). Moreover, the mutant was produced by substituting the base of the binding site (p50A-stb _mut : SEQ ID NO: 85). FIG. 17 is an aptamer that binds to p50 used in the experiment. In preparing the mutant, the substituted base is indicated by an arrow in FIG.

FIG. 18 is a graph comparing the translation efficiency of each switch plasmid. The prepared switch plasmid was co-introduced into HEK293FT cells together with a trigger plasmid expressing p50. Translation efficiency was obtained by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when p50 was introduced and the value when not introduced. Furthermore, the control switch plasmid (FIG. 4; No aptamer) was used as a reference. Error bars show mean ± standard error (n = 3, 3 independent experiments, averaged from 3 samples each time). As a result, stronger translational inhibition was observed in p50A-stb, which is considered to be more structurally stable than p50A. Therefore, even with an aptamer obtained artificially, a highly sensitive mRNA switch can be prepared by the same approach.

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[6] Huang, D.-B. et al. Crystal structure of NF- B (p50) 2 complexed to a high-affinity RNA aptamer. Proc. Natl. Acad. Sci. 100, 9268-9273 (2003).

Claims (10)

1. A method for identifying an endogenous protein comprising the following steps:
   (1) a step of introducing into a desired cell a cell endogenous protein-responsive mRNA or aptamer sequence specific to a cell endogenous protein and a marker gene sequence operably linked thereto, or a vector encoding the mRNA; 2) A step of identifying a cellular endogenous protein based on the translation amount of the marker gene.
2. A method for identifying a cell comprising the following steps:
   (1) A step of introducing a cell endogenous protein-responsive mRNA containing an aptamer sequence specific to a cell endogenous protein and a marker gene sequence operably linked thereto or a vector encoding the mRNA into a desired cell;
   (2) A step of identifying cells expressing the protein based on the translation amount of the marker gene.
3. A structure-stabilized LIN28A aptamer obtained by adding one or more of the following modifications based on the RNA secondary structure formed by the natural LIN28A aptamer sequence represented by SEQ ID NO: 1:
   (1) AU base pairs constituting a stem including a 5 ′ terminal base and a 3 ′ terminal base are replaced with GC base pairs;
   (2) Replace the AU base pair constituting the stem located between the two loops with a GC base pair; or (3) Add 1 to 5 base pairs to the stem located between the two loops.
4. Structure-stabilized LIN28A aptamer represented by any of SEQ ID NOs: 2 to 4.
5. Based on the RNA secondary structure formed by the natural U1A aptamer sequence represented by SEQ ID NO: 6, the structure-stabilized U1A aptamer is modified as follows:
   (1) 1 to 15 AU base pairs or GC base pairs are added to a stem structure containing a 5 ′ terminal base and a 3 ′ terminal base, or 1 to 15 AU base pairs are added to a stem existing between two boxes. Alternatively, a GC base pair is added; or a structure-stabilized U1A aptamer obtained by adding the following modifications based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8:
   (2) Add 1 to 15 AU base pairs or GC base pairs to the stem containing the 5 ′ terminal base and the 3 ′ terminal base.
6. Based on the RNA secondary structure formed by the p50A aptamer sequence represented by SEQ ID NO: 83, the structure-stabilized p50A aptamer is modified as follows:
   1 to 20 AU base pairs or GC base pairs are added to the stem structure containing the 5 ′ terminal base and the 3 ′ terminal base.
7. Structure-stabilized U1A aptamer represented by any of SEQ ID NOs: 7, 9, 10, and 84.
8. An mRNA comprising the structure-stabilized aptamer sequence according to any one of claims 3 to 7 and a marker gene sequence operably linked thereto.
9. A method for identifying the differentiation state of a cell, comprising the following steps:
   (1) Introduction of an mRNA containing an aptamer sequence specific to a protein expressed by undifferentiated cells and a marker gene sequence operably linked thereto, or a vector encoding the mRNA into a cell group that can contain pluripotent stem cells And (2) identifying the differentiation state of the cell based on the translation amount of the marker gene.
10. The method according to claim 9, wherein the aptamer sequence is a natural LIN28A aptamer sequence or the structure-stabilized aptamer sequence according to claim 3 or 4.

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