JP6775911B2 - Methods and devices for determining cell characteristics, and disease testing methods - Google Patents

Description

Embodiments of the present invention relate to methods and devices for determining cell characteristics, and methods for testing diseases.

Biopsy, also called biopsy, is a widely used clinical test. In biopsy, first, a tissue or cell or the like is collected as a sample from a subject having or suspected of having a disease, and a sample is prepared. Next, the pathological characteristics of the obtained specimen are determined. In the case of cells, the specimen is prepared by smearing, fixing, degreasing, staining, encapsulating, etc. the collected cells on a slide glass. In the case of tissues, specimens are generally prepared by fixation with formalin or ethanol, paraffin embedding, slicing, staining, or the like.

For example, typical staining methods for observing morphological features include Papanicolaou staining, Giemsa staining, and PAS staining. In these staining methods, nuclei, cytoplasm, polysaccharides and the like are dyed separately. In addition, by using an immunostaining method that stains a specific protein using an antibody, it is possible to determine the presence or absence of the stained specific protein. Thereby, the cell characteristics can be determined. Immunostaining methods include, for example, AFP staining, PSA staining, HER-2 staining and Ki-67 staining. Observe the lesions under a microscope for cells and tissues stained by these methods.

Biomarkers that use cancer growth as an index include Ki67 and HER2. Ki67 is a cell cycle-related nuclear protein whose antibody is used in immunostaining as a marker for cell proliferation and cell cycle. HER2 is an oncogene having a structure similar to that of the human epithelial cell growth factor receptor gene. Immunostaining for HER2 protein and HER2 gene amplification tests have been performed on specimens of formalin-fixed paraffin-embedded tissue and are used to predict the prognosis and efficacy of breast cancer.

In the current pathological examination, for example, in the case of cancer, those that have low amplification ability and appear to be benign but are actually malignant, or those that appear morphologically malignant but are actually benign. There are some situations where the results include false negatives or false positives.

Special Table 2010-524856

BMC Developmental Biology (2012) 10:121 NMR in Biomedicine (2013) 26 (7) 872-884

It is an object of the embodiment to provide a method and an apparatus for determining cell characteristics with higher accuracy, and a method for testing a disease based on the method and apparatus.

To solve the above problems, embodiments provide a method of determining cell properties. This method involves (1) introducing a self-replicating reporter vector into a test cell that is activated under specific conditions to produce a detectable signal, and (2) culturing the test cell to obtain a reporter vector. Self-renewal, (3) detecting a detectable signal generated from a test cell for each cell, (4) observing the morphology of the test cell from which the signal was obtained, (5) (3) ) And (4), the characteristics of the test cell are determined based on the results obtained.

The figure which shows an example of the reporter vector which concerns on embodiment. The figure which shows an example of the self-replication vector which concerns on embodiment. The figure which shows a further example of the self-replication vector which concerns on embodiment. A scheme showing an example of the method according to the embodiment. The schematic diagram which shows an example of the apparatus which concerns on embodiment. The schematic diagram which shows an example of the apparatus which concerns on embodiment. A scheme showing an example of the method according to the embodiment. A scheme showing an example of the method according to the embodiment. The schematic diagram which shows the reporter vector used in an experiment. The figure which shows the experimental result. The schematic diagram which shows the reporter vector used in an experiment. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. A scheme showing the manufacturing process of the reporter vector used in the experiment. A graph showing the experimental results. The schematic diagram which shows the reporter vector used in an experiment. The schematic diagram which shows the reporter vector used in an experiment. A graph showing the experimental results. The schematic diagram which shows the primer used in an experiment and the region amplified by them. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. The schematic diagram which shows the reporter vector used in an experiment. The schematic diagram which shows the primer used in an experiment and the region amplified by them. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. A graph showing the experimental results. A scheme showing the manufacturing process of the reporter vector used in the experiment. A scheme showing the experimental process. The schematic diagram which shows the reporter vector used in an experiment. A graph showing the experimental results.

Hereinafter, embodiments will be described in detail.

One embodiment is a method of determining the cell characteristics of a test cell based on information on gene expression and expression control and morphological information.

The cell characteristics are, for example, an index of the health condition of the test cells. For example, by knowing the cell characteristics, it is possible to know whether the test cell is a normal or intact cell, an abnormal or impaired cell, or a cell in an intermediate state. In addition, it is possible to test for a specific disease by determining the cell characteristics. Examples of specific diseases may be cancer, mental illness, lifestyle-related diseases, neurological diseases, autoimmune diseases, cardiovascular diseases, etc., or pre-illness states such as pre-cancerous state, pre-diabetic state, etc. There may be. It is also possible to know whether or not the test cell is in a specific state by determining the cell characteristics. Further, by knowing the cell characteristics, for example, it is possible to know whether or not the cells obtained by inducing differentiation from stem cells have the desired characteristics.

The information on gene expression and expression control may be, for example, information on the promoter transcription activity of the test cell obtained by using the reporter vector. Promoter transcriptional activity is an indicator of the presence or absence and magnitude of expression of a specific gene. Furthermore, the promoter transcription activity can be used as an index of the methylation state of the genomic nucleic acid contained in the test cell, for example, the presence / absence and / or degree of methylation.

From such information on promoter transcriptional activity, information on the expression and regulation of expression of a specific gene can be obtained. Information on promoter transcriptional activity can be obtained using a reporter vector that is activated under specific conditions, which will be described in detail later, and expresses a detectable signal.

In the method for determining cell characteristics according to the embodiment, such a reporter vector is introduced into a test cell, and a detectable signal (hereinafter, also simply referred to as “signal”) generated by activation of the reporter vector is first. Detect as information. Thereby, information on gene expression or expression control is obtained as the first result. Further, in this method, morphological information is obtained as second information at the same time as or after the detection, prior to the detection of the signal from the reporter vector. Here, it is preferable that the second information is obtained for the test cell from which the first information was obtained. For example, the second information may be obtained by observing the morphology of the cells in which the signal from the reporter vector was detected. In this way, morphological information is obtained as a second result. Cell characteristics are determined based on the first and second results. As a result, information on the genes of the cells to be tested and morphological information can be obtained at the same time while the cells to be tested are alive. In this way, by obtaining information about cells in a living state, it becomes possible to know the state of cells as they are more accurately. It is also possible to obtain information from the test cells over time.

1. 1. Reporter vector Information on the transcriptional activity of the promoter of the test cell is obtained by using the reporter vector. The reporter vector includes, for example, a transcriptional regulatory sequence that is activated under specific conditions and exhibits promoter activity, and a reporter gene that is functionally linked downstream of the transcriptional regulatory sequence. In the reporter vector of the embodiment, under certain predetermined conditions, the promoter activity of the transcription control sequence is activated, and the reporter gene downstream thereof is transcribed and translated. As a result, the reporter protein encoded by the reporter gene is expressed.

An example of a reporter vector will be further described with reference to FIG. Reporter vector 1 contains a reporter gene expression unit 2. The reporter gene expression unit 2 includes a transcription control sequence 4, a reporter gene 5 encoding a reporter protein functionally linked downstream thereof, and a transcription termination signal sequence 6 functionally linked downstream thereof.

Here, "functionally linked" means a state in which each gene is linked in a state in which it can exert a desired function. For example, in the case of a protein-encoding sequence, it means that the amino acid sequences encoded by the respective base sequences are correctly linked. In other words, it means that the amino acid codon frame is not deviated and the peptide having the desired activity is synthesized in the cell into which the base sequence is introduced. Also here, the term "functionally concatenated" may be used interchangeably with the term "operably concatenated".

The activation of the promoter activity of the transcription control sequence 4 is controlled by the interaction between the sequence on the transcription control sequence 4 and the transcription factor. The transcription control sequence may be a promoter sequence in which promoter activity is activated under specific conditions, or may include a promoter sequence in which promoter activity is activated under specific conditions. The transcription control sequence 4 may be selected in advance as desired.

A "specific condition" in which promoter activity is activated is a type of cell to be detected, a condition that reflects the condition or condition of the cell to be detected, in other words, an indicator of such condition or condition. It may be a condition. The specific conditions may be arbitrarily selected depending on the purpose. For example, the specific condition may be a condition that reflects a specific health condition, for example, a condition suffering from a specific disease. Examples of specific illnesses may be, for example, cancer, psychiatric disorders, lifestyle-related disorders, neurological disorders, autoimmune disorders, and cardiovascular disorders. Such specific conditions may be conditions that are indicators of pre-disease status, eg, pre-cancerous status. Alternatively, it may be a condition that is an indicator of whether or not the organ that has been induced to differentiate from stem cells in regenerative medicine is normal. For example, it may be the methylation state of the genomic nucleic acid contained in the cell, eg, the presence or absence and / or degree of methylation. It may also be a condition relating to the cell itself, eg, the "state" inside and / or outside the cell, the "environment" surrounding the cell. Such conditions may be arbitrarily determined in advance. For example, it may be an index showing signs of a specific disease, an index showing the onset of a specific disease, an index showing the degree of progression of the onset of a specific disease, an index showing the severity of a specific disease, or the like. For example, it may be a condition for an intracellular substance whose content changes in relation to disease onset, disease presence or disease progression. For example, such conditions include the presence of a particular gene, the expression of a particular gene, intracellular and extracellular pH values, information about oxidation-reduction, the presence of a particular ion, the presence of an enzyme, the presence of an enzyme substrate, the presence of a particular substance. For existence and the like, the presence or absence of their existence, the magnitude of the quantitative value for their existence, their existence distribution, and / or the change in the existence state may be used. In addition, the specific conditions may be, for example, conditions that are indicators of the situation regarding drug sensitivity or drug adaptability of the test cells.

Examples of the target nucleic acid sequence may be, for example, the promoter sequence of the human nicotine amide phosphoribosyl transposase gene, the promoter sequence of the Arf6 gene, the promoter sequence of the Ki67 gene, the promoter sequence of the Her2 gene, the promoter sequence of the Glut1 gene, and the like. However, it is not limited to these.

The reporter gene 5 may be any gene encoding a protein that produces a detectable signal. The detectable signal may be any detectable signal generated from the reporter protein itself, the reaction between the reporter protein and any substance, the binding between the reporter protein and any substance, or the like.

The reporter gene 5 encoding the reporter protein may be any reporter protein that produces a detectable signal. The reporter gene 5 may be, for example, a luciferase gene, a β-galactosidase gene, a nitrogen monoxide synthase gene, a xanthin oxidase gene, a blue fluorescent protein gene, a green fluorescent protein gene, a red fluorescent protein gene, and a heavy metal binding protein gene. .. Each of these genes encodes luciferase, β-galactosidase, nitrogen monoxide synthase, xanthine oxidase, blue fluorescent protein, green fluorescent protein, red fluorescent protein and heavy metal binding protein, and activation of the reporter vector produces these proteins. Express. A preferred example in one embodiment is a gene encoding a luminescent enzyme protein, including, for example, a luciferase gene, a β-galactosidase gene, and the like.

An example of a reporter vector expressing a heavy metal binding protein as a detectable signal is disclosed in JP-A-2012-200245.

The signal obtained from the reporter gene may be any signal that can be detected by any device. Further, such a signal may be detected by using any device selected according to the type of the signal. Examples of the device may be a light emission measuring device, a fluorescence measuring device, an X-ray measuring device, an electron spin resonance device, a nuclear medicine diagnostic device, a magnetic resonance imaging device, and the like. In addition, the result obtained by detecting the signal may indicate the presence or absence of the signal, the magnitude of the signal, or both.

Preferred examples of detectable signals are optical signals such as light emission, fluorescence and color development. As used herein, "luminescence" refers to chemiluminescence, bioluminescence or biochemical luminescence. When the signal is an optical signal, the signal may be detected by an optical detection device. For example, when the detectable signal is light emission, the signal may be detected by a light emission measuring device capable of detecting the light emission, for example, a light emission microscope. Luminescence is preferably used in one embodiment because of the ease of introduction of the reporter vector into the test cells and its low cytotoxicity.

The transcription termination signal sequence 6 may be a sequence that terminates transcription of the gene upstream thereof, and may be, for example, a poly (A) addition signal. The poly (A) addition signal may be one that functions to terminate transcription of a mammalian gene. Examples include the late poly (A) addition signal of the SV40 virus (SEQ ID NO: 1), the poly (A) addition signal of the bovine growth hormone gene (SEQ ID NO: 2), and the like. However, the poly (A) addition signal that can be used in carrying out this embodiment is not limited to this, and a modified gene sequence is used as long as the function as the poly (A) addition signal is not impaired. You may use it. The nucleotide sequences shown in SEQ ID NOs: 1 and 2 are shown in Table 1.

The reporter vector may be a self-replicating vector. When using a self-replicating vector, embodiments include, for example; preparing a self-replicating reporter vector that is activated under certain conditions and expresses a detectable signal, said. Introducing a reporter vector into a test cell, culturing the test cell and self-replicating the reporter, detecting the detectable signal generated from the test cell for each cell, and transmitting the signal. Observing the resulting morphology of the cell and determining the properties of the test cell based on the results for the detectable signal and the results for the morphology.

A self-replicating reporter vector (hereinafter referred to as a self-replicating vector) includes a self-replication initiation unit that self-replicates the reporter vector in addition to the reporter gene expression unit. The self-replication initiation unit may include a self-replication initiation sequence that signals the reporter vector to initiate self-replication.

The self-replicating vector will be further described with reference to FIG. The self-replicating vector 21 contains the same configuration as the reporter vector shown in FIG. 1, except that it contains a replication initiation unit.

The self-replicating vector 21 contains a reporter gene expression unit 2 and a replication initiation unit linked downstream thereof. The example shown in FIG. 2 is an example in which the replication initiation unit includes the replication initiation sequence 3. The replication origin sequence 3 initiates self-renewal in the cell into which the self-replication vector 21 has been introduced by binding of the corresponding replication initiation protein.

The self-replicating vector performs self-renewal in the nucleus of the test cell under the condition that the replication initiation protein is expressed in the test cell. The replication initiation sequence 3 may be, for example, a sequence that is activated by binding of a replication initiation protein and initiates self-renewal in a mammalian cell, and may be a replication initiation sequence known per se. The replication initiation sequence 3 may be, for example, a replication initiation sequence derived from Simian virus 40 (SEQ ID NO: 3), Epstein-Barr virus or mouse polyoma virus, and one example thereof is shown in Table 2. Shown.

As the self-replicating vector, for example, a plasmid vector known per se may be used. For example, an example of a plasmid vector of this self-replicating vector is disclosed in Japanese Patent Application Laid-Open No. 2013-42721.

The self-replication of the self-replication vector is initiated by the binding of the replication initiation protein to the replication initiation sequence 3. The replication origin protein 7 may be present in the nucleus of the test cell together with the self-replication vector so that it can bind to the replication origin sequence 3. For example, the replication initiation protein 7 may be expressed from a self-replicating vector used as a reporter vector, may be expressed from a further vector, or may be expressed by a test cell.

For example, a replication initiation protein unit may be present to supply the replication initiation protein 7 into the nucleus of the test cell. The replication initiation protein unit may include, for example, a promoter, a replication initiation protein gene encoding a replication initiation protein functionally linked downstream thereof, and a transcription termination signal sequence functionally linked downstream thereof. Alternatively, the replication initiation protein unit may consist of these sequences.

The replication initiation protein unit may be present on the nucleic acid of the self-replicating vector 21, or may be present on a nucleic acid different from that of the self-replicating vector 21. If the replication initiation protein unit is present on a nucleic acid different from the self-replication vector, the replication initiation protein unit may be present on the chromosome of the test cell or on another nucleic acid contained in the test cell. You may.

When the replication initiation protein unit is contained in the self-replication vector as a reporter vector, the expression of the reporter gene expression unit 2 and the replication initiation protein unit contained therein is controlled together by one transcription control sequence 4. They may be regulated by two independent transcription control sequences, respectively. When their expression is regulated by two transcription control sequences, for example, the replication initiation protein unit may be present on the nucleic acid of the self-replication vector 1 independently of the reporter gene expression unit 2. In that case, the promoter that controls the expression of the replication initiation protein gene is preferably a constitutively expressed promoter.

The replication initiation protein gene may be, for example, the large T antigen gene of Simian virus 40, the EBNA-1 gene of Epstein-Barr virus, the large T antigen gene of mouse polyoma virus, or the like. Examples of preferred large T antigen genes are SEQ ID NO: 4 (wild type, Table 3-1 and Table 3-2), SEQ ID NO: 5 (mutant, Table 4-1 and Table 4-2) or SEQ ID NO: 6 (mutation). By type, nucleic acid shown in Table 5-1 and Table 5-2), or by a sequence in which one or several bases in the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 are deleted, substituted or added. It may be a nucleic acid shown and having a DNA replication initiation function. Here, the DNA replication initiation function means encoding a protein capable of initiating replication. Examples of the replication initiation protein gene are shown in Table 3-1 and 3-2, Table 4-1 and Table 4-2, Table 5-1 and Table 5-2.

