Patent Application
20170204469
This application is based upon and claims the benefit of priority from Japanese Patent Application No. 2016-008125, filed Jan. 19, 2016, the entire contents of which are incorporated herein by reference.
Embodiments described herein relate generally to a reporter vector for evaluating characteristics of a subject cell, an assay kit, method and device therefore.
The first cause of death in our country is cancer. Cancer is a group of diseases with a common feature of formation of a malignant tumor, and various tumors as many as over two hundreds types have been identified up to now. The common features of the malignant tumors includes uncontrollable reproduction of cells, potential of invading neighboring tissues and metastaticity to any tissues in the whole body.
In the treatment of cancer, for example, a malignant tumor, it is important to select an appropriate therapy according to the character of the tumor (namely, the characteristics of the oncocyte). Therefore, it is necessary to evaluate the characteristics of an oncocyte, i.e., the benign or malignancy of the tumor. In the case of a malignant tumor, it is preferable to be able to evaluate the aggression thereof, that is, for example, invasive and/or metastatic potential.
For example, when a change in expression of a specific gene exhibits a high correlation with the characteristics of an oncocyte, the gene can be used as a tumor marker. By detecting the tumor marker, the characteristics of the tumor relevant to a genetic expression can be distinguished. For example, if a specific tumor marker is detected in blood, other body fluids or extracted oncocyte, existence of oncocyte with a high correlation to the marker is suggested. A well-known example of the tumor markers is the gene Ki-67 used for evaluating the proliferativity of oncocyte.
There is a method of measuring the promoter activity of a tumor marker with use of a reporter gene. According to this method, a vector in which a promoter of the tumor marker and a reporter gene are connected together is introduced to a subject cell, and the signal from the reporter gene is detected or measured, based on which the cell characteristics of the subject cell are evaluated. One of such promoter assays is the valuation method which locates a sequence for self-replication of a vector in a downstream of a promoter reporter gene. However, more excellent evaluation methods are in further demand.
In general, according to one embodiment, a reporter vector comprises a first reporter gene expression unit, a second reporter gene expression unit and a replication initiation sequence. The first reporter gene expression unit includes a promoter sequence of a cell characteristics marker, a first reporter gene existing in a downstream thereof, and a first transcription termination sequence existing in a further downstream thereof. The second reporter gene expression unit includes a promoter sequence exhibiting constitutive activity, a second reporter gene existing in a downstream thereof, a bicistronic expression sequence existing in a further downstream thereof, a replication initiation protein gene existing in a further downstream thereof, and a second transcription termination sequence existing in a further downstream thereof. The replication initiation sequence binds to a replication initiation protein synthesized from the replication initiation protein gene, thereby initiating replication of the reporter vector.
An embodiment will now be described with reference to accompanying drawings.
The embodiment provides a reporter vector for evaluating the characteristics of a subject cell, an assay kit, a method and a device therefore.
The reporter vector of this embodiment comprises, for example, at least two reporter genes, that is, a first reporter gene to be expressed by activation of a promoter sequence of a marker for cell characteristics, a second reporter gene to be expressed by activation of a promoter sequence exhibiting a constitutive activity, and a replication initiation unit for reproducing the vector itself within the subject cell into which the vector is introduced. The reporter vector may include a further reporter gene other than the first reporter gene or the second reporter gene. Thus, the reporter vector may be a self-replicating multi-reporter vector. A self-replicating multi-reporter vector may be, for example, a self-replicating dual reporter vector when including two reporter genes. Moreover, when three reporter genes are included, it may be a self-replicating triple reporter vector. However, the reporter vector is not limited to these.
By the evaluation of the characteristics of a subject cell, it is determined whether or not the conditions of the “status” or “characteristics” of the cell itself, for example, the inside or outside of the cell, or the “environment” surrounding the cell match the specific predetermined conditions. Such conditions may be judged based on whether or not there are indexes which indicate a sign of a specific disease, onset of a specific disease, the grade of progression of the onset of a specific disease and/or the severity of a specific disease. Such conditions may be those of a substance in the cell, whose content changes with the onset, existence or degree of progress of a disease, for example. Such conditions may be concrete indexes, for example, the existence of a specific gene, the expression of a specific gene, the pH value inside or outside the cell, information of the redox, the existence of a specific ion, the existence of an enzyme, the existence of an enzyme substrate, the existence of a specific substance, or the like, whether or not it exists, the degree of the quantitative value of the existence thereof, the distribution of the existence thereof, and/or the change in existential state, etc.