The combination of the replication initiation protein gene and the replication initiation sequence may be any combination capable of initiating replication. Examples of such combinations are:
A combination in which the gene encoding the replication initiation protein is the large T antigen gene of Simian virus 40, and the replication initiation sequence is the replication initiation sequence from Simian virus 40;
A combination in which the replication initiation protein gene is the EBNA-1 gene of Epstein-Barr virus and the replication initiation sequence is the replication initiation sequence from Epstein-Barr virus;
A combination in which the replication initiation protein gene is a large T antigen gene of mouse polyoma virus and the replication initiation sequence is a replication initiation sequence from mouse polyoma virus. Combinations such as these are preferred, but not limited to.

Further, the self-replicating vector may be a bicistronic expression vector. In the bicistronic expression vector, by using the bicistronic expression sequence, the transcriptional control sequence 4 simultaneously regulates the expression of two genes contained downstream thereof, that is, the expression of the reporter gene and the expression of the replication initiation protein. To. That is, in such a self-replicating vector, the reporter gene expression unit 2 and the replication initiation protein unit are controlled and expressed by the same promoter. An example of such a bicistronic expression self-replicating vector will be described with reference to FIG. The self-replicating vector 31 contains a transcription control sequence 4, a reporter gene 5, IRES 32, a replication initiation protein gene 33, a transcription termination signal sequence 34, and a replication initiation sequence 3. The transcription control sequence 4, the reporter gene 5, the IRES 32, the replication initiation protein gene 33, and the transcription termination signal sequence 34 are functionally linked in this order from upstream to downstream. The IRES32 is an internal ribosomal entry site and is an example of a sequence for bicistronic expression. An example of a bicistronic expression sequence is, for example, the internal ribosome entry sequence of brain myocardial virus (SEQ ID NO: 7).

An example of this is shown in Table 6.

The sequence of the reporter gene 5 is transcribed by the transcription control sequence 4 activated by the presence of IRES32 depending on the degree of modification of the base in the sequence of the transcription control sequence 4, followed by the replication initiation protein gene 33. Transcribed. After the sequence of the replication initiation protein gene 33 is read, transcription is stopped by the presence of the transcription termination signal sequence 34. The replication initiation protein produced by being transcribed and translated from the replication initiation protein gene 33 binds to the replication initiation sequence 3, and the replication of the self-replication vector 31 is initiated.

The transcription control sequence 4 may be a sequence different from the sequence contained in the genome of the test cell, or may be a sequence having the same identity as a specific nucleic acid sequence on the genome of the test cell. A particular nucleic acid sequence on the genome may be a partial sequence in the genome.

When the transcription control sequence 4 is a sequence having identity with a part of the test cell's genome, such a self-replicating vector is used as a reporter vector to obtain the epigenetic properties of the test cell. It may be determined as a cell characteristic. Such a self-replicating vector contains, as a transcription control sequence, a target nucleic acid sequence having a base that has identity with a part of the genome of the test cell and the functional group can be substituted. This self-replicating vector self-replicates when the introduced cells are in a state where they can divide, that is, in a cell cycle other than the quiescent phase. At the time of this self-replication, the status of functional group substitution in the sequence of a part of the genome of the test cell having the same identity as the target nucleic acid sequence is set as an epigenetic property on the base in the target nucleic acid sequence of the self-replicating vector. It becomes the same situation as.

The following describes an example in which epigenetic information is information on methylation at a specific sequence of the genome. Table 7 shows the correlation between the reporter activity of the reporter vector and methylation.

The correlation shown in Table 7 is an example in which the signal of the reporter vector is increased by demethylation of the corresponding sequence of the reporter vector and decreased by methylation.

This reporter vector can be used, for example, to detect cancer. In the case of cancer cells, demethylation occurs at specific sequences on the genome.

Epigenetic information in such cells is transcribed into the corresponding sequence contained in the reporter vector during its self-renewal. For example, if the corresponding sequence of the reporter vector is premethylated, demethylation will occur during self-replication. At this time, the detectable signal from the reporter vector becomes large. For example, it becomes possible to detect cancer by using the detection of such a large signal as an index. Here, the "corresponding sequence" is a sequence homologous to a specific sequence on the genome, that is, a sequence in which the modification to be detected is located.

For example, a reporter vector containing many methyl groups is prepared (A-1). It is assumed that a specific sequence on the genome is rich in methyl groups (A-2-1). In this case, when self-replicating, there are no or few functional group substitutions in the vector (A-3-1). In this case, the detection signal is small (A-4-1). Reporter activity is relatively low (A-5-1). On the other hand, it is assumed that a specific sequence on the genome does not contain many methyl groups (A-2-2). In this case, when self-replicating, the functional group is substituted in the vector and demethylation occurs (A-3-2). In this case, the detection signal is large (A-4-2). Reporter activity is relatively high (A-5-2).

Alternatively, for example, a reporter vector containing a small number of methyl groups is prepared (B-1). It is assumed that a specific sequence on the genome is rich in methyl groups (B-2-1). In this case, upon self-replication, the functional groups in the vector are substituted and methylation occurs (B-3-2). In this case, the detection signal is small (B-4-2). Moreover, in this case, the signal becomes smaller with the passage of time. Reporter activity is relatively low (B-5-2). On the other hand, it is assumed that a specific sequence on the genome does not contain many methyl groups (B-2-2). In this case, when self-replicating, there are no or few functional group substitutions in the vector (B-3-2). In this case, the detection signal is large (B-4-2). Reporter activity is relatively high (B-5-2).

The correlation between the reporter activity of the reporter vector and methylation has been shown above, but this correlation is shown for illustration purposes and is not limited thereto.

The transcription control sequence 4 may be a target nucleic acid sequence, which is a nucleic acid sequence for which epigenetic information should be obtained, or may be a sequence containing the target nucleic acid sequence. The target nucleic acid sequence has the same identity as the specific nucleic acid sequence possessed by the test cell. The target nucleic acid sequence then contains a modified base as a base to which the functional group can be substituted. The transcription control sequence 4 has a promoter activity that depends on the degree of modification of the target nucleic acid sequence in the modified base. Activation of the promoter activity of transcription control sequence 4 is controlled by modification of the modified base on the target nucleic acid sequence. Such modified bases are targets for modification. The transcription control sequence 4 and the target nucleic acid sequence may be selected in advance as desired. From the epigenetic information obtained regarding the target nucleic acid sequence, epigenetic information of a specific nucleic acid sequence in the test cell can be obtained.

Modification of the target nucleic acid sequence is, for example, methylation or alkylation. In this case, the modified state of the target nucleic acid sequence is whether or not a methyl group and / or an alkyl group, which are functional groups, are bound to the bases constituting the target nucleic acid sequence. .. The "epigenetic information" of the test cell may be the presence or absence of such functional groups. Alternatively, it may be information on how much functional group is present. Alternatively, it may be information such as a change in the state of such modification, i.e., the presence or absence or degree of functional group substitution. Moreover, any combination of them may be used. Modification of the target nucleic acid sequence may be, for example, modification of cytosine and / or guanine in the main chain of the target nucleic acid sequence. If more than one cytosine and / or guanine is present in the target nucleic acid sequence, it may be a modification for one or more of them. For example, modification of the target nucleic acid sequence may be methylation of cytosine and / or guanine in the target nucleic acid sequence.

Promoter activation of transcription control sequence 4 is controlled by the state of modification of the modified base on the target nucleic acid sequence. In other words, the promoter activity of the transcription control sequence 4 is activated depending on the degree of modification of the base in the sequence of the target nucleic acid sequence.

The promoter activity of the transcription control sequence 4 is activated, for example, when the degree of modification of the target nucleic acid sequence in the modified base is low, or when the modification is present, or when no modification is present. Any of these may be used, such as being activated. The transcription control sequence 4 having such promoter activity is "activated depending on the degree of modification of the base in the sequence of the target nucleic acid sequence" or "active depending on the degree of substitution of the functional group in the base". This is an example of a transcription control sequence that is "converted".

The sequence having a promoter activity depending on the degree of modification in the modified base in the sequence of the target nucleic acid sequence may be, for example, a sequence derived from a test cell, or an artificially synthesized sequence. For example, an example of such a sequence may be the 5'upstream region (-1651 to + 32bp) (SEQ ID NO: 8) of the Glial fibrillary acidic protein (GFAP) gene. The 5'upstream region sequence of the GFAP gene is activated in the absence of methylation of the target nucleic acid sequence. Alternatively, in the 5'upstream region (-617 to +61 bp) (SEQ ID NO: 9) of the cytokeratin 19 (CK19) gene or the 5'upstream region (-540 to +115 bp) (SEQ ID NO: 10) of the cyclooxygenase 2 (COX2) gene. There may be. Examples of further target nucleic acid sequences include the human nicotine amide phosphoribosyl transposase gene (NAMPT gene) promoter sequence (SEQ ID NO: 11), CK19 gene promoter sequence (SEQ ID NO: 12), GFAP gene promoter sequence, SOX2 gene promoter sequence and Examples thereof include, but are not limited to, the COX2 gene promoter sequence, the Arf6 gene promoter sequence, the Ki67 gene promoter sequence, the Her2 gene promoter sequence, and the Glut1 gene promoter sequence. Examples of these sequences are shown in Table 8, Table 9, Table 10, Table 11-1, Table 11-2, and Table 12. These target nucleic acid sequences may be used as transcription control sequences.

The reporter gene 5 is functionally linked downstream of the transcription control sequence 4. That is, the transcription control sequence 4 is functionally linked upstream of the reporter gene 5. The reporter gene 5 is transcribed by activating the transcription control sequence 4. Reporter protein is expressed by this transcription.

When the self-replicating vector 31 containing the target nucleic acid sequence is introduced into a cell, the modified base in the target nucleic acid sequence may or may not be modified.

When the modified base in the target nucleic acid sequence of the self-replicating vector 31 is not modified, the self-replicating vector 31 introduced into the test cell behaves as follows. First, when the modified state of the target nucleic acid present in the test cell is high, the modified base in the target nucleic acid sequence is highly modified. Therefore, the promoter activity is not activated, and the reporter gene and the replication initiation protein gene are not expressed. Therefore, the self-replication of the self-replication vector 31 is stopped. On the other hand, when the modified state of the target nucleic acid present in the test cell is low or not modified, the modified state of the modified base is kept low. Therefore, the promoter activity is activated. When in the activated state, the IRES32-linked reporter gene 5 and the replication-initiating protein gene 33 are transcribed and translated into bicistronic mRNA. When IRES32 is integrated between two genes, the two genes are transcribed as a single bicistron mRNA. The ribosome not only translates the reporter gene 5 from bicistron mRNA, but also binds to the position of the IRES and translates the replication initiation protein gene. The transcription termination signal sequence 34 is a base sequence encoding a signal for terminating transcription between the reporter gene 5 and the replication initiation protein gene 33. The replication initiation protein transcribed from the replication initiation protein gene 33 and translated by the components of the test cell recognizes the replication initiation sequence 3 and binds to it. Furthermore, a plurality of proteins involved in DNA replication contained in the test cell bind to each other. The bound proteins cause self-renewal of the self-replication vector 31 in the test cells.

When the modified base in the target nucleic acid sequence of the self-replicating vector 31 is modified, the self-replicating vector 31 introduced into the test cell behaves as follows. First, when the modified state of the target nucleic acid present in the test cell is high, the promoter activity is not activated because the degree of initial modification of the modified base in the target nucleic acid sequence is maintained. Therefore, the reporter gene and the replication initiation protein gene are not expressed. Therefore, the self-replication of the self-replication vector 31 is stopped. On the other hand, when the modified state of the target nucleic acid present in the test cell is low or not modified, the modified state of the target nucleic acid sequence at the modified base is reduced or eliminated, so that the transcription control sequence 4 is present. Be activated. When the promoter activity is in the activated state, the IRES32-linked reporter gene 5 and the replication initiation protein gene 33 are transcribed and translated into bicistronic mRNA. When IRES32 is integrated between two genes, the two genes are transcribed as a single bicistron mRNA. The ribosome not only translates the reporter gene 5 from bicistron mRNA, but also binds to the position of the IRES and translates the replication initiation protein gene. The transcription termination signal sequence 34 is a base sequence encoding a signal for terminating transcription between the reporter gene 5 and the replication initiation protein gene 33. The replication initiation protein transcribed from the replication initiation protein gene 33 and translated by the components of the test cell recognizes the replication initiation sequence 3 and binds to it. Furthermore, a plurality of proteins involved in DNA replication contained in the test cell bind to each other. The bound proteins cause self-renewal of the self-replication vector 31 in the test cells.

Reporter gene 5 is expressed when the degree of modification of the target nucleic acid sequence in the modified base is low. Furthermore, the self-replication of the self-replication vector 31 is started by reading the sequence existing downstream of it. The replication product produced by the self-replication of the self-replication vector 31 has the same sequence as the self-replication vector 31 in terms of sequence. However, the modified state of the target nucleic acid present in the test cell is reflected in the modified state of the target nucleic acid sequence in the modified base. As a result, when the degree of modification of the target nucleic acid sequence in the modified base is high, the promoter activity is not activated. Therefore, the reporter gene 5 is not expressed. And the sequences existing downstream from it are not read. Therefore, the self-replication of the self-replication vector 31 is stopped.

In the above, a vector in which the reporter gene 5 and the replication initiation protein gene 33 are linked by IRES32 and the expression of the gene is controlled by the target nucleic acid sequence as one transcription control sequence 4 has been described. However, the expression of the reporter gene 5 and the replication initiation protein gene 33 may be controlled by the respective transcription control sequences without using a bicistronic expression sequence such as IRES.

With respect to the target nucleic acid sequence, the "sequence having identity" with respect to the specific nucleic acid sequence possessed by the test cell is the same sequence as the specific sequence on the genome of the test cell, or is specified on the genome. It is sufficient that the sequence is substantially the same as the sequence of, for example, a sequence in which one or several bases are substituted, deleted or added, and has a promoter activity that is activated under specific conditions. Here, the terms "having identity" and "having homology" may be used interchangeably. For example, "having identity", "homologous" and "homologous" may be sequences that are 90% or more or 95% or more identical to a specific sequence. In addition, the "specific sequence" may be any sequence that carries epigenetic information in the genome. A "sequence identical to a sequence on the genome" is a plurality of sequences having the same identity to two or more specific sequences that are not continuous on the genome, even if the sequence is the same as a specific sequence that is continuous on the genome. The array may be an integrated array.

For example, the target nucleic acid sequence may be a sequence homologous to a sequence obtained by integrating two or more sequences that are homologous to two or more sequences existing at a plurality of positions on the genome. A sequence homologous to the converted sequence may be included. In other words, assuming that each of two or more sequences existing at multiple positions on the genome is one unit, the base sequence of the target nucleic acid sequence integrates a sequence homologous to each of these multiple units. It may consist of a sequence homologous to the resulting sequence, or may include a sequence homologous to such an integrated sequence. That is, the target nucleic acid sequence may be composed of two or more units, corresponding to units that are homologous sequences on the genome.

In the target nucleic acid sequence, for example, one unit is homologous to a portion of a particular promoter sequence on the genome and the other unit comprises a modified base capable of controlling activation of that promoter sequence. It may be homologous to the sequence. For example, a portion of a particular promoter sequence on the genome and a sequence capable of controlling activation of this promoter sequence are not necessarily contiguous on the genome and include additional sequences in between. Sometimes. In addition, when selecting a specific sequence on the genome, a sequence adjacent to the sequence to be selected may be further included in the target nucleic acid sequence. However, if such an additional sequence is a sequence that has no effect on the regulation of activation of the promoter sequence, it does not necessarily have to be included in the target nucleic acid sequence.

The transcription control sequence 4 may contain any additional sequence in addition to the target nucleic acid sequence having identity to the sequence on the genome of the test cell. When containing any additional sequence, as long as transcriptional control sequence 4 has promoter activity, any additional sequence is between any of the bases that make up a sequence homologous to the sequence on the genome of the test cell. It may be contained adjacent to a sequence having the same sequence on the genome of the test cell.

In the above reporter vector, promoter activity means inducing transcription of a gene operably linked downstream of the promoter sequence. When the transcription control sequence 4 exhibits promoter activity, the promoter activity is a sequence of a part of the transcription control sequence 4, for example, a target nucleic acid sequence contained therein, even if the promoter activity is derived from the entire sequence of the transcription control sequence 4. It may be derived from. Also, for example, the transcription control sequence 4 may contain a minimal promoter. The promoter activation of the transcription control sequence 4 is the presence or absence of binding of a transcription factor to a part of the sequence on the transcription control sequence 4 and the state of modification of the modified base, that is, the substitution of the functional group in the base in which the functional group can be substituted. It is controlled by the degree.

The self-replicating vector contains, in the transcription control sequence or a part thereof, a sequence having the same identity as a specific sequence to be observed, that is, a target nucleic acid sequence. By having such a structure, when the reporter vector self-replicates, the modified state on a specific sequence of the genome and the modified state on the identical sequence in the reporter vector become the same. The reporter vector produces a detectable signal depending on the presence of the functional group copied to the reporter vector. At this time, the presence / absence, magnitude, or amount of the signal differs depending on the presence or absence of the functional group. In this way, the reporter vector is capable of reporting the modification status on a particular sequence of the genome.

The reporting function of such a reporter vector is obtained only when the reporter vector self-replicates. Therefore, the reporter vector is introduced into the nucleus of the test cell placed under conditions under which self-renewal of the reporter vector is initiated. Then, after the time required for the reporter vector introduced into the nucleus of the test cell to self-replicate has elapsed, the signal generated by the reporter vector in the test cell is detected. The detection of the signal may be performed on the presence or absence of the signal, or on the magnitude or quantity of the signal. The results obtained by the detection are epigenetic information of the test cells.

Here, "cell epigenetic information" refers to whether or not a functional group is bound to a base constituting a specific sequence on the genome of a test cell, whether or not there is a substitution, or not. It may be information about. The detectable signal varies in presence or absence or magnitude depending on the presence or absence of the functional group copied to the reporter vector.

2. Method for Determining Cell Characteristics of Test Cell The cell characteristics of the test cell are determined based on the information on gene expression and expression control obtained for the test cell and the morphological information. The information on gene expression and expression control and the morphological information may be performed at the same time, and the morphological information may be obtained before or after the information on gene expression and expression control. The detection and morphological information of the detectable signal may be obtained for each individual test cell, or may be obtained as the result of averaging the results obtained for a plurality of test cells.

An example of a method for determining the cell characteristics of the test cells will be described with reference to FIG. First, a sample is prepared (S1). The specimen may be blood, urine, stool, semen, saliva, tissue obtained by biopsy, oral mucosa, body cavity fluid, sputum and the like. Next, the test cells are separated from the sample (S2). For example, treatment with a proteolytic enzyme, treatment with filter filtration, etc. may be included. Next, for the test cells obtained by isolation, information on the transcriptional activity of the test cells is obtained. A reporter vector having a desired target nucleic acid sequence is prepared (S3). Next, the reporter vector is introduced into living test cells (S4). The introduction of the reporter vector into the test cells may be carried out by, for example, any known method such as electroporation, sonoporation, or lipofection, or a combination thereof. Then, under the condition that the test cells can be visually recognized one by one, the state in which the reporter vector biosynthesizes the reporter protein according to the preset conditions is maintained in the test cells (S5). For example, the test cell is cultured for an arbitrary time in a state where the replication initiation protein is present in the nucleus of the test cell. The arbitrary time may be, for example, the time for cell division and proliferation. During this culture period, the test cells are observed as necessary. Next, the amount of reporter protein is measured (S6). If necessary, separately, the results obtained from the control cells by the same method, that is, the detected amount of the reporter protein and the results obtained by (S6) are compared (S7). For example, when the reporter protein mass of the test cell is larger than the reporter protein mass of the control cell, it is determined that the transcriptional activity in the homologous sequence of the target nucleic acid sequence is high. When the reporter protein mass of the test cell is smaller than the reporter protein mass of the control cell, it is determined that the transcriptional activity in the homologous sequence of the target nucleic acid sequence is low. The reporter protein mass of the control cell may be measured in advance.

On the other hand, for the same cells whose reporter protein mass was measured, images of the test cells are acquired by a non-invasive optical method (S8). Further, it is compared with the image of the control cell (S9). Thereby, the morphological characteristics of the test cells are determined. For example, when the ratio of the minor axis to the major axis of the test cell (for example, the aspect ratio) is significantly different from the ratio of the minor axis to the major axis of the control cell, it is determined that the morphology of the test cell has changed.

Here, the step of obtaining the data of the control cell may be performed every time the test is performed, or the data of the target cell may be obtained in advance and used as the reference data from the target cell. In addition, the order in which the data on the transcriptional activity characteristics and the data on the morphological characteristics are obtained may be in any order. That is, the data on the morphological characteristics may be obtained after obtaining the data on the transcriptional activity characteristics, or the data on the morphological characteristics may be obtained before obtaining the data on the transcriptional activity characteristics. Alternatively, data on morphological properties may be obtained before and after obtaining data on transcriptional activity properties. Further, these steps may be repeated or may be performed over time.

Next, with respect to the test cells, the cell characteristics of the test cells are determined based on two indexes of transcriptional activity and morphology (S10). The cell characteristics of the test cells may be determined by comparing with the data from the control cells obtained for each test, by comparing with the data from the control cells prepared in advance, or by comparing with the data from the control cells prepared in advance. It may be performed by comparison with the reference value prepared based on the data from the control cells prepared in advance.

The method for detecting the reporter protein expressed from the reporter vector will be described below. As the reporter gene, a luminescent protein gene, a fluorescent protein gene, a coloring protein gene, an active oxygen producing enzyme gene, a heavy metal binding protein gene, or the like can be selected. A luminescent protein gene is a gene encoding an enzyme protein that catalyzes a luminescent reaction. Examples of the luminescent protein gene include a luciferase gene. Luciferase, which is a translation product of the luciferase gene, undergoes a luminescence reaction using luciferin, which is a kind of substrate.

Further chromogenic protein genes include, for example, the β-galactosidase gene. Β-galactosidase, which is a translation product of the β-galactosidase gene, is 5-bulmo 4-chloro-3-indrill-β-D-galactopyranoside (X-Gal) or o-nitrophenyl-β-D-galactopyrano. The color development reaction is carried out using a substrate such as sid (ONPG).

Specifically, when the reporter gene is a luciferase gene, the luminescence intensity may be measured after the substrate is added to the test cells and the protein from the reporter gene is synthesized. When the reporter gene is a fluorescent protein gene, for example, the test cell may be irradiated with light of a specific wavelength, and the intensity of fluorescence generated from the fluorescent protein in the test cell may be measured by a fluorescence measuring device. The active oxygen-producing enzyme gene is a gene encoding an enzyme that produces active oxygen. Examples of the active oxygen-producing enzyme gene include a nitric oxide synthase gene and a xanthine oxidase gene. Nitric oxide synthase, which is a translation product of the nitric oxide synthase gene, and xanthine oxidase, which is a translation product of the xanthine oxidase gene, generate active oxygen. Active oxygen can be detected by an electron spin resonance apparatus (ESR apparatus) or the like. In the method using an ESR device, the amount of active oxygen generated may be measured directly, or may be measured using a specific spin trap agent. When using a spin trapping agent, first, the active oxygen-producing enzyme is trapped with the spin-trapping agent, and then the amount of active oxygen generated from the trapped active oxygen-producing enzyme is measured. The heavy metal binding protein gene is a gene encoding a protein that specifically binds to a heavy metal. The amount of the protein bound to the heavy metal is determined by, for example, a magnetic resonance imaging device, a nuclear medicine diagnostic device, or an X-ray computed tomography device. It should be done.

The detection of the amount of heavy metal-binding protein generated is performed, for example, as follows. First, a measurable heavy metal is added to the culture medium of the test cells in advance. Next, after washing the test cells as needed, the heavy metal-binding protein is extracted from the test cells, and the obtained heavy metal-binding protein is quantified. Alternatively, a heavy metal is added to the culture medium of the test cells. Next, the test cells are imaged with an image diagnostic apparatus, and the heavy metal-binding protein to which the heavy metal is bound is detected or quantified from the obtained image. The heavy metal binding protein may be expressed inside the test cell or may be expressed on the outer surface of the test cell. For example, in MRI imaging, a gadolinium compound is preferably used because of its high contrast effect. The gadolinium compound may be, for example, a metal compound consisting of gadolinium, gadolinium ion, gadolinium complex, salts thereof and derivatives thereof, derivatives containing any of these, or analogs of gadolinium compounds.

In such a method for determining the cell characteristics of the test cells, the promoter activity characteristics of the test cells and the morphological characteristics of the same test cells are determined, thereby determining the overall cell characteristics of the test cells. judge. This makes it possible to determine the characteristics of cells with higher accuracy.

In the above example, comparison with control cells is performed in (S7) and (S9). However, the promoter activity characteristics of the test cells were specified in (S1) to (S6), and the morphological characteristics were specified in (S8) without comparing the control cells. From these results, the test cells of the test cells were identified. The characteristics may be determined.

For example, the determination of cell characteristics of a test cell depends on the constitution of a pre-designed reporter vector, that is, whether or not the cell is under a specific condition according to a preselected "specific condition". This may be done by identifying the cell and by identifying the morphological features.

Conventionally, when a reporter vector is used to determine cell properties, the morphology of the cells to be observed has not been emphasized. This is considered to be due to the same reason that morphology is not emphasized in PCR and FACS. However, when determining cell characteristics from only the information obtained using a reporter vector, for example, the frequency of expression of the reporter gene may not be sufficient. According to the embodiment, the cell characteristics can be determined more efficiently and more accurately by simultaneously observing the result of the promoter assay and the cell morphology and thereby determining the cell characteristics of the test cells.

Information on gene expression and expression control can be obtained using a reporter vector as described above and using a detectable signal as an index. Morphological information may be obtained by observing the desired morphological features of the test cells using a detectable device. Observation of morphological features may be performed by detecting predetermined feature points regarding cell contour, cell size, nucleus contour, nucleus size, number of nuclei, and the like. Detection of these feature points may be performed, for example, on individual cells contained in the obtained bright-field image obtained by imaging a bright-field image under a microscope. Further, the feature point to be detected may be obtained as the brightness indicated in combination with the position information in the image such as a bright field image. Further, it may be obtained as, for example, a table including position information in an image and brightness for each position. Further, the feature point may be, for example, a numerical value obtained by analyzing numerical values such as position information and brightness in an image by a predetermined analysis method.

Morphological features include, for example, the area of the test cell, the ratio of the area of the nucleus to the area of the test cell, the ratio of the minor axis to the major axis of the test cell, the number of nuclei in the test cell, or the side of the test cell. It may be a feature of the edge portion, for example, a contrast ratio of the edge portion. These are obtained by non-invasive optical imaging methods. For example, such information may be obtained by a hyperspectral imaging method, a phase contrast microscopy method, a differential interference contrast method, or the like.

For example, when morphological observation is performed in a bright field, it is preferable to irradiate light having a wavelength in the near infrared region. For example, the light having a wavelength in the near infrared region is preferably near infrared light having a wavelength of 700 nm to 750 nm or multispectral light having a wavelength of 350 nm to 750 nm. For example, the test cell may be irradiated with light having a wavelength in the near infrared region, and the reflection or absorption of the light, for example, the reflectance or absorption rate may be used as an index to obtain morphological information of the test cell. ..

By determining the cell characteristics of the test cells from the results of the promoter assay and the morphological characteristics, it becomes easier to avoid false positives and false negatives, and it becomes possible to perform pathological examinations with higher accuracy. In addition, cells having cell characteristics related to, for example, diseases can be detected more efficiently than in the past.

A method for determining the cell properties of a test cell is to detect a signal from a reporter vector and obtain a morphological feature of the test cell, for example, a CCD image sensor or a CMOS image such as a luminescent microscope or a fluorescence microscope. This may be performed using a device equipped with a fluorescence imaging device having an imaging function such as a sensor, a light emission imaging device, a fluorescence light emission imaging device, a magnetic resonance imaging device, a nuclear medicine diagnostic device, an X-ray computer tomography device, or the like. .. These devices further provide a detector capable of revealing, for example, the contours of the test cells, the contours of each, and predetermined morphological features in order to observe the morphological features of the test cells. You may have.

In one embodiment, the detection of the signal from the reporter vector and the acquisition of morphological features may be performed on one test cell or on a cell-by-cell basis for multiple test cells. It may be performed for each group for a group containing a plurality of test cells. For example, when an luminescent image is imaged using a luminescent microscope to detect a signal from a reporter protein, the bright field image is captured in the same field of view before or after imaging the luminescent image in addition to the luminescent image. Morphological features may be obtained. By superimposing (merging) the luminescent image and the bright-field image thus obtained, gene expression or expression control is related to one test cell or a plurality of test cells for each cell or group. Information and morphological information can be obtained.

The signal from the reporter protein is generated in the test cell through the following process, for example. From the time of introduction of the reporter vector into the test cell, the transcription control sequence is activated according to the environment in the test cell, and after the promoter activity is shown, the reporter protein is released by transcription of the reporter gene and translation of the transcript. Express. If desired, for example, if the reporter proteins are luciferases, they will react after the addition of the substrate. Therefore, the signal from the reporter protein may be measured after the predetermined time required for it to be observed. For example, it may be performed once or multiple times at any time between 8 hours and 72 hours, and once or multiple times at any time between 8 hours and 48 hours or 48 hours and 72 hours. It may be done.

Acquisition of morphological features may be before or after detecting the signal from the reporter protein, or may be simultaneous with signal detection. Also, the detection or measurement of the signal from the reporter protein may be performed at one time point, at multiple time points or continuously. Similarly, for the acquisition of morphological features, the acquisition points may be performed at one time point, at a plurality of time points, or continuously.

The nucleus may be stained to obtain morphological features. Nucleus staining may be carried out using a dye, for example, a fluorescent dye. Morphological features may be obtained from unstained test cells or from stained test cells, or one test cell may be obtained both unstained and stained. When staining the nucleus, a fluorescent dye such as DAPI, Hoechst 33258 or Hoechst 33342 may be incorporated into the nucleus of the test cell prior to observing the nucleus. In this case, for example, using a device capable of capturing a bright-field image, a luminescent image, and a fluorescent image, the test cell is first imaged with a bright-field image, then with a luminescent image, and then with a fluorescent dye. A fluorescence image may be taken after adding to the sample and staining the nucleus. It is also possible to perform a more accurate and rapid pathological examination by superimposing the bright-field image, the luminescent image, and the fluorescence image on each other.

The introduction of the reporter vector into the test cells is preferably performed before imaging the bright field image. Further, a bright field image, a light emitting image and a fluorescence image may be taken for each of the plurality of fields of view, and the results obtained from each of the plurality of fields of view may be averaged. Further, for example, an arbitrary field of view may be observed and arbitrarily imaged at a low magnification first, and a part of a region included therein may be observed and arbitrarily imaged at a higher magnification. Also, for example, even if it is calculated how many cells among all the test cells contained in the sample satisfy a specific condition and how many cells are morphologically abnormal. Good.

For example, in a preferred embodiment, an unstained test cell group is imaged in the bright field, then a luminescent image of the same test cell group is imaged in the dark field, and then the nucleus of the test cell group is fluorescent. After staining with the dye, a fluorescent image of the same cell group to be examined is taken. In this way, the cell characteristics of the test cells may be determined from all the results obtained by imaging the bright field image, the luminescent image, and the fluorescence image in this order. Further, the cell characteristics may be determined using a merged image obtained by superimposing a bright field image, a luminescent image, and a fluorescence image on each other. According to such an embodiment, it is possible to perform a more accurate and rapid pathological examination.

By such a method, it is possible to obtain information on the cell characteristics of the test cells as alive as possible. This makes it possible to perform a more accurate pathological examination.

Based on the results regarding the test cells obtained by such a method for determining cell characteristics, it is also possible to determine the size of the possibility that the test cells are a specific disease of the target from which the test cells are derived. In addition, a specific disease of the target from which the test cells are derived may be diagnosed based on the results regarding the test cells obtained by the method for determining the cell characteristics.

3. 3. Disease Testing Method A specific disease may be tested using a sample from a subject by determining cell characteristics. Testing methods for such diseases are also provided as embodiments. For example, according to embodiments, it is possible to know whether a test cell has cell properties associated with a particular disease. For example, even if a specific cell characteristic is linked to a specific disease in advance and the test cell is determined to have the specific cell characteristic, it is predicted that the target is likely to have the specific disease. Good.

The disease testing method may include the method for determining cell characteristics as described above, and the specific condition for activating the promoter activity may be a condition related to the disease to be tested. The disease-related condition may be a condition that is an indicator of the pre-stage or onset of a specific disease.

Examples of specific diseases may be cancer, mental illness, lifestyle-related diseases, neurological diseases, autoimmune diseases, cardiovascular diseases, etc., or pre-illness states such as pre-cancerous state, pre-diabetic state, etc. There may be, but it is not limited to these.

The "subject" may be an individual such as a mammal including mainly humans, livestock, pet animals and industrial animals, or may be an organ, organ, tissue or cell group obtained from the subject. The "test cell" may be a cell in a state contained in a subject, a cell in a state contained in a tissue, or an isolated cell. Preferably, the "test cell" is an isolated cell.