For example, the evaluation of the characteristics of a subject cell may be evaluating, in the case of evaluation of the characteristics of a cancer cell or oncocyte, the tumor characteristics of the subject cell, that is, for example, whether it is metastatic or invasive, or its degree, and whether the cell is normal or abnormal. When a subject cell is or may be abnormal, the evaluation of the characteristics of a subject cell may include evaluating the degree of the abnormality, or the degree of malignancy. The evaluation of the characteristics of an oncocyte may be evaluating, for example, whether or not the oncocyte is metastatic or invasive, the degree of the metastaticity of the oncocyte, the degree of malignancy or non-malignancy of the oncocyte, or the degree of the normality or abnormality of the oncocyte.
Note that the term “cancer cell”, “oncocyte”, “cancer” and “tumor” are used interchangeably in this specification.
1. Reporter Vector
The reporter vector according to the embodiment may be, for example, a self-replicating multi-reporter vector for evaluating the characteristics of a subject cell.
The first reporter gene expression unit 13 includes a promoter sequence of a cell characteristics marker 10, a first reporter gene 11 locating downstream, and a first transcription termination sequence 12 locating further downstream. The promoter sequence of the cell characteristics marker 10, the first reporter gene 11, and the first transcription termination sequence 12 are functionally linked. Thus, the expression of the first reporter gene 11 is indicated by activation of the promoter sequence of the cell characteristics marker 10. These sequences will be described in detail later.
The second reporter gene expression unit 25 includes a promoter sequence exhibiting constitutive activity 20, a second reporter gene 21 locating downstream, a bicistronic expression sequence 22 locating further downstream, a replication initiation protein gene 23 locating further downstream, and a second transcription termination sequence 24 locating further downstream. The promoter sequence exhibiting constitutive activity 20, the second reporter gene 21, the bicistronic expression sequence 22, the replication initiation protein gene 23 and the second transcription termination sequence 24 are linked functionally. Thus, the second reporter gene 21 and the replication initiation protein gene 23 are expressed by activation of the promoter sequence exhibiting constitutive activity 20. These sequences will be described in detail later.
The replication initiation unit includes a replication initiation sequence 30 and a replication initiation protein gene 23 included in the second reporter gene expression unit. The replication initiation sequence 30 links to a replication initiation protein synthesized from the replication initiation protein gene 23, by which the replication of the self-replicating dual reporter vector 1 is started. The replication initiation sequence 30 may exist in any region of the sequence of the self-replicating dual reporter vector except the region where the first reporter gene expression unit 13 and the second reporter gene expression unit 25 exist.
Here, the expression “link functionally” is meant such a state that each nucleotide sequence included in a reporter vector links to be able to exhibit its target function. For example, in the case of a sequence encoding a protein, the amino acid sequences encoded by the base sequences are linked each other correctly. In other words, there is no misalignment between the frames of the amino acid codons, and a peptide which exhibits the target activity is synthesized in the cell into which the base sequence is introduced. Moreover, the expression “link functionally” is used interchangeably with “link operatably”.
The first reporter gene expression unit 13, the second reporter gene expression unit 25 and the replication initiation sequence 30 are included on one reporter vector 1. The second reporter gene expression unit 25 may exist downstream the first reporter gene expression unit 13 and vice versa.
The transcription directions of the first reporter gene expression unit 13 and the second reporter gene expression unit 25 may be the same. But, the transcription directions of the first reporter gene expression unit 13 and the second reporter gene expression unit 25 may be in reverse to each other. An example of such a self-replicating dual reporter vector is shown in
The first reporter gene expression unit 13, the second reporter gene expression unit 25 and the replication initiation sequence 30 in the reporter vector may be arranged thereon clockwise in this order, that is, from 5′ to 3′, or may be in an opposite direction, but it is preferable to be arranged in the direction from 5′ to 3′. Or the order may be the first reporter gene expression unit 13, the replication initiation sequence 30, and the second reporter gene expression unit 25.
The first reporter gene expression unit and the second reporter gene expression unit in the reporter vector may be arranged thereon clockwise in this order, that is, from 5′ to 3′, or may be in an opposite direction, but it is preferable that the two genetic expression units be arranged in the direction from 5′ to 3′.
Although omitted from
The further reporter gene may be a well-known reporter gene including a reporter gene similar to the first reporter gene or the second reporter gene, which will be described later. It is preferable that the signal obtained from the reporter protein expressed from the reporter gene and the signal obtained from the reporter protein expressed from the first or second reporter gene be identifiably different from each other.