Further, according to the embodiment, the disease may be further diagnosed for the subject based on the cell characteristics determined by the above embodiment. Diagnosis may be made depending on what particular cellular conditions or conditions in which the pre-designed reporter vector is activated suggest.

The method for determining the cell characteristics of the test cells is performed in vitro for the collected test cells. That is, the method may be performed on the test cells collected from the subject. It can also be used as a method for diagnosing a disease in a subject based on the genetic information on the transcriptional activity of cells and the information on the morphology obtained by the method. The diagnostic method introduces a self-replicating vector into an individual, organ, organ, tissue, cell group and / or single cell to be detected. After that, the signal from the reporter protein may be detected and morphological information may be obtained.

4. Drug screening method A method for determining the cell characteristics of a test cell can also be used to screen a drug for treating or preventing a specific disease. For example, if a drug-treated cell group treated with a drug to be screened and a pathological cell group not treated with a drug are prepared, the cell characteristics of each of these two cell groups are determined, and the obtained results are compared. Good. The pathological cell group may have cell characteristics characteristic of cells having a pathological condition to be treated, that is, pathological cell characteristics. Drug screening may be performed using whether or not such pathological cell characteristics have been treated by drug treatment as an index. Further, in addition to the drug-treated cell group and the pathological cell group, a normal cell group may be prepared and the cell characteristics of the normal cell may be determined. The cellular properties of normal cells may be used as a control when screening for drugs. The cellular properties of normal cells may be determined in parallel with drug screening or may be determined in advance.

5. Device for determining cell characteristics of test cells A device for determining cell characteristics of test cells according to an embodiment will be described with reference to FIG. FIG. 5 is a block diagram showing an outline of the device 70. The device 70 is an example of a device that measures the transcriptional activity and morphological characteristics of a test cell and determines the cell characteristics based on the measurement.

The device 70 includes a cell separation unit 71, a DNA introduction unit 72, a measurement unit 73, and a stage 74 continuously arranged below them. The cell separation unit 71, the DNA introduction unit 72, and the measurement unit 73 share one stage 74 and are communicated in this order by the stage 74. In other words, the cell separation unit 71, the DNA introduction unit 72, and the measurement unit 73 are arranged above the stage 74 in this order from upstream to downstream.

The cell separation unit 71 includes a separator 75 for separating cells to be tested from a sample obtained from a subject. The separator 75 is a receptor 76a that receives a sample obtained from a subject, a processor 76b that digests the sample received by the receptor 76a, and cells to be tested from the processed sample processed by the processor 76b. It is provided with a separation unit 77 for separating the above. The receptor 76a, the processor 76b, and the separation unit 77 are formed by stacking three hollow tubular bodies having different inner diameters in this order from top to bottom and liquid-tightly integrating them with each other. Each boundary between the receptor 76a, the processor 76b, and the separation portion 77 is entwined with a valve so as to be openable and closable. The receiver 76a is a tapered tubular body with an open top and has a lid that can be opened and closed. At the boundary between the receptor 76a and the processor 76b, the valve closes liquid-tightly. When this valve closes, it becomes the bottom of receptor 76a. The processor 76b is a tubular body continuous with the lower end of the receptor 76a. When the valve at the boundary with the receptor 76a is closed liquidtightly, the processor 76b is covered. When the valve at the boundary with the separating portion 77 is closed, the processor 76b becomes bottomed. The separation portion 77 is a tubular body whose inner wall is liquidtightly continuous from the inner wall of the processor 76b, and a valve is provided at the lower end thereof. The valve closes liquidtightly and the separation part 77 becomes bottomed. A separating material is fixed inside the separating portion 77. When the device 70 is used, the container 78 is placed on the stage 74 below the bottom of the separation portion 77. The container 78 is a container 78 that receives a sample containing the separated test cells 79 from the separation unit 77 and stores the sample. Here, the processor 76b may be provided with a heating and / or constant temperature mechanism for performing a desired treatment such as digestion and biochemical reaction on the sample contained therein. Further, the separating material may be a member for separating and collecting a sample containing a test cell from a sample descended from the processor 76b, or a contaminant may be removed from the sample descended from the processor 76b. It may be a member to be removed. The separating material may spread along a surface intersecting the central axis of the separating portion 77 and may be fixed over the circumference of the inner wall of the separating portion 77. The separating material may be, for example, a filter, a membrane and / or a magnet.

The sample containing the test cells 79 separated by the separator 75 is housed in a container 78 placed on the stage 74. The container 78 containing the test cells is sent to the DNA introduction unit 72.

The DNA introduction unit 72 includes a control unit 80 for introducing a reporter vector into the test cells housed in the container 78, and an introduction probe 81. The introduction of the reporter vector into the test cells may be carried out by a method known per se. The control unit 80 and the introduction probe 81 may be devices that utilize any introduction mechanism for achieving the selected introduction method. For example, the DNA introduction unit 72 may be a device that performs electroporation. In that case, the control unit 80 may be a pulse generator, the introduction probe 81 may be an electrode probe, and the electroporator may be configured by these. Further, the DNA introduction unit 72 may be provided with a nozzle for supplying a reagent containing a reporter vector to the container 78 (not shown). Further, the DNA introduction unit 72 may include a liquid feeding system for feeding the reagent to the nozzle and a reagent storage container for supplying the reagent to the liquid feeding system (not shown).

Further, the DNA introduction unit 72 may function as an incubator for culturing the test cells 79 in the container 78 under a predetermined culture condition for a predetermined time. The culture of the DNA introduction unit 72 may be controlled by the control unit 80.

In the DNA introduction unit 72, the container 78 containing the test cells into which the reporter vector has been introduced is then sent to the measurement unit 73.

The measurement unit 73 is installed in a dark box 82 that covers a part of the stage 74 from the upper part and the lower part, a light source 83 arranged inside the dark box 82, a window that opens on the stage surface below the light source 83, and below the window. It includes an image pickup unit 88 to which the objective lens 87 is attached, a control unit 85 that communicates with the light source 83 and the image pickup unit 88, and a display unit 84 and a storage unit 86 that communicate with the control unit 85, respectively. For example, the light source 83, the objective lens 87, and the imaging unit 88 of the measurement unit 73 may be included in a microscope equipped with a CCD camera, a CMOS camera, or the like. Further, the control unit 85, the display unit 84, and the storage unit 86 may be a computer including these.

An apparatus for determining the cell characteristics of the test cells according to the embodiment will be described with reference to FIG. FIG. 6 is an example of the device 70 shown in FIG. 5, which differs only in the configuration of the stage. The device 70 includes a cell separation unit 71, a DNA introduction unit 72, a measurement unit 73, and stages 74a, 74b, 74c arranged below each of these units, respectively. As described above, in the device 70 of FIG. 6, a stage is provided for each unit. The movement between the units, that is, between the stages may be performed manually, or each movement may be performed by a robot arm or the like.

An example of determination of cell characteristics by the device 70 shown in FIG. 5 will be described with reference to FIG. First, the sample is brought into the cell separation unit (S20). Next, the sample is processed in the processing unit (S21). The test cells are separated from the sample treated in S21 at the separation unit (S22). The isolated test cells are seeded in a container (S23). A container containing the test cells is sent from the cell separation unit to the DNA introduction unit (S24). A reporter vector is introduced into the test cells (S25). The test cells are cultured for a predetermined time (S26). A bright field image is taken (S27). The luminescence image is taken (S28). The cell characteristics are determined based on the bright field image and the luminescent image (S29). For example, the determination of such cell characteristics may be performed as follows. First, the container 78 is placed on the stage 74 below the separation portion 77 of the cell separation unit 71. Next, a sample that has been pretreated, such as mince or homogenize, is brought into the receptor 76a from the upper opening, if desired. Along with this, reagents necessary for processing in the processing unit 76b are added. The reagent may be added from the reagent addition port provided in the processor 76b so as to be openable and closable. The lid of the receptor 76a is closed and the valve at the boundary with the processor 76b is opened. The digestion process is performed in the processor 76b according to preset conditions. The processor 76b may include a temperature controller to create an environment that satisfies preset conditions. Next, the valve at the boundary between the processor 76b and the separation unit 77 is released, and the sample processed in the processor 76b is sent to the inside of the separation unit 77. By passing through the separating material of the separating unit 77, impurities contained in the sample are removed. The sample containing the isolated test cells is sent to the container 78 and contained. Here, a reaction stop reagent for stopping the reaction carried out in the processor 76b prior to passing through the separating material may be added to the sample. For this addition, the reaction stop reagent may be stored in the separation unit 77 in advance, and an openable / closable reagent addition port for adding such a reagent is provided on the wall surface at any position of the separator 75. May be.

The container 78 is sent to the DNA introduction unit 72 while still containing the sample containing the test cells 79. A reagent containing a reporter vector is added to the container 78 from the nozzle of the DNA introduction unit 72. The introduction probe 81 is brought into contact with the sample in the container 78, and the reporter vector is introduced into the test cell 79 under the control of the control unit 80. Under the control of the control unit 80, the test cells 79 are cultured under a predetermined culture condition for a predetermined time.

After that, the container 78 is sent to the measuring unit 73. The measuring unit 73 measures the reporter protein mass and morphological characteristics of the test cells 79 housed in the container 78. All procedures in the measurement unit 73 are controlled by the control unit 85 according to a table in which a program and a plurality of information stored in advance in the storage unit 86 are collected. For example, the measurement unit 73 controls light irradiation from the light source 83, control of irradiation conditions such as irradiation time and irradiation interval, control of imaging and imaging conditions by the imaging unit 88, image processing and image processing of the captured image, and the like. Will be done. Further, the control unit 85 also determines the cell characteristics by referring to the table including the information in which the image and the cell characteristics are associated with each other according to the program stored in the storage unit 86 in advance.

After culturing the test cells in the DNA introduction unit 72, the container 78 is sent to the measurement unit 73. After that, the light from the light source 83 is irradiated into the container 78. The light that has passed through the container 78 is collected by the objective lens 87, and the imaging unit 88 obtains a bright field image from the light. Then, in the dark box 82, an optical signal (here, light emission as an example) from the reporter protein introduced into the test cells is imaged by the imaging unit 88 through the objective lens 87. The light irradiation of the light source 83 is stopped, the light emitted from the test cells is collected by the objective lens 87, and the imaging unit 88 obtains a light emitting image from the light. The bright field image and the light emitting image obtained by the imaging unit 88 are image-processed by the control unit 85, superposed (merged), and displayed on the display unit 84. The control unit 85 determines the cell characteristics of the test cells based on the bright-field image and the luminescent image and the table stored in the storage unit 86 at the same time as or before and after the display of the image on the display unit 84. judge. The result of this determination is shown on the display unit 84 together with the merged image.

Here, an example in which light emission is generated as an optical signal from the reporter protein is shown, but even for a detectable signal in which a reporter protein different from the light emission is generated, the detection device constituting the imaging unit 88 is replaced. It can be measured in the same way.

Further, although not shown in the figure, the measurement unit 73 may further include a processing unit that performs computer processing, image processing, and the like, or an analysis unit that performs characteristic determination. These processing units and analysis units may perform the computer processing and image processing performed by the control unit 85 and the characteristic determination described above.

In the above embodiment, the example in which the DNA introduction unit 72 cultures the test cells is shown, but the test cells may be cultured in the measurement unit 73. In that case, the device 70 may further include a member that enables cell culture. Alternatively, the test cells may be cultured in both the DNA introduction unit 72 and the measurement unit 73.

Each unit may have its own control unit to control the movement of each unit of the device 70, and all the units included in the device 70 may be performed by one control unit, for example, the control unit 85. .. Further, the movement between the units of the container 78 mounted on the stage 74 may be performed by the container moving mechanism mounted on the stage 74. Such a mechanism may be performed, for example, by a robot arm provided in the device 70 and whose movement is controlled by a control unit provided in the device 70. Alternatively, a belt conveyor may be attached to the stage 74.

Further, the addition of the reporter vector to the container 78 may be performed at any time before the container 78 is sent to a predetermined position of the DNA introduction unit 72. In that case, the device 70 may further provide a reporter vector addition mechanism at a desired position upstream of the DNA introduction unit 72.

Further, FIG. 8 shows an example of a method in which the signal from the reporter protein is luminescence and the nucleus is observed by staining the nucleus with a fluorescent dye. Up to the imaging of the luminescent image (S28), the same method as shown in FIG. 7 may be performed. After imaging the luminescent image, a fluorescent dye for staining the nucleus is added into the container 78 (S29A). Next, a fluorescence image is taken of the nucleus of the test cell (S30A). Then, the cell characteristics are determined based on the obtained bright-field image, luminescent image, and fluorescence image (S31A). In addition, the bright field image, the luminescent image, and the fluorescent image may be shown merged with each other.

With such a device, the cell characteristics of the test cells are determined. According to this device, it is possible to evaluate the cell characteristics of the test cells with higher accuracy. In addition, since the inspection can be performed with throughput, the inspection can be easily performed, and it is possible to prevent the sample from being mistaken and contamination.

In conventional cytodiagnosis, cell fixation is usually performed. For example, in the case of HER-2 staining, a formalin-fixed paraffin-embedded specimen is used. Fluorescence in situ hybridization (FISH) is performed on the fixed cells to detect the copy number of the HER-2 gene, thereby obtaining genetic information on the test cells. Such formalin-fixed paraffin-embedded specimens do not test for promoter activity of test cells. In the embodiment, since the test cells are tested alive, it is possible to more accurately test the state of the subject. In addition, since the diagnosis is made from multiple aspects of promoter activity and morphological characteristics, more accurate results can be obtained.

In addition, it is also possible to obtain epigenetic information by using self-replicating vectors, whereby a subject suffers from a particular disease, even at the early or pre-disease stage, for example. It becomes possible to judge the possibility more accurately. Changes in epigenetic regulation are caused by changes in regulation such as DNA modification, chemical modification of histones, and non-coding RNA. For example, the presence or absence of modification such as methylation of a specific base sequence and the change in the amount of modification are one of useful epigenetic information. By determining cellular properties from such epigenetic information and morphological properties, embodiments, the subject suffers from a particular disease, even at the early or pre-disease stage, for example. It becomes possible to more accurately determine whether or not it is present.

[Example]
Example 1 Analysis of breast cancer cells using a reporter vector incorporating the NAMPT gene (1) Construction of a reporter vector As a target nucleic acid sequence, the promoter region (-2702- + 276bp, SEQ ID NO: 11) of the nicotinamide phosphoribosyltransferase (Nampt) gene is used. The incorporated reporter vector was prepared. The promoter region of the Nampt gene was amplified by PCR using the human genome as a template to obtain a nucleic acid fragment consisting of the target nucleic acid sequence. PCR was performed using PrimeStar GLX DNA polymerase (TaKaRa BIO). The primer sequences used for PCR are shown below.

Forward primer: 5'-GGGACGCGTCCATGTTGCCCAGGCTGGTCTCA-3'(SEQ ID NO: 13)
Reverse primer: 5'-CGGCTCGAGAGCTCCCTGGCGCGGCTGCGAGGAA-3' (SEQ ID NO: 14).

The nucleic acid fragment obtained above was incorporated into PGV-B2 (TOYO B-Net) to prepare a reporter vector: pNampt-L. PGV-B2 is a vector in which a luciferase gene (reporter gene) and a transcription termination signal sequence are provided in this order from upstream to downstream. A nucleic acid fragment having a CK19 promoter region obtained by PCR was incorporated upstream of the luciferase gene of PGV-B to prepare a reporter vector: pNampt-L (FIG. 9).

(2) Lipofection (introduction of reporter DNA into cells)
To 500 μL of Opti-MEM (Life Technologies), 2.5 μg of reporter vector: pNampt-L and 2.5 μg of enhancer reagent (Plus reagent, Life Technologies) were added. This was incubated at room temperature for 5 minutes. To this solution, 6.25 μL of Lipofectamine LTX (Life Technologies) was added and the mixture was well turbid. It was then incubated at room temperature for 25 minutes. Transfer the solution to a 35 mm culture dish and gently add 2.0 mL of a suspension of human mammary gland-derived malignant tumor cells: BT-549 (purchased from ATCC) or benign tumor cells: MCF-10A (purchased from ATCC). Mixed. Then, the cells were cultured in an incubator at 37 ° C. and a 5% CO ₂ atmosphere.