By disposing the transcription termination sequence upstream the first reporter gene expression unit 13 and the second reporter gene expression unit 25, leak of a genetic expression from the first reporter gene expression unit 13 or the second reporter gene expression unit 25, and unexpected leak of the genetic expression from an arbitrary sequence on the plasmid can be decreased. Thus, the background (noise) caused by the leakage can be decreased, and the sensitivity and precision of the signal detection can be improved. The transcription termination sequence will be described in detail later.
An example of the configuration elements of a reporter vector of the embodiment will be described.
1-1. First Reporter Gene Expression Unit
The promoter sequence of the cell characteristics marker 10 includes a promoter of the cell characteristics marker. The promoter of the cell characteristics marker may be a promoter of the cell characteristics marker originated from the subject cell. The cell characteristics marker is a gene which is expressed in the cell suffered from a specific disease, or is expressed more greatly or less in the cell suffered from a specific disease as compared with a normal cell. The cell characteristics marker to be expressed in a cell suffered from a specific disease may be, for example, a gene to be expressed only in the cell suffered from the specific disease. Each of these genes is expressed by activation of a promoter included therein. Thus, the activation of the promoter of a cell characteristics marker corresponds to the characteristics of the subject cell, that is, for example, the amount of expression of the cell characteristics marker according to the state of the cell itself or the environment surrounding the cell.
When the cell characteristics to be evaluated are tumor characteristics, The promoter sequence of the cell characteristics marker 10 may be a tumor marker. The tumor marker may be, for example, a tumor marker well-known by itself. The promoter of such a cell characteristics marker may include a gene promoter of, for example, NAMPT, Ki67, fos, myc or the like. The type of tumor to be detected may be, for example, epithelial tumor, non-epithelial tumor, hemopoietic tissue tumor, fetal tumor or any of combinations of these. The epithelial tumor may be, for example a mammary tumor or a lung tumor.
When the object to be detected is a cell relating to bone dybolism such as arthritis and osteoporosis, for example, a gene promoter of NFATC1 or CICC4, may be used. Or the object to be detected is a cell exposed to the oxidant stress, which may elicite various diseases, for example, a gene promoter of catalase or SOD may be used.
The promoter sequence of the cell characteristics marker 10 may be a polynucleotide including such a promoter as described above. The promoter sequence of the cell characteristics marker 10 may include a linker sequence in addition to the above-described promoters.
The promoter sequence of the cell characteristics marker 10 may be, for example, the sequence of the promoter of NAMPT gene represented by SEQ ID NO:1 shown in TABLE 1.
In a further embodiment, The promoter sequence of the cell characteristics marker 10 may include a plurality of types of promoters. For example, The promoter sequence of the cell characteristics marker 10 may include a promoter of another gene in addition to the sequence of the promoter of the NAMPT gene.
The first reporter gene 11 may be any gene which encodes a reporter protein producing a detectable signal and can express a reporter protein by activation of the promoter sequence of the cell characteristics marker 10.
The detectable signal may be fluorescence, photogenesis, coloration or the like, or may be presentation of a substance such as protein. Or it may only be a signal detectable by, for example, a well-known method itself. The term “photogenesis” used here refers to chemoluminescence, bioluminescence or biochemical photogenesis.
The detectable signal may be a signal emitted from a reporter protein itself. Or, the signal concerned may be emitted from a reporter protein or any substance resulting from the reaction of the reporter protein and substances, for example, an enzyme reaction. Or, the signal concerned may be a signal produced from a reporter protein or any substance resulting from the binding of the reporter protein and the substances.
The reporter protein may be, for example, blue fluorescent protein, green fluorescent protein, red fluorescent protein, luciferase, β-galactosidase, nitric-monooxide synthetic enzyme, xanthine oxidase, or a heavy metal-binding protein.
A reporter gene which produces a signal from the reporter protein itself may be, for example, a gene which encodes a fluorescent protein such as a blue fluorescent protein, green fluorescent protein or red fluorescent protein.
The reporter gene which produces a signal from the reaction of a reporter protein and any substance may be, for example, a gene encoding a luminescent enzyme protein such as a firefly luciferase gene, a renilla luciferase gene or NanoLuc (registered trademark) luciferase gene, or a gene encoding an active oxygen generation enzyme such as a xanthine-oxidase gene or a nitric-monooxide synthetase gene, or a gene encoding a chromogenic enzyme protein such as a β-galactosidase gene or a chloramphenicol acetyltransferase gene.