(3) Judgment of cell characteristics The luminescence imaging of the reporter vector-introduced cell line was performed by the LUMINOVIEW luminescence imaging system (LV200, Olympus). Twenty-four hours after the lipofection shown in Example 1 (2) above, the luminescent substrate VivoGlo luciferin (Promega) was added to the medium of pNampt-AmpL-introduced BT-549 and pNampt-AmpL-introduced MCF-10A (final concentration 200 μM). The culture dish was set at a predetermined position in LUMINOVIEW, and signals from cells luminescent by transcriptional activation of the reporter vector were acquired by luminescence imaging. Imaging of luminescent cells was performed under dark field conditions at a magnification of 20 times and a luminescence detection time of 20 minutes. Cell morphology was obtained by imaging under bright field conditions. Further, the two images were superposed by the image processing software Metamorph. Many luminescent cells were observed in human mammary gland-derived malignant tumor cells BT-549 (Fig. 10 (A)), but few luminescent cells were observed in human mammary gland-derived benign tumor cells MCF-10A (Fig. 10 (A)). 10 (B)). Further, it was confirmed that the luminescence in FIG. 10B was from a region other than the cell. In addition, the morphology of the human mammary gland-derived malignant tumor cell BT-549 and the human mammary gland-derived benign tumor cell MCF-10A could be observed well. That is, in the benign tumor cells MCF-10A derived from the human mammary gland, the observed cell morphology in the visual field was constant. In contrast, the human mammary gland-derived malignant tumor cell BT-549 had an amorphous cell morphology contained in the observed visual field as compared with the human mammary gland-derived benign tumor cell MCF-10A. According to the embodiment, it was proved that the presence or absence of breast cancer characteristics can be determined for the test cells from the reporter vector activity characteristics and morphological characteristics (Fig. 10).

Example 2 Analysis of liver cancer cells using a reporter vector incorporating the CK19 gene (1) Preparation of a self-amplifying reporter vector As a target nucleic acid sequence, the promoter region of the cytokeratin 19 (CK19) gene (-845- + 61bp, SEQ ID NO: 12) A self-amplifying reporter vector incorporating the above was prepared. The nucleic acid fragment of the promoter region of the CK19 gene was obtained by amplification by PCR using the human genome as a template. The obtained nucleic acid fragment was used as a nucleic acid fragment having a target nucleic acid sequence. PCR was performed using PrimeStar GLX DNA polymerase (TaKaRa BIO). The primer sequences used for PCR are shown below.

Forward primer: 5'-TGGGCTAGCCCAAGAAGCTGGTTCTGAGAGG-3'(SEQ ID NO: 15)
Reverse primer: 5'-GGACTCGAGGGCGAGGCGGAGCACGGACGGAG-3' (SEQ ID NO: 16).

The target nucleic acid sequence obtained above was incorporated into a non-amplified reporter vector: PGV-B2 (TOYO B-Net) or a self-amplified reporter vector: pM53-R550K to prepare pCK19-L and pCK19-AmpL, respectively. .. PGV-B2 is a vector in which a luciferase gene (reporter gene) and a transcription termination signal sequence are provided in this order from upstream to downstream, and pM53-R550K is a luciferase gene (reporter gene), IRES, and a replication initiation protein gene. This is a vector in which a transcription termination signal sequence and a replication initiation sequence are provided in this order from upstream to downstream. A nucleic acid fragment having a CK19 promoter region obtained by PCR was incorporated upstream of the luciferase gene of PGV-B2 and pM53-R550K to prepare a non-amplified reporter vector: pCK19-L and a self-amplified reporter vector: pCK19-AmpL. (Fig. 11).

(2) Electroporation (introduction of reporter DNA into cells)
It was suspended in human hepatoma cell line Huh-7 at a density of 2x10 ⁶ cells / mL Opti-MEM (Life Technologies ). To 100 μL of this cell suspension, 10 μg of a reporter vector (pCK19-AmpL or pCK19-L) was added, mixed well, and then transferred to a cuvette electrode for electroporation. For electroporation, using CUY21EDIT II (Bex Co., Ltd.), poration pulse 150V, pulse width 2.5ms, pulse number 1 time, polarity +, driving pulse 10V, pulse width 50ms, pulse interval 50ms, pulse number 10 This was performed under the conditions of a capacitor of 1416 μF and a polarity of +/-. This introduced the reporter vector into Huh-7. After electroporation, the medium was added to the cell suspension and transferred to a 35 mm culture dish, and the cells were cultured in an incubator at 37 ° C. and a 5% CO ₂ atmosphere.

(3) Luminescence imaging The luminescence imaging of the reporter vector-introduced cell line was performed by a LUMINOVIEW luminescence imaging system (LV200, Olympus). Two hours after the electroporation of Example 2 (2)], the luminescent substrate VivoGlo luciferin (Promega) was added to the medium of pCK19-AmpL-introduced Huh-7 and pCK19-L-introduced Huh-7 (final concentration 200 μM). The culture dish was set at a predetermined position in LUMINOVIEW, and signals from cells luminescent by transcriptional activation of the reporter vector were acquired by luminescence imaging. The luminescent cells were imaged under dark field conditions at a magnification of 20 times and a luminescence detection time of 10 seconds. In addition, photography was performed intermittently for 64 hours at intervals of 30 minutes. Cell morphology was obtained by imaging under bright field conditions. After that, the two images were superposed by the image processing software Metamorph. 12-1 and 12-2 show images obtained by imaging cells whose transcription was activated by each vector at 0, 24, 48, and 64 hours. The pCK19-AmpL-introduced Huh-7 showed strong luminescence after 24 hours, the intensity of which luminescence increased over time (FIGS. 12-3 (b-2), 12-4 (b-3) and (Fig. 12-3 (b-3)). b-4)). On the other hand, the emission of the pCK19-L-introduced Huh-7 was much weaker than that of the pCK19-Ampl-introduced Huh-7, and no increase in the emission intensity was observed with the passage of time (FIG. 12-1 (a-). 1) and (a-2), FIGS. 12-2 (a-3) and (a-4)).

Example 3 Analysis of morphological characteristics by hyperspectral imaging method The following experiments were conducted to clarify the effect of the wavelength of the irradiated light on the imaging accuracy. Specifically, the morphological characteristics of breast cancer cells and hepatocellular carcinoma cells were observed by a hyperspectral imaging method or a method using an inverted microscope. The cells used as imaging targets are the six types shown in Table 13.

The imaging apparatus used for observation by the hyperspectral imaging method is as follows. A hyperspectral camera (HSC1072, manufactured by Hokkaido Satellite Co., Ltd.) was connected to an upright microscope (Axio Imager M2: manufactured by ZEISS) from which the IR cut filter of the halogen light source had been removed in advance. This imaged the subject. As the analysis software, HSD Analysiser Ver4 (manufactured by Hokkaido Satellite Co., Ltd.) was used.

First, each of the above six types of cells was cultured on a resin chamber slide (Vermanox treatment). At the time of imaging, the culture solution was removed and sealed with a cover glass together with a small amount of phosphate buffer (PBS).

The results of imaging each cell by the hyperspectral imaging method are shown in FIGS. 13 to 18. FIG. 13 shows a 10-fold observation of the Huh-7-P strain, which is a human liver cancer cell. 13 (a), 13 (b) and 13 (c) are views of the same field of view of the same sample irradiated with light containing different wavelengths.

FIG. 13A is a diagram taken by irradiating light including all wavelengths between 350 nm and 1050 nm without splitting into a specific wavelength. FIG. 13B is a diagram taken by spectroscopy and irradiating with light containing only the wavelengths of the visible region, 400 nm to 700 nm. FIG. 13 (c) is a diagram imaged by spectroscopically irradiating light containing only a wavelength between 700 nm and 750 nm, which is a wavelength in the near infrared region. From the results shown in FIGS. 13 (a), 13 (b) and 13 (c), near-infrared region image (700 nm to 750 nm)> all-wavelength image (350 nm to 1050 nm)> visible region image (400 nm to 700 nm). It can be seen that it is possible to clearly identify the edge of the cell in the order of. That is, in order to obtain morphological information of cells, it is preferable to take a near-infrared region image (700 nm to 750 nm) or an all-wavelength image (350 nm to 750 nm), and further, a near infrared region image (700 nm to 750 nm). ) Is more preferable.

The results of FIGS. 13 (a), 13 (b) and 13 (c) were processed by analysis software (HSD Analysister Ver4), and the obtained results are shown in FIG. FIG. 14 shows the spectral intensities obtained when observing the cell edge and the cytoplasm using wavelengths of 400 nm to 1000 nm, respectively. In FIG. 14, the horizontal axis represents the wavelength (nm) and the vertical axis represents the spectral intensity. From the graph showing the relationship between the wavelength and the spectral intensity, the wavelength region in which the cell edge and the cytoplasm can be distinguished more accurately was examined. As a result, it was found that the wavelength at which the cell edge and the cytoplasm can be discriminated most accurately is 700 nm to 750 nm. The wavelength region in which higher discrimination accuracy can be obtained was 700 nm to 750 nm.

15 (a) and 15 (b) are images of different human liver cancer cell lines, namely Huh-7-P and Huh-7-02, taken with an inverted microscope. In the case of any cell line, the peripheral part and the nucleus of each cell were clearly imaged. It was observed that both Huh-7-P and Huh-7-02 were cells having a small aspect ratio without long protrusions. As described above, it was possible to satisfactorily observe the morphological characteristics of the cells of different cell lines of liver cancer cells.

16 (a), 16 (b) and 16 (c) are diagrams showing the results of imaging the same field of view of Huh-7-P with light of different wavelengths by the hyperspectral imaging method. 16 (a), 16 (b) and 16 (c) are the results of imaging with light containing 350 nm to 1050 nm, 400 nm to 700 nm and 700 nm to 750 nm, respectively. 16 (d), 16 (e) and 16 (f) are diagrams showing the results of imaging the same field of view of Huh-7-02 with light of different wavelengths. 16 (d), 16 (e) and 16 (f) are the results of imaging with light including wavelengths of 350 nm to 1050 nm, 400 nm to 700 nm and 700 nm to 750 nm, respectively. In the case of both cell lines, the result of imaging with light having a wavelength of 700 nm to 750 nm was the best and morphological information could be clearly obtained. For example, for individual cells, the cell edge and cytoplasm could be distinguished more accurately. From these results, it is clear that it is possible to obtain clearer cell morphological information by using the hyperspectral imaging method than by using a bright-field image taken by a general inverted microscope. It became.

Next, morphological features were observed for human breast cancer cell lines, BT549, MDA-MD-231, T47D and MCF7.

17 (a), 17 (b), 17 (c) and 17 (d) are views of MCF7, T47D, BT549 and MDA-MD-231 imaged using an inverted microscope, respectively. From these figures, MCF7 and T47D both had a small aspect ratio, and some cells showed paving stones. On the other hand, BT549 and MDA-MD-231 had a long and thin shape with a large aspect ratio. On the other hand, as shown in Table 13, BT549 and MDA-MD-231 are highly invasive type cancer cells, and MCF7 and T47D are low infiltration type cells. These results indicate that the highly invasive type breast cancer cells have a large aspect ratio, and the low infiltration type breast cancer cells have a small aspect ratio. Using the size of such aspect ratio as an index of cell morphological characteristics, more accurate determination of cell characteristics from this index and information on gene expression and expression regulation, in this case, in highly infiltrating type breast cancer cells It is possible to determine whether the cells are present or low infiltration type breast cells.

FIG. 18 is a diagram showing the results of observation by the hyperspectral imaging method at wavelengths separated so as to include 700 nm to 750 nm. 18 (a), 18 (b), 18 (c) and 18 (d) show the results of the low infiltration types MCF7 and T47D, and the high infiltration types BT549 and MDA-MD-231, respectively. .. This result more clearly shows that the low infiltration type cells show a round morphology with a small aspect ratio, and the high infiltration type cells show a spindle shape with a large aspect ratio. This figure also clearly shows the shape of the nucleus. From these results, it is clear that it is possible to obtain clearer cell morphological information by using the hyperspectral imaging method than by using a bright-field image taken by a general inverted microscope. Is.

Example 4 Epigenetic analysis Preparation of plasmid-type self-replicating vector A plasmid-type self-replication vector containing a methylated target nucleic acid sequence was prepared as outlined in FIG. The GFAP gene promoter sequence was selected as the target nucleic acid sequence. A GFAP gene promoter sequence was prepared as a target nucleic acid sequence by amplifying the template nucleic acid and cleaving it with a restriction enzyme. The obtained GFAP gene promoter sequence is a nucleic acid sequence containing a modified base and having promoter activity. Cleavage with a restriction enzyme was performed, and then this target nucleic acid sequence was methylated with the methylase SssI. The resulting methylation target nucleic acid sequence was incorporated into the pM53-R550K vector by ligase ligation. The pM53-R550K vector is a vector in which a reporter gene, an IRES, a replication initiation protein gene, a transcription termination signal sequence, and a replication initiation sequence are provided in this order from upstream to downstream. The obtained vector was used as a plasmid-type self-replicating vector containing a methylated target nucleic acid sequence. Details of each process are described below.

(1) Preparation of GFAP gene promoter sequence, which is a target nucleic acid sequence PCR was performed using mouse genomic DNA as a template. The amplified sequence in the mouse genomic DNA is the sequence shown in SEQ ID NO: 8 shown in Table 8.

The nucleotide sequences of the primers used for PCR are the following SEQ ID NOs: 17 and 18;
Forward primer: 5'-CGACGCGTGTCTGTAAGCTGAAGACCTGGC-3'(SEQ ID NO: 17)
Reverse primer: 5'-AAAAGTACTCCTGCCCTGCCTCTGCTGGCTC-3' (SEQ ID NO: 18).

PCR was performed using mouse genomic DNA as a template using these forward and reverse primers to obtain a nucleic acid fragment having the GFAP gene promoter sequence shown in SEQ ID NO: 8. The obtained GFAP gene promoter sequence was used as a target nucleic acid sequence containing a modified base and having a promoter activity depending on the degree of modification. This GFAP gene promoter sequence is the 5'upstream region (-1651 bp to + 32 bp) of the mouse GFAP gene. Next, the GFAP gene promoter sequence was cloned into a PGV-B2 (TOYO B-Net) vector.

The PGV-B2 (TOYO B-Net) vector was cleaved with the restriction enzyme SmaI. This was dephosphorylated. The phosphorylated nucleic acid fragment represented by SEQ ID NO: 8 was ligated to this vector with T4 ligase. As a result, a PGV-B2-GFAPp vector was prepared.

The obtained PGV-B2-GFAPp vector is used as a material for obtaining the GFAP gene promoter sequence in the following steps, and at the same time, it is used as a PGV-B2-GFAPp vector in a detection test described later for comparison. used. In addition, a methylated PGV-B2-GFAPp vector is also produced by methylating the GFAP gene promoter sequence contained in the PGV-B2-GFAPp vector by a step described later. This is also used in the detection test described later for comparison. None of the methylated or unmethylated PGV-B2-GFAPp vectors are self-replicating vectors.

To obtain the GFAP gene promoter sequence, the PGV-B2-GFAPp vector was digested with restriction enzymes MluI and XhoI. As a result, a GFAP gene promoter sequence to which the restriction enzyme MluI and the restriction enzyme XhoI recognition sequence were added was prepared. This sequence was used in subsequent steps as the target nucleic acid sequence.

(2) Methylation of modified base The GFAP gene promoter sequence was methylated, and the modified base contained therein was methylated.

For 40 μL of the GFAP gene promoter sequence nucleic acid solution prepared in (1) above, 16 μL of 10xNE Buffer2, 8 μL of methylase SssI (New England Biolab Japan Co., Ltd.), S-adenosylmetryionine (SAM) (New). England Biolabs Japan, Ltd.) 8μL, and ddH ₂ O was added 88μL. As a result, a reaction solution was prepared. This was subjected to an enzymatic reaction at 37 ° C. for 6 hours. After the methylation reaction, the cells were incubated at 65 ° C. for 20 minutes using a heat block. This inactivated Sss I. Further, this was purified using a QIA quick PCR purification kit (Qiagen Co., Ltd.). As a result, a purified GFAP gene promoter sequence was obtained. The base to be methylated in the obtained sequence is methylated.

On the other hand, for the above reaction solution, a purified GFAP gene promoter sequence in which the methylated base was not methylated was prepared as an unmethylated control sequence by the same method as above except that SssI was not added.

In addition, methylation of the GFAP gene promoter sequence contained in the PGV-B2-GFAPp vector obtained in (1) above was also carried out in the same manner as above. As a result, a methylated PGV-B2-GFAPp vector was obtained.

(3) Functional linkage between the plasmid-type self-replicating vector pM53-R550K and the purified target nucleic acid sequence GFAP gene promoter sequence The self-replicating vector pM53-R550K vector was cleaved with restriction enzymes MluI and XhoI. In response, the purified GFAP gene promoter sequence obtained in (2) above was ligated with T4 ligase. As a result, a pM53-R550K-GFAPp vector was obtained. This pM53-R550K-GFAPp vector contains a target nucleic acid sequence. The target nucleic acid sequence is methylated.

The unmethylated control sequence obtained in (2) above was also incorporated into the vector by the same method. As a result, an unmethylated pM53-R550K-GFAPp vector was obtained as a control vector.

Example 5 Detection of methylation and demethylation of target nucleic acid sequence To confirm that the degree of promoter activation changes depending on the state of base modification of the target nucleic acid sequence contained in the pM53-R550K-GFAPp vector. Was tested.

(1) Vectors used in the test The methylated plasmid-type self-replicating vector pM53-R550K-GFAPp vector obtained by the method of Example 4 above and the unmethylated plasmid-type self-replication vector pM53-R550K-GFAPp vector. And were used. These are self-replicating plasmid vectors.

For comparison, the methylated PGV-B2-GFAPp vector obtained by the above method and the unmethylated PGV-B2-GFAPp vector were used. These are plasmid vectors that cannot self-replicate.

As a vector for the internal standard, a β-galactosidase expression vector pcDNA4 / V5-His / lacZ vector (Life Technologies) was used.

(2) Introduction of vector into glioma C6 cells (a) Introduction of vector into cells pcDNA4 / V5-His / lacZ vector, methylated or unmethylated PGV-B2-GFAPp vector and methylated or unmethylated The pM53-R550K-GFAPp vector was introduced into each glioma C6 cell. This introduction was carried out by the lipofection method. For each introduction of these vectors, Lipofectamine 2000 (Life Technologies) was used as an introduction reagent. The operation of lipofection was performed according to the manual by the reagent manufacturer. The outline is as follows. In a microtube, 0.45 μL of cationic lipid (lipofectamine 2000) suspended in 25 μL of Opti-MEM was mixed with 25 μL of Opti-MEM containing either vector. This formed a lipofectamine / vector complex. Here, the amount of each vector contained in 25 μL of Opti-MEM is as follows. For pM53-R550K-GFAPp or PGV-B2-GFAPp, 25 μL of Opti-MEM contained 0.066 μg of DNA. For pcDNA4 / V5-His / lacZ, 25 μL of Opti-MEM contained 0.033 μg of DNA.

(B) Seeding After forming a lipofectamine / vector complex for each vector, 50 μL of each complex was added to a culture plate (96-well plate) per well. Further, 100 μL of a glioma C6 cell solution (5 × 10 ⁵ cells / mL) suspended in RPMI1640 medium (containing 10% immobilized FCS) was inoculated per well.

(C) Culturing After that, the plate was gently stirred for 1 minute to mix the reaction solution and the cell solution. Then, adhesion culture was carried out at 37 ° C. and 5% CO ₂ atmosphere for 24 hours and at 37 ° C. and 5% CO ₂ atmosphere.

(2) Demethylation of Modified Base by 5-aza-2-deoxycytide After introducing each vector into glioma C6 cells, a demethylation reaction was carried out on the target nucleic acid sequence contained in each vector. The demethylation reaction was carried out using 5-aza-2-deoxycytide (5-aza-dC) (Funakoshi Co., Ltd.), which is an inhibitor of methyltransferase.

The 5-aza-dC reagent was prepared as follows. 5-aza-dC was added to PBS to a concentration of 500 μM to prepare a 5-aza-dC diluted solution. This 5-aza-dC dilution was added to fresh RPMI1640 medium to make a solution containing 5 μM 5-aza-dC. The resulting solution was used as a 5-aza-dC reagent.

Demethylation was performed as follows. First, after culturing (c), the medium in each well was removed. Then, 200 μL of 5-aza-dC reagent was added. As a control for not performing the demethylation reaction, 200 μL of RPMI1640 medium supplemented with PBS was added instead of the 5-aza-dC reagent. This was further subjected to adherent culture for 48 hours at 37 ° C. in a 5% CO ₂ atmosphere.

(3) Reporter gene assay (luciferase assay)
The medium was removed from the culture plate 48 hours after the addition of 5-aza-dC. Each cell was washed twice with PBS and then luciferase (reporter protein) was extracted from each cell. Specifically, the extraction was performed as follows. A cell lysate (PicaGene Cell lysis buffer LCβ, TOYO B-Net), which is an extraction reagent, was added, and the suspension of cells and the cell lysate was incubated for 15 minutes at room temperature. The suspension was then centrifuged at 15,000 rpm for 5 minutes with a centrifuge. Thereby, cell residues were removed from the suspension. As a result, a supernatant was obtained. A luciferase substrate solution (PicaGene LT2.0, TOYO B-Net) was added to this supernatant. Next, a luciferase substrate solution (luciferin solution) as a detection reagent was added. The generated emission intensity was measured with a luminometer (Mithras LB940, Berthold).

(4) Results The results are shown in FIGS. 20 (a) and 20 (b). These graphs represent changes in reporter protein signal intensity from vector-introduced cells due to demethylation.

FIG. 20 (a) shows the measured data for cells into which the PGV-B2-GFAPp vector has been introduced. A PGV-B2-GFAPp vector containing an unmethylated target nucleic acid sequence was introduced into the left end of FIG. 20 (a), and the signal intensity obtained from cells that had not undergone demethylation treatment was described as 100%. In the center, a PGV-B2-GFAPp vector containing a methylation target nucleic acid sequence is introduced, and the relative signal intensity obtained from cells that have not undergone demethylation treatment is shown. This relative signal intensity was 3.4%. At the right end, a PGV-B2-GFAPp vector containing a methylated modified base was introduced, and the relative signal intensity obtained from cells subjected to demethylation treatment is shown. This relative signal intensity was 2.4%.

FIG. 20B shows measured data for cells into which the pM53-R550K-GFAPp vector has been introduced. The pM53-R550K-GFAPp vector containing an unmethylated modified base was introduced into the left end of FIG. 20 (b), and the signal intensity obtained from cells that had not undergone demethylation treatment was described as 100%. In the center, a pM53-R550K-GFAPp vector containing a methylated modified base was introduced, and the relative signal intensity obtained from cells that had not undergone demethylation treatment is shown. This relative signal intensity was 34.1%. At the right end, a pM53-R550K-GFAPp vector containing a methylated modified base was introduced, and the relative signal intensity obtained from cells subjected to demethylation treatment is shown. This relative signal intensity was 74.6%.

As described above, the PGV-B2-GFAPp vector, which is a non-replicating plasmid vector, could not detect the demethylation reaction that occurred in the cell for the target nucleic acid sequence. On the other hand, by using the pM53-R550K-GFAPp vector, which is a self-replicating plasmid vector, the demethylation reaction that occurred in the cell with respect to the target nucleic acid sequence could be detected. The homology between the GFAP gene promoter sequence, which is the target nucleic acid sequence, and the corresponding sequence in the glioma C6 cell, which is the test cell, was 90.26%.

From these results, it was clarified that the following can be achieved by using the pM53-R550K-GFAPp vector, which is a plasmid-type self-replicating vector which is an example of the embodiment. That is, it was clarified that its use makes it possible to detect demethylation in the modified base using the signal intensity of the reporter protein as an index. That is, these results indicate that the degree of promoter activation varies depending on the state of modification of the modified base contained in the pM53-R550K-GFAPp vector. This difference in the degree of activation could be detected using the signal intensity of the reporter protein as an index. Thus, according to the embodiment, it is possible to obtain epigenetic information about a specific sequence by utilizing a self-replicating vector having a target nucleic acid sequence homologous to the specific sequence of the cell. In addition, when the pM53-R550K-GFAPp vector, which contains a sequence homologous to a specific sequence of the test cell and is a plasmid-type self-replication vector, self-replicates in the test cell, the following occurs. That is, the modified state of the modified base in the specific sequence of the test cell is reflected in the modified state of the modified base in the target nucleic acid sequence on the pM53-R550K-GFAPp vector.

From these results, it was clarified that by using the self-replicating vector, it is possible to obtain epigenetic information on a specific sequence of the test cell using the signal intensity of the reporter protein as an index.

Conventionally, in order to obtain epigenetic information on the genome by a foreign nucleic acid, it is essential that the foreign nucleic acid is replicated at the same time as the genome is replicated and undergoes an epigenetic modification reaction. Therefore, it is difficult to determine the characteristics of cells using a reporter vector that is not integrated into the genome. However, according to the embodiment, it is possible to obtain epigenetic information such as the modification state of the nucleic acid of interest without incorporating a foreign nucleic acid into the genome.

Example 6 Comparison of methylation rate of CK19 gene promoter region (target nucleic acid sequence) in genomic DNA and reporter vector A self-replication vector and a replication initiation protein gene expression vector (vector having a replication initiation protein unit) were constructed. The promoter region of the CK19 gene (-617 to +61 bp, SEQ ID NO: 9) was incorporated into the reporter vector as the target nucleic acid sequence, and the methylation rate of the target nucleic acid sequence of the reporter vector was compared with the genomic DNA of the test cell into which the vector was introduced. ..

(1) Construction of reporter vector The promoter region (-617 to +61, SEQ ID NO: 9) of the CK19 gene, which is the target nucleic acid sequence, was amplified by PCR using human genomic DNA as a template. PCR was performed using PrimeSTAR GLX DNA polymerase (TaKaRa BIO). The base sequence of the primers used for PCR is shown below.

Forward primer: 5'-GCCTGGTCAACATGGTGAAAC-3'(SEQ ID NO: 19)
Reverse primer: 5'-TGGGCTAGCCCAAGAAGCTGGTTCTGAGAGG-3' (SEQ ID NO: 20).

The target nucleic acid sequence obtained by PCR was incorporated upstream of the luciferase gene of the reporter vector having the replication initiation sequence of Simian virus 40 to prepare the reporter vector p19sLo shown in FIG.

(2) Construction of replication initiation protein gene expression vector The replication initiation protein gene was a large T antigen gene (SV40LT) of Simian virus 40. The gene was amplified by PCR using PrimeSTAR GLX DNA polymerase (TaKaRa BIO) and then integrated downstream of the cytomegalovirus early promoter of the gene expression vector. As a result, the replication initiation protein (SV40LT) gene expression vector pCMV-LT shown in FIG. 22 was prepared. The base sequence of the primers used for PCR is shown below.

Forward primer: 5'-GGGGTACCAGATGGATAAAGTTTTAAACAGAGAGGAA-3' (SEQ ID NO: 21)
Reverse primer: 5'-GGGAATTCTTATGTTTCAGGTTCAGGTTCAGGGGGAG-3' (SEQ ID NO: 22).

(3) Transfection To 100 μL of Opti-MEM (Life Technologies), 0.5 μg of the reporter vector was added alone or together with 0.4 μg of the reporter vector and 0.1 μg of the replication initiation protein expression vector pCMV-LT. Then, 0.5 μL of enhancer reagent (Plus reagent, Life Technologies) was added. This was incubated at room temperature for 5 minutes. To this solution, 1.25 μL of Lipofectamine LTX was added and the mixture was well turbid. It was then incubated at room temperature for 25 minutes. The solution was transferred to a 24-well plate, 200 μL of Huh-7 cell suspension was added and mixed gently. Then, the cells were cultured in an incubator at 37 ° C. and a 5% CO ₂ atmosphere.

(4) Preparation of genomic DNA and reporter vector from cells 96 hours after transfection, the culture medium was removed from the 24-well plate, and the cells were washed with phosphate buffered saline (PBS). Then, 200 μL of Trypsin-EDTA was added, and the mixture was allowed to stand at room temperature for 5 minutes. 300 μL PBS was added and cells were collected in 1.5 mL tubes. The cells were then precipitated by centrifugation. The cells were suspended in 200 μL of PBS and a solution containing genomic DNA and a reporter vector was obtained using DNeasy Blood & Tissue Kit (Qiagen).

(5) Detection of genomic DNA methylation rate CCGG sequence methylation rate contained in the promoter region (-617 to +61 bp, SEQ ID NO: 9) of the CK19 gene of genomic DNA (presence or absence of 5-methylation of the second cytosine) Was investigated as follows. That is, it was investigated by a methylation detection method using a methylation-sensitive restriction enzyme HpaII and a methylation-insensitive restriction enzyme MspI. HpaII and MspI are restriction enzymes that recognize a common CCGG sequence. HpaII can not cleave CCGG sequence containing methylated cytosine ^(C m CGG), MspI cleaves CCGG sequence irrespective of the methylation cytosine. The solution containing the genomic DNA and reporter vector prepared in (4) above was cleaved with the restriction enzyme HpaII or MspI. Then, it was purified by QIAquick PCR purification kit (Qiagen). This was used as restriction enzyme-treated DNA. Using this DNA and the restriction enzyme-untreated DNA as templates, PCR was performed using the following primers. As a result, the region containing the CCGG sequence of the COX2 gene promoter region of genomic DNA was amplified (Fig. 24).

Forward primer 1'(G1) 5'-TCAGAGGGGACAAAAGGGGAGTTGG-3'(SEQ ID NO: 23)
Reverse primer 1 (C1); 5'-AGAGGCCCCTGCCCTCCAGAGGT-3' (SEQ ID NO: 24)
Forward Primer 2 (C2); 5'-GCAAATTCCTCAGGGCTCAGATA-3'(SEQ ID NO: 25)
Reverse primer 2'(G2);5'-CCAGGCCTCCGAAGGACGACGTGGC-3'(SEQ ID NO: 26)
The PCR reaction solution was electrophoresed on an agarose gel (containing ethidium bromide). Then, the PCR amplification product in the gel was visualized by UV irradiation and a photograph was taken. Then, each band intensity in the obtained photograph was quantified by the image analysis software ImageJ. Furthermore, the ratio of CCGG sequence methylation to the total DNA of A and B was calculated from the obtained numerical values and the following formula. Here, "A" and "B" shown in the figure showing the results are the regions in which "A" is amplified by the forward primer 1'(G1) and the reverse primer 1 (C1) as shown in FIG. Refers to A. Similarly, "B" refers to region B amplified by the forward primer 2 (C2) and the reverse primer 2'(G2).

These results are shown in FIGS. 23 (a) and 23 (b). Here, in each of the photographs obtained by the above shooting, the background is black and the band is white. The band intensity of this photograph was quantified by image analysis software as it was. However, if the image is shown as it is in the drawing, the band is difficult to see. Therefore, in order to make the band easier to see, an image obtained by importing the photograph obtained by the above shooting into a computer and using image processing software to invert the black and white color so as not to impair the band intensity and the relativity with other data. (Fig. 23 (a)). Hereinafter, all the photographs taken in the same manner were similarly inverted in black and white.

In FIG. 23a, lane N is a band of PCR amplification products of restriction enzyme-untreated DNA and represents the total amount of DNA subjected to PCR. Lane H is a band of PCR amplification products of HpaII-treated DNA. This represents the amount of DNA in which the second cytosine of CCGG is methylated among the DNAs subjected to PCR. Lane M is a PCR amplification product of MspI-treated DNA and represents the detection background. As shown in the results of FIG. 23a, a band of PCR amplification product was detected in lane H of CCGG sequences A and B, and the band intensity was higher in B than in A.

The results regarding the rate of CCGG sequence methylation obtained by the above calculation are shown in FIG. 23b. As shown in the results of FIG. 23b, the rates of CCGG sequence methylation of A and B were 2% for A and 87% for B, respectively.

Here, in the above formula, the methylation rate is calculated from the signal intensity of the band of the PCR amplification product, but the methylation rate is similarly calculated from the signal intensity of the amplification products obtained by other amplification methods. May be done. Further, the method for calculating the methylation rate is not limited to the above formula alone.

(6) Detection of Methylation Rate of Non-Replicating Reporter Vector The methylation rate of the non-replicating reporter vector was determined by the method shown in (3) above. Human liver cancer cell line Huh-7 was transfected with the reporter vector pC2sLo or p19sLo alone. The methylation rate was detected using the DNA solution extracted after 96 hours. For replication of the reporter vector, the replication initiation sequence and the replication initiation protein must coexist in the same cell. Therefore, when p19sLo is introduced into cells alone, p19sLo does not replicate inside the cells. CCGG sequence methylation rate (presence or absence of 5-methylation of second cytosine) contained in the promoter region (-617 to +61 bp, SEQ ID NO: 9) of the CK19 gene of the reporter vector p19sLo by the same method as in (5) above. I examined. The DNA prepared in (4) above was cleaved with the restriction enzyme HpaII or MspI. Then, it was purified by QIAquick PCR purification kit (Qiagen). This was used as restriction enzyme-treated DNA. Using these DNAs and DNAs untreated with restriction enzymes as templates, PCR was performed using the following primers. As a result, the region containing the CCGG sequence of the CK19 gene promoter region of p19sLo was amplified (Fig. 24). Here, "A" refers to the region A amplified by the forward primer 1'(P1) and the reverse primer 1 (C1) as shown in FIG. 24. Here, "B" refers to the region B amplified by the forward primer 2 (C2) and the reverse primer 2'(P2), as shown in FIG.