The gene which produces a signal from the binding of a reporter protein and any substance may be a gene encoding a heavy metal binding protein. An example of the gene expresses a heavy metal binding protein as a detectable signal is disclosed in JP 2012-200245 A.
It is more preferable that the first reporter gene 11 be a gene encoding a luminescent enzyme protein such as a luciferase gene and a β-galactosidase gene.
The first transcription termination sequence 12 is a sequence for terminating the transcription of the gene upstream. The first transcription termination sequence 12 may be a sequence including, for example, a poly(A) sequence, and may be, for example, a poly(A) addition signal sequence.
The poly(A) addition signal sequence may be, for example, a poly(A) addition signal sequence of simian virus (SV) 40, a poly(A) addition signal sequence of a bovine growth hormone gene, or an artificially synthesized poly(A) addition signal. The poly(A) addition signal of SV40 virus, poly(A) addition signal of bovine growth hormone gene, or artificially synthesized poly(A) addition signal sequence may include, for example, base sequences represented by SEQ ID NO: 2, SEQ ID NO 3, and SEQ ID NO 4 provided in TABLE 2, respectively. Note that the poly(A) addition signal sequences usable in the embodiment are not limited to these, but unless the functions as the poly(A) addition signal sequences are impaired, those with a modified gene sequence may be used.
1-2. Second Reporter Gene Expression Unit
A promoter sequence exhibiting constitutive activity 20 contains a promoter exhibiting constitutive activity in a subject cell. Such a promoter may be activated when the transcription of the gene constitutively expressed is initiated within the subject cell.
The promoter sequence exhibiting constitutive activity 20 may include a linker sequence and the like in addition to the sequence having promoter activity. The promoter sequence exhibiting constitutive activity 20 may be, for example, cytomegalovirus (CMV) promoter, thymidine kinase (tk) promoter of human herpesvirus, a promoter of a ubiquitin gene family origin or a polypeptide chain elongation factor (EF1α) gene promoter. The CMV promoter and tk promoter of human herpesvirus may include, for example, sequences represented by SEQ ID NO 5 and SEQ ID NO 6 provided in TABLE 3, respectively.
A second reporter genes 21 may be similar to the first reporter gene 11 except that it can express a reporter protein by activation of the promoter sequence exhibiting constitutive activity 20. It is preferable that the signal obtained from the reporter protein expressed from the first reporter gene and the signal obtained from the reporter protein expressed from the second reporter gene 21 be identifiably different from each other.
A bicistronic expression sequence 22 may be a base sequence including a sequence which enables bicistronic expression in the subject cell. It can be achieved by binding ribosome to the inside of mRNA to initiate the translation of the protein without depending on 5′ terminal cap structure of mRNA. The bicistronic expression means translating two types of proteins encoded by two genes existing upstream and downstream the bicistronic expression sequence 22 with one mRNA and express them simultaneously.
Therefore, with the bicistronic expression sequence 22 existing between the second reporter gene 21 and the replication initiation protein gene 23, the expressions of the second reporter gene 21 and the replication initiation protein gene 23 can be simultaneously controlled by the activation of the promoter sequence exhibiting constitutive activity 20.
The bicistronic expression sequence 22 may be, for example, internal ribosomal entry site (IRES) sequence. The IRES sequence may be of encephalomyocarditis virus or the like. The IRES sequence of the encephalomyocarditis virus may include, for example, a base sequence represented by SEQ ID NO: 7 provided in TABLE 4.
The replication initiation protein gene 22 may be a gene encoding a protein which initiates the self-replication of the reporter vector 1 in a subject cell. The replication initiation protein gene 22 may be, for example, a wild type large T-antigen gene of simian virus 40, EBNA-1 gene of an Epstein-Barr virus, or a large T-antigen gene of murine polyomavirus. The replication initiation protein gene 22 may be, for example, a base sequence represented by SEQ ID NO: 8 provided in TABLE 5, or SEQ ID NO: 9 provided in TABLE 6, or SEQ ID NO: 10 provided in TABLE 7. The replication initiation protein gene 22 may be a nucleic acid having a DNA replication initiation function, represented by a sequence of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in which one or several bases are deleted, substituted or added. Here, the DNA replication initiation function is defined by encoding a protein which can initiate replication.