Forward primer 1'(P1) 5'-TAGGCTGTCCCCAGTGCAAGT-3' (SEQ ID NO: 27)
Reverse primer 1 (C1) 5'-AGAGGCCCCTGCCCTCCAGAGGT-3' (SEQ ID NO: 24)
Forward Primer 2 (C2) 5'-GCAAATTCCTCAGGGCTCAGATA-3' (SEQ ID NO: 25)
Reverse primer 2'(P2) 5'-AATGCCAAGCTTACTTAGATCG-3' (SEQ ID NO: 28).

The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 25 (a). From the results of FIG. 26a, no PCR amplification product was detected in lanes H of A and B. Therefore, the methylation rate of the CCGG sequence was 0% for both A and B (FIG. 25 (b)). In addition, methylation of A and B did not occur in the non-replicating reporter vector.

(7) Detection of methylation rate of replication reporter vector The methylation rate of the replication reporter vector was detected by co-introducing the reporter vectors p19sLo and pCMV-LT into Huh-7 and using the DNA extracted 96 hours later. When P19sLo and pCMV-LT are co-introduced into a cell, the replication initiation sequence and the replication initiation protein coexist in the cell, so that p19sLo replicates in the cell. PCR was performed using the same primers as in (6) above, using the restriction enzyme-treated DNA obtained by treating the DNA with HpaII or MspI and the restriction enzyme-untreated DNA as templates. Thereby, the region containing the CCGG sequence of the CK19 gene promoter region of p19sLo was amplified. The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 26 (a). As a result, PCR amplification products were detected in lanes A and B. The methylation rate of the CCGG sequence was 0% for A and 16% for B (FIG. 26 (b)). Furthermore, the methylation rate of the CK19 gene promoter region CCGG sequence of only the reporter vector replicated in the cell was investigated by combining the DpnI method (detection method of the vector replicated in the cell) and the methylation detection method by HpaII and MspI. (Fig. 27 (a)). As a result, PCR amplification products were detected in lanes A and B. The methylation rate of each CCGG sequence was 0% for A and 25% for B (FIG. 27 (b)). The CK19 gene promoter region of the reporter vector was methylated in approximately the same proportion as the CK19 gene promoter region (target nucleic acid sequence) of genomic DNA.

Example 7 Comparison of methylation rate of COX2 gene promoter region (target nucleic acid sequence) in genomic DNA and reporter vector A self-replication vector and a replication initiation protein gene expression vector (vector having a replication initiation protein unit) were constructed. The promoter region of the COX2 gene (-540 to +115 bp, SEQ ID NO: 10) was incorporated into the reporter vector as a target nucleic acid sequence. The methylation rates of the genomic DNA of the test cells into which this vector was introduced and the target nucleic acid sequence of the reporter vector were compared.

(1) Construction of reporter vector The promoter region (-540 to +115 bp, SEQ ID NO: 10) of the COX2 gene, which is the target nucleic acid sequence, was amplified by PCR using human genomic DNA as a template. PCR was performed using PrimeSTAR GLX DNA polymerase (TaKaRa BIO). The base sequence of the primers used for PCR is shown below. Also,
Forward primer; 5'-CTTAACCTTACTCGCCCCAGTCT-3'(SEQ ID NO: 29)
Reverse primer; 5'-AGGCTCGAGCGCGGGGGTAGGCTTTGCTGTCTGAG-3' (SEQ ID NO: 30).

The target nucleic acid sequence obtained by PCR was incorporated upstream of the luciferase gene of the reporter vector having the replication initiation sequence of Simian virus 40. As a result, the reporter vector pC2sLo shown in FIG. 28 was prepared.

(2) Replication initiation protein gene expression vector The same replication initiation protein (SV40LT) gene expression vector pCMV-LT as in (2) of Example 6 above was used.

(3) Methylation of reporter vector A reaction solution (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM DTT, 160 μM S-adenosylmethionine) consisting of a reporter vector and a reaction buffer was prepared. In response, DNA methyltransferase: SssI CpG Methyltransferase (New England Biolabs) was added and reacted at 37 ° C. overnight to methylate the cytosine of the CG sequence of the reporter vector.

(4) Transfection Transfection was performed in the same manner as in (3) of Example 6.

(5) Preparation of genomic DNA and reporter vector from cells The preparation of genomic DNA and reporter vector from cells was carried out in the same manner as in (4) of Example 6 above.

(6) Detection of genomic DNA methylation rate The CCGG sequence methylation rate (presence or absence of 5-methylation of the second cytosine) contained in the COX2 gene promoter (-540 to +115 bp, SEQ ID NO: 10) of genomic DNA is as follows. I checked it like this. That is, it was investigated by a methylation detection method using a methylation-sensitive restriction enzyme HpaII and a methylation-insensitive restriction enzyme MspI. The solution containing the genomic DNA and reporter vector prepared in (5) was cleaved with a restriction enzyme HpaII or MspI, and then purified with a QIAquick PCR purification kit (Qiagen), which was used as a restriction enzyme-treated DNA. This DNA and the restriction enzyme-untreated DNA were used as templates, and PCR was further performed using the following primers. As a result, region A and region B containing the CCGG sequence of the COX2 gene promoter region of genomic DNA were amplified (Fig. 29).

Forward Primer 1 (C1) 5'-GGAAGCCAAGTGTCCTTCTGC-3'(SEQ ID NO: 31)
Reverse primer 1 (C2) 5'-GGGCAGGGTTTTTTACCCAC-3'(SEQ ID NO: 32)
Forward Primer 2 (C3) 5'-AGCTTCCTGGGTTTCCGATTT-3'(SEQ ID NO: 33)
Reverse primer 2'(G2) 5'-GCCAGGTACTCACCTGTATGGCTG-3' (SEQ ID NO: 34).

Here, as shown in FIG. 29, A refers to the region amplified by the forward primer 1 (C1) and the reverse primer 1 (C2), and B refers to the forward primer 2 (C3) and the reverse primer 2'(. The region amplified by G2) is shown.

The PCR reaction solution was electrophoresed on an agarose gel (containing ethidium bromide). Then, the PCR amplification product in the gel was visualized by UV irradiation and a photograph was taken. The results are shown in FIG. 30 (a). From the results of FIG. 30 (a), a band of PCR amplification product was detected in lane H of CCGG sequences A and B. The band strength was higher in A than in B. The rates of CCGG sequence methylation of A and B calculated by the same method as in (5) of Example 6 were 29% and 7%, respectively (FIG. 30 (b)).

(7) Detection of methylation rate of non-replicating unmethylated reporter vector The methylation rate of the non-replicating reporter vector is determined by the method shown in (3) above, with or alone from the reporter vector pC2sLo. Huh-7 was transfected. It was detected using the extracted DNA solution 96 hours later. The CCGG sequence methylation rate contained in the promoter region (-540 to +115 bp, SEQ ID NO: 10) of the COX2 gene of the reporter vector pC2sLo was examined by the same method as in (5) above. The DNA prepared in (5) above was cleaved with the restriction enzyme HpaII or MspI. Then, it was purified by QIAquick PCR purification kit (Qiagen). This was used as restriction enzyme-treated DNA. Using these DNAs and DNAs not treated with restriction enzymes as templates, PCR was performed using the following primers. The region containing the CCGG sequence of the COX2 gene promoter region of pC2sLo was amplified (Fig. 29). Here, "A" and "B" shown in the figure showing the results, as shown in FIG. 29, "A" is a region A amplified by the forward primer 1 (C1) and the reverse primer 1 (C2). Point to. Similarly, "B" refers to region B amplified by forward primer 2 (C3) and reverse primer 2'(P2).

Forward Primer 1 (C1) 5'-GGAAGCCAAGTGTCCTTCTGC-3'(SEQ ID NO: 31)
Reverse primer 1 (C2) 5'-GGGCAGGGTTTTTTACCCAC-3'(SEQ ID NO: 32)
Forward Primer 2 (C3) 5'-AGCTTCCTGGGTTTCCGATTT-3'(SEQ ID NO: 33)
Reverse primer 2'(P2), 5'-AATGCCAAGCTTACTTAGATCG-3' (SEQ ID NO: 35).

The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 31 (a). From the results of FIG. 31 (a), no PCR amplification product was detected in lanes H of A and B. The rate of methylation of the CCGG sequence was 0% for both A and B, and methylation of A and B did not occur in the non-replicating reporter vector (Fig. 31 (b)).

(8) Detection of methylation rate of replication unmethylated reporter vector For the methylation rate of the replication reporter vector, the reporter vector pC2sLo and the replication initiation protein gene expression vector pCMV-LT were co-introduced into Huh-7. It was detected using the extracted DNA solution 96 hours later. Using the restriction enzyme-treated DNA obtained by treating the DNA with HpaII or MspI and the restriction enzyme-untreated DNA as templates, PCR was performed with the same primers as in (7) above. Thereby, the region containing the CCGG sequence of the COX2 gene promoter region of pC2sLo was amplified. The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 32 (a). As a result, PCR amplification products were detected in lanes A and B. The rate of CCGG sequence methylation was 14% for A and 24% for B (FIG. 32 (b)). Furthermore, by combining the DpnI method (detection method of intracellularly replicated vector) and the methylation detection method by HpaII and MspI, the methylation rate of the COX2 gene promoter region CCGG sequence of only the reporter vector replicated intracellularly can be determined. It was investigated (FIG. 33 (a)). As a result, PCR amplification products were detected in lanes A and B. The methylation rate of each CCGG sequence was 57% for A and 23% for B (FIG. 33 (b)). The COX2 gene promoter region of the replicated reporter vector was methylated in approximately the same proportion as the COX2 gene promoter region (target nucleic acid sequence) of genomic DNA.

(9) Detection of Methylation Rate of Non-Replicating Methylated Reporter Vector To detect the methylation rate of non-replicating methylated reporter vector, the methylated reporter vector pC2sLo was alone transfected into the human hepatoma cell line Huh-7. did. After 96 hours, the DNA solution extracted by the method (5) above was used. DNA was cleaved with the restriction enzymes HpaII or MspI. Then, it was purified by QIAquick PCR purification kit (Qiagen) to be used as restriction enzyme-treated DNA. Using these DNAs and DNAs not treated with restriction enzymes as templates, PCR was performed using the following primers. Thereby, the region containing the CCGG sequence of the COX2 gene promoter region of pC2sLo was amplified. The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 34 (a). From the results of FIG. 34 (a), PCR amplification products were detected in lanes H of A and B. The methylation rate of the CCGG sequence was 100% for A and 86% for B (FIG. 34 (b)). The methylation rate of the non-replicating methylation reporter vector did not match the methylation rate of the genomic DNA of the host cell.

(10) Detection of Methylation Rate of Replication Methylation Reporter Vector To detect the methylation rate of the replication methylation reporter vector, the reporter vector pC2sLo and the replication initiation protein gene expression vector pCMV-LT were co-introduced into Huh-7. , The DNA solution extracted after 96 hours was used. A restriction enzyme-treated DNA treated with HpaII or MspI and a restriction enzyme-untreated DNA were used as templates. PCR was performed with the same primers as in (6) above. Thereby, the region containing the CCGG sequence of the COX2 gene promoter region of pC2sLo was amplified. The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 35 (a). As a result, PCR amplification products were detected in lanes A and B. The rate of CCGG sequence methylation was 45% for A and 79% for B (FIG. 35 (b)). Furthermore, by combining the DpnI method (detection method of intracellularly replicated vector) and the methylation detection method by HpaII and MspI, the methylation rate of the COX2 gene promoter region CCGG sequence of only the reporter vector replicated intracellularly can be determined. It was investigated (FIG. 36 (a)). As a result, PCR amplification products were detected in lanes A and B. The methylation rate of each CCGG sequence was 95% for A and 35% for B (FIG. 36 (b)). Comparing the reporter vector replicated in the cell with the methylation rate of the CCGG sequence in the COX2 gene promoter region of the genomic DNA, B was strongly demethylated and the methylation rate was high in A.

Example 8 Detection of demethylation of target nucleic acid sequence (COX2 gene promoter region) by luciferase assay The methylated / unmethylated reporter vector pC2sLo alone or the replication initiation protein expression vector pCMV-LT is used in the same manner as in Example 7 above. Was transfected into the human liver cancer cell line Huh-7. After culturing for 72 hours, the reporter activity derived from pC2sLo was measured. The culture was removed from the 24-well plate and washed with PBS. Then, 150 μL of cell lysate (Promega) was added, and the cells were allowed to stand at −80 ° C. for 30 minutes or more to freeze. After thawing the cell lysate at room temperature, the lysate was collected in a centrifuge tube. The cell residue was precipitated by this centrifugation. 10 μL of the supernatant was dispensed into a 96-well plate (Nunc). To this, 50 μL of Luciferase substrate solution (One-Glo Luciferase Assay System, Promega) was added, and the luminescence intensity of the cell lysate was measured. The results are shown in FIG. The vertical axis of the graph of FIG. 37 shows the recovery rate of the emission intensity. The recovery rate of this luminescence intensity is an index of demethylation. The recovery rate of the luminescence intensity is the ratio of the luminescence intensity obtained from the cells into which the methylated reporter DNA is introduced, when the luminescence intensity obtained from the cells into which the unmethylated reporter vector is introduced is 100%. The left side of the graph shows the recovery rate of the luminescence intensity of the non-replicating reporter vector, and the right side shows the recovery rate of the replication reporter vector. From this graph, it was found that the recovery rate of luminescence intensity was higher in the replication reporter vector than in the non-replication reporter vector. This made it possible to detect demethylation of the target nucleic acid sequence with high sensitivity in the replication reporter vector. From this result, it was clarified that by using a reporter vector that replicates in cells, it is possible to obtain epigenetic information on a specific sequence of a test cell using the reporter activity as an index.

Example 9 A self-replicating vector having two types of reporter gene expression units (a unit containing a target nucleic acid sequence in which a functional group has been substituted and a unit containing a target nucleic acid sequence in which a functional group has not been substituted) and a replication initiation sequence (1). ) Preparation of vector A self-replicating vector was constructed. The reporter vector pNL1.1 (Promega) incorporating shrimp-derived luciferase was cleaved with the restriction enzymes KpnI and BamHI. The DNA ends were blunted with T4 DNA polymerase. Then, a DNA fragment consisting of eviluciferase and SV40 transcription termination sequence was purified. This fragment was ligated with p19sLo cleaved with the restriction enzyme SspI with T4 DNA ligase to construct a self-replicating vector p19sLo-NL. The COX2 gene promoter (SEQ ID NO: 10, Table 10) in which CpG cytosine was methylated was incorporated into this p19sLo-NL.

The COX2 gene promoter was amplified by PCR using the human genome as a template. The primer sequences used for PCR are shown below.

Forward primer; 5'-GGGGCTAGCCTTAACCTTACTCGCCCCAGTCT-3'(SEQ ID NO: 36)
Reverse primer; 5'-AGGCTCGAGCGCGGGGGTAGGCTTTGCTGTCTGAG-3' (SEQ ID NO: 30).

The PCR amplification product (COX2 gene promoter) was purified. Then, a reaction solution (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM DTT, 160 μM S-adenosylmethionine) consisting of the amplification product and the reaction buffer was prepared. DNA methyltransferase: SssI CpG Methyltransferase (New England Biolabs) was added thereto, and the mixture was reacted overnight at 37 ° C. As a result, cytosine in the CG sequence of the COX2 gene promoter was methylated. This methylated DNA fragment (methylated COX2 gene promoter) was ligated with a T4 DNA ligase to the self-replicating vector p19sLo-NL treated with restriction enzymes KpnI and XhoI. As a result, self-replication including two types of reporter gene expression units (including a reporter gene expression unit containing a methylated COX2 gene promoter and a reporter gene expression unit containing a non-methylated CK19 gene promoter) and a replication initiation sequence. The vector p19sLo-mC2sNL was constructed (Fig. 38).

(2) Transfection To 100 μL of Opti-MEM (Life Technologies), 0.5 μg of the reporter vector alone or 0.4 μg of p19sLo-mC2sNL and 0.1 μg of the replication initiation protein expression vector pCMV-LT were added together. .. Then, 0.5 μL of enhancer reagent (Plus reagent, Life Technologies) was added, and the mixture was incubated at room temperature for 5 minutes. To this solution, 1.25 μL of Lipofectamine LTX was added and the mixture was well turbid. It was then incubated at room temperature for 25 minutes. The solution was transferred to a 24-well plate. 200 μL of a suspension of Huh-7 cells was added thereto, and the mixture was gently mixed. This introduced the vector into the cells (Fig. 39).

Example 10 Self-replication vector having a reporter gene expression unit, a replication initiation gene expression unit, and a replication initiation sequence (1) Preparation of a vector A self-replication vector was constructed. The structure of this reporter vector is shown in FIG. The reporter vector 91 contains a reporter gene expression unit 2, a replication initiation protein unit 92, and a replication initiation sequence 3 in one nucleic acid chain. The reporter gene expression unit 2 includes a transcription control sequence 4, a reporter gene 5 encoding a reporter protein functionally linked downstream thereof, and a transcription termination signal sequence 6 functionally linked downstream thereof. The replication initiation protein unit 92 includes a constitutively expressed promoter 94, a replication initiation protein gene 95 functionally linked downstream thereof, and a transcription termination signal sequence 96 functionally linked downstream thereof. The replication initiation sequence 3 is linked downstream of the transcription termination signal sequence 96 of the replication initiation protein unit 92. This reporter vector was prepared as follows. The replication initiation protein gene expression unit was cleaved from pCMV-LT (Example 6 (2)) with restriction enzymes BglI and BamHI. The DNA ends were blunted with T4 DNA polymerase. Then, the DNA fragment of the replication initiation protein gene expression unit was purified. This DNA fragment was ligated to pC2sLo (Example 7 (1)) cleaved with the restriction enzyme SspI to construct a self-replicating vector pC2sLo-CMVLT.

(2) Transfection To 100 μL of Opti-MEM (Life Technologies), 0.5 μg of pC2sLo-CMVLT was added. Then, 0.5 μL of enhancer reagent (Plus reagent, Life Technologies) was added, and the mixture was incubated at room temperature for 5 minutes. To this solution, 1.25 μL of Lipofectamine LTX was added and the mixture was well turbid. It was then incubated at room temperature for 25 minutes. The solution was transferred to a 24-well plate. 200 μL of a suspension of Huh-7 cells was added thereto, and the mixture was gently mixed. Then, the cells were cultured in an incubator at 37 ° C. and a 5% CO ₂ atmosphere.

(3) Preparation of genomic DNA and reporter vector from cells The genomic DNA and reporter vector were prepared in the same manner as in (1) of Example 10 above.

(4) Detection of methylation rate of reporter vector For the methylation rate of the reporter vector, the self-replicating vector pC2sLo-CMVLT was introduced into Huh-7, and the DNA solution extracted 72 hours later was used to determine the methylation rate of Example 7 (7) above. ) Was investigated in the same way. The solution prepared in (3) above was cleaved with the restriction enzyme HpaII or MspI. It was then purified by the QIAquick PCR purification kit (Qiagen). Using these DNAs and DNAs not treated with restriction enzymes as templates, PCR was performed with the same primers as in 6 (7) above. The region containing the CCGG sequence of the COX2 gene promoter region (-540 to +115 bp, SEQ ID NO: 10) of pC2sL-CMVLT was amplified (FIG. 29). The results of agarose gel (containing ethidium bromide) electrophoresis of the PCR reaction solution are shown in FIG. 41 (a). From the results of FIG. 41 (a), PCR amplification products were detected in lanes H of A and B. The methylation rate of the CCGG sequence was 9% for A and 0.5% for B (FIG. 41 (b)). In the COX2 gene promoter region of the self-replicating vector pC2sLo-CMVLT, the methylation rate of A was high, as in the COX2 gene promoter region of genomic DNA (Example 7 (6), FIG. 30).

According to these embodiments or examples, it is possible to provide a method and an apparatus for determining cell characteristics with higher accuracy.

Although some embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other embodiments, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are also included in the scope of the invention described in the claims and the equivalent scope thereof.
Hereinafter, the inventions described in the claims of the original application of the present application will be added.
[1]
(1) Introducing a self-replicating reporter vector into a test cell that is activated under specific conditions to generate a detectable signal.
(2) Culturing the test cells and self-replicating the reporter vector.
(3) To detect the detectable signal generated from the test cell for each cell,
(4) Observing the morphology of the test cell from which the signal was obtained, and
(5) To determine the characteristics of the test cell based on the result obtained in (3) above and the result obtained in (4) above.
A method for determining cell characteristics including.
[2]
(1) Introducing a reporter vector into test cells:
Here, the reporter vector is
(A) (A-a) Transcription control sequences exhibiting promoter activity under specific conditions,
(A-b) A reporter gene encoding a reporter protein that is functionally linked downstream of the transcriptional control sequence, expressed by activation of the transcriptional control sequence, and produces a detectable signal.
(Ac) A transcription termination signal sequence functionally linked downstream of the reporter gene,
Reporter gene expression unit, including
(B) A replication initiation unit that is located on the same nucleic acid as the reporter gene expression unit and contains a replication initiation sequence that initiates self-replication of the reporter vector by binding to the replication initiation protein.
Including:
(2) Culturing the test cells under the condition that the replication initiation protein is expressed:
(3) To detect the reporter protein expressed in the test cell for each cell:
(4) Observing the morphology of the cell in which the reporter protein was detected in (3) above:
(5) To determine the characteristics of the test cells based on the results obtained in (3) and the results observed in (4):
A method for determining cell characteristics including.
[3]
The reporter vector further
(C) The replication initiation protein unit
The replication-initiating protein unit contains
(CA) An IRES sequence functionally linked downstream of the reporter gene,
(C-b) With the replication initiation protein gene encoding the replication initiation protein functionally linked downstream of the IRES sequence.
Including
The method according to [2], wherein the transcription termination signal sequence is functionally linked downstream of the replication initiation protein gene.
[4]
The reporter vector further
(D) A replication initiation protein unit on the same nucleic acid strand as the nucleic acid strand in which the reporter gene expression unit is arranged.
The replication-initiating protein unit comprises
(Da) Constitutively expressed constitutive promoters:
(Db) With the replication initiation protein gene encoding the replication initiation protein operably linked downstream of the constitutive promoter:
The method according to [2], which comprises.
[5]
The reporter vector copies the state of modification of a particular sequence on the genome of the test cell onto the corresponding sequence by substitution of a functional group during self-renewal of the test cell in the nucleus. To any one of [1] to [4], which is characterized by producing a detectable signal by promoter activity activated under specific conditions, depending on the presence of the copied functional group. The method described.
[6]
The transcriptional control sequence of the reporter vector contains a target nucleic acid sequence having identity to a specific sequence on the genome of the test cell, which contains a base in which the functional group can be substituted, and the substitution of the functional group. The method according to any one of [2] to [4], which has a promoter activity that is activated depending on the degree.
[7]
The method according to [5] or [6], wherein the functional group is a methyl group or an acetyl group.
[8]
Observing the morphology of the cells is the area of the test cell, the ratio of the area of the nucleus to the area of the test cell, the ratio of the minor axis to the major axis of the test cell, the number of nuclei in the test cell. The method according to any one of [1] to [7], which comprises obtaining the contrast ratio of the marginal portion of the test cell.
[9]
1) The replication initiation protein gene is a large T antigen gene of Simian virus 40, and the replication initiation sequence is a replication initiation sequence from Simian virus 40:
2) The replication initiation protein gene is the EBNA-1 gene of Epstein-Barr virus, and the replication initiation sequence is the replication initiation sequence from Epstein-Barr virus: or
3) The replication initiation protein gene is a large T antigen gene of mouse polyoma virus, and the replication initiation sequence is a replication initiation sequence from mouse polyoma virus:
The method according to any one of [2] to [7].
[10]
The large T antigen gene is deleted or substituted with the nucleic acid represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or one or several bases in the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Alternatively, the method according to [9], wherein the nucleic acid is indicated by an added sequence and has a DNA replication initiation function.
[11]
Any of [4] to [10], wherein the constitutive promoter is selected from the group consisting of a cytomegalovirus promoter, a Raus sarcoma virus promoter, a Simian virus early promoter, and a Simian virus late promoter. The method described in one.
[12]
The method according to any one of [1] to [11], wherein the detection of the detectable signal and the observation of the morphology of the cells are performed by a non-invasive optical imaging method.
[13]
The method according to [12], wherein observing the morphology of the cells is performed by phase contrast microscopy, differential buffer microscopy, or hyperspectral imaging.
[14]
The method according to [12], wherein the detection of the detectable signal is performed using the emission intensity as an index, and observation of the morphology of the cells is performed by imaging in a bright field.
[15]
Further, determining the characteristics of the test cell, including staining the nucleus of the test cell and observing the stained nucleus, is based on the results obtained by observing the nucleus. The method according to any one of [1] to [14].
[16]
The detection of the detectable signal is performed by imaging the light emitting field, the observation of the morphology of the cells is performed by imaging in the bright field, and the observation of the nucleus is performed by imaging the fluorescence field. The method according to [15].
[17]
By observing the morphology of the cells, the test cells are irradiated with light having a wavelength in the near infrared region, and information on the characteristics of the peripheral portion of the test cells can be obtained by using the reflection or absorption of the light as an index. The method according to any one of [1] to [16], which is characterized by the above.
[18]
The method according to [17], wherein the wavelength in the near infrared region is a wavelength of 700 nm to 750 nm.
[19]
The specific condition for activating the promoter is any one of [2] to [18], which is a condition suggesting that the test cell is a cancer cell or a precancerous cell. The method described.
[20]
A method for testing a disease, which comprises the method according to any one of [1] to [19], wherein the specific condition is a condition related to the disease.
[21]
The test method according to [20], wherein the disease is cancer or precancerous.
[22]
(1) The state of modification in a specific sequence on the genome of the test cell is copied onto the corresponding sequence by substitution of a functional group during self-renewal in the nucleus of the test cell, and the copy is copied. A gene transfer site that introduces a reporter vector that emits light by promoter activity activated under specific conditions into the nucleus of a test cell, depending on the presence of functional groups.
(2) A culture unit for self-replicating the reporter vector by culturing the test cells treated by the gene transfer unit and dividing the test cells.
(3) A bright-field image is taken of the test cells cultured in the culture section, and a luminescence image including a luminescence signal generated for each cell due to the expression of the reporter gene is imaged in the same field of view. Imaging unit and
(4) A determination unit for determining the cell characteristics of each cell of the test cell based on the bright field image and the luminescent image.
A cell characteristic determination device comprising.

Claims (17)

(1) Introducing a self-replicating reporter vector containing the promoter sequence of the NAMPT gene into the test cells to generate a detectable signal.
(2) Culturing the test cells and self-replicating the reporter vector.
(3) Detecting the detectable signal generated from the living test cell for each cell, and
(4) In the same visual field as the visual field where the signal was obtained in (3), the morphology of the living test cells was observed under bright visual field conditions having a wavelength of 700 nm to 750 nm.
(5) The test cell based on both the signal detected in (3) above (first result) and the morphology of the test cell observed in (4) (second result). To determine the characteristics of
Including
The second result is the magnitude of the aspect ratio of the test cell when compared with the control cell.
In (5), it is determined from the first result and the second result that the aspect ratio is larger that the test cell is or is likely to be a highly infiltrating breast cancer cell. Or, from the first result and the second result that the aspect ratio is smaller, the living including determining that the test cell is or is likely to be a low infiltration breast cancer cell. A method that assists in determining the characteristics of cells. (1) Introducing a reporter vector into test cells:
Here, the reporter vector is
(A) (A-a) The promoter sequence of the NAMET gene and
(A-b) A reporter gene encoding a reporter protein that is functionally linked downstream of the transcriptional control sequence, expressed by activation of the transcriptional control sequence, and produces a detectable signal.
(Ac) A transcription termination signal sequence functionally linked downstream of the reporter gene,
A reporter gene expression unit containing the above, and (B) a replication initiation unit containing a replication initiation sequence that is located on the same nucleic acid as the reporter gene expression unit and initiates self-renewal of the reporter vector by binding to the replication initiation protein.
Including:
(2) Culturing the test cells under the condition that the replication initiation protein is expressed:
(3) To detect the signal from the reporter protein expressed in the living test cell for each cell:
(4) 700 nm to 750 nm in the same field of view as the field of view in which the signal was obtained in (3) above.
Observing the morphology of the test cells that lived under bright field conditions of the wavelength of:
(5) The test cell based on both the signal detected in (3) above (first result) and the morphology of the test cell observed in (4) (second result). To determine the characteristics of:
Including
The second result is the magnitude of the aspect ratio of the test cell when compared with the control cell.
In (5), it is determined from the first result and the second result that the aspect ratio is larger that the test cell is or is likely to be a highly infiltrating breast cancer cell. Or, from the first result and the second result that the aspect ratio is smaller, the living including determining that the test cell is or is likely to be a low infiltration breast cancer cell. A method for determining the characteristics of cells. The reporter vector further
(C) The replication initiation protein unit is contained, and the replication initiation protein unit is
(CA) An IRES sequence functionally linked downstream of the reporter gene,
(C-b) Containing a replication initiation protein gene encoding a replication initiation protein operably linked downstream of the IRES sequence.
The method according to claim 2 , wherein the transcription termination signal sequence is functionally linked downstream of the replication initiation protein gene. The reporter vector further
(D) The replication initiation protein unit is contained on the same nucleic acid chain as the nucleic acid chain in which the reporter gene expression unit is arranged, and the replication initiation protein unit is
(Da) With a constitutive promoter that is constitutively expressed:
(Db) With the replication initiation protein gene encoding the replication initiation protein operably linked downstream of the constitutive promoter:
2. The method according to claim 2 , wherein the method comprises. The reporter vector copies the state of modification of a particular sequence on the genome of the test cell onto the corresponding sequence by substitution of a functional group during self-renewal of the test cell in the nucleus. The invention according to any one of claims 1 to 4 , wherein a detectable signal is generated by the promoter activity activated under specific conditions, depending on the presence of the copied functional group. Method. The transcription control sequence of the reporter vector contains a target nucleic acid sequence having identity to a specific sequence on the genome of the test cell, which contains a base in which the functional group can be substituted, and the substitution of the functional group. The method according to any one of claims 2 to 4 , characterized in having a promoter activity that is activated depending on the degree. The method according to claim 5 or 6 , wherein the functional group is a methyl group or an acetyl group. 1) The replication initiation protein gene is a large T antigen gene of Simian virus 40, and the replication initiation sequence is a replication initiation sequence from Simian virus 40:
2) The replication initiation protein gene is the EBNA-1 gene of Epstein-Barr virus, and the replication initiation sequence is the replication initiation sequence from Epstein-Barr virus: or 3) The replication initiation protein gene is , A large T-Barr gene of the mouse polyoma virus, wherein the replication initiation sequence is the replication initiation sequence from the mouse polyoma virus:
The method according to any one of claims 2 to 7 , wherein the method is characterized by the above. The large T antigen gene is deleted or replaced with the nucleic acid represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or one or several bases in the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Alternatively, the method according to claim 8 , wherein the nucleic acid is indicated by an added sequence and has a DNA replication initiation function. Any one of claims 4 to 9 , wherein the constitutive promoter is selected from the group consisting of a cytomegalovirus promoter, a Raus sarcoma virus promoter, a Simian virus early promoter and a Simian virus late promoter. The method described in. The method according to any one of claims 1 to 10 , wherein the detection of the detectable signal and the observation of the morphology of the cells are performed by a non-invasive optical imaging method. The method according to claim 11 , wherein observing the morphology of the test cells is performed by phase contrast microscopy, differential buffer microscopy, or hyperspectral imaging. The method according to claim 11 , wherein the detection of the detectable signal is performed using the emission intensity as an index, and observation of the morphology of the test cells is performed by imaging in a bright field. Further, the determination of the characteristics of the test cell, which includes staining the nucleus of the test cell and observing the stained nucleus, is based on the results obtained by observing the nucleus. The method according to any one of claims 1 to 13 , wherein the method is performed. The detection of the detectable signal is performed by imaging the light emitting field, the observation of the morphology of the test cells is performed by imaging in the bright field, and the observation of the nucleus is performed by imaging the fluorescence field. The method according to claim 14 . The information observing the form of the test cell, the irradiating light of a wavelength in the near infrared region to the test cell, on the characteristics of the peripheral portion of the test cell of the reflection or absorption as an index of the optical The method according to any one of claims 1 to 15 , wherein the method is obtained. (1) The state of modification in a specific sequence on the genome of the test cell is copied onto the corresponding sequence by substitution of a functional group during self-renewal in the nucleus of the test cell, and the copy is copied. A gene transfer site that introduces a reporter vector that emits light by the promoter activity of the NAMET gene into the nucleus of the test cell, depending on the presence of the functional group.
(2) A culture unit for self-replicating the reporter vector by culturing the test cells treated by the gene transfer unit and dividing the test cells.
(3) A bright-field image was taken of the living test cells cultured in the culture section under bright-field conditions having a wavelength of 700 nm to 750 nm, and further, cells derived from the expression of the reporter gene in the same field of view. An imaging unit that captures a luminescent image including a luminescent signal generated for each
(4) The magnitude of the aspect ratio of the test cell when compared with the control cell (second result) obtained from the bright field image and the luminescence signal (first result) obtained from the luminescence image. Based on both of the above, from the first result and the second result that the aspect ratio is larger, it is determined that the test cell is a highly invasive breast cell or is likely to be. , Or, based on the first result and the second result that the aspect ratio is smaller, it is provided with a determination unit for determining that the test cell is or is likely to be a low-invasive breast cancer cell. Cell characteristic determination device.

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