A second transcription termination sequence 24 may be a sequence for terminating the transcription of the second reporter gene 21 and the replication initiation protein gene 23 gene which may exist upstream, and may be a sequence similar to that of the first transcription termination sequence 12.
1-3. Replication Initiation Sequence
A replication initiation sequence 30 may be a base sequence including a sequence which initiates the replication of the reporter vector 1 by binding replication initiation protein thereto. The replication initiation protein may be a replication initiation protein expressed from the replication initiation protein gene 23, or a replication initiation protein existing in a subject cell.
The replication initiation sequence 30 can be selected appropriately based on the origin organism of the replication initiation protein gene 23, the type of the replication initiation protein expressed thereby, etc.
For example, when the replication initiation protein gene 23 is a large T-antigen gene of simian virus 40, a replication initiation sequence from the simian virus 40 may be used as the replication initiation sequence 30. In that case, the replication initiation sequence 30 may include a base sequence of SEQ ID NO: 11 provided in TABLE 8.
When the replication initiation protein gene 23 is EBNA-1 gene of Epstein-Barr virus, a replication initiation sequence from the Epstein-Barr virus may be used as the replication initiation sequence 30.
When the replication initiation protein gene 23 is a large T-antigen gene of murine polyomavirus, a replication initiation sequence from the murine polyomavirus may be used as the replication initiation sequence 30.
Although these examples are preferable, the embodiment is not limited to these.
With the above-described configuration, the first signal from the first reporter gene according to the quantity of the cell characteristics marker in the subject cell, and the second signal from the second reporter gene according to the quantity of the gene having the constitutive activity are simultaneously obtained from the same subject cell. For example, the second signal can be used for standardization of the first signal. For example, the relative value of the first signal may be computed by dividing the first signal by the second signal. Thus, the dispersion in the amount of the reporter vector introduced by the subject cell can be corrected, thereby making it possible to estimate the cell characteristics of the subject cell with high precision.
By being expressed the replication initiation protein gene by the promoter with constitutive activity, the reporter vector itself is cumulatively replicated. Thus, the first signal and the second signal can be amplified. Consequently, the cell characteristics of a subject cell can be estimated with precision higher than those of the conventional techniques.
A further example of the reporter vector of the embodiment is shown in
A reporter vector 3 includes a first reporter gene expression unit, a second reporter gene expression unit and a replication initiation sequence. The promoter of a cell characteristics marker included in the first reporter gene expression unit may be a promoter of the NAMPT gene represented by SEQ ID NO: 1. The first reporter gene may be NanoLuc (registered trademark) luciferase gene, and the first transcription termination sequence may be a poly(A) addition signal sequence of bovine growth hormone gene, represented by SEQ ID NO: 3.
The promoter sequence exhibiting the constitutive activity included in the second reporter gene expression unit is a CMV promoter represented by SEQ ID NO: 5. The second reporter gene may be a firefly luciferase gene and the bicistronic expression sequence may be the IRES sequence of the encephalomyocarditis virus represented by SEQ ID NO 7. The replication initiation protein gene is a large T-antigen gene of SV40 represented by SEQ ID NO 10, and the transcription termination sequence may be a late poly(A) addition signal sequence of SV40, represented by SEQ ID NO 2.
Moreover, the replication initiation sequence may be a replication initiation sequence of SV40, represented by SEQ ID NO 11.
Note that the reporter vector is not limited to those described above.
2. Assay Kit for Evaluating Cell Characteristics of Subject Cell
As a further embodiment, an assay kit for evaluating the cell characteristics of a subject cell which contains the reporter vector of the above embodiment can be provided.
The assay kit contains at least one kind of reporter vector of those discussed above in Section 1.
The assay kit may contain a carrier which supports a reporter vector, and/or any reagent necessary for evaluating the characteristics of the subject cell.
The carrier may be, for example, a buffer solution, liposome, cationic lipid, cationic polymer, calcium chloride or nanoparticle of apatite.
The reagent may be, for example, an excipient, a stabilizer, a diluent and/or an auxiliary material, etc.
The assay kit may contain a substance necessary for detecting of a signal selected according to the types of the first and/or second reporter genes of the reporter vector included in the assay kit.
The substance necessary for detecting of the signal may be a substance which produces a signal when reacting with a reporter protein (for example, substrate), an additive which detects the substance produced by the reaction, or a substance which produces a signal when binding to a reporter protein.
The reporter vector, carrier and reagent can be contained each separately or in any combination in a container or containers, to be provided.
3. Method of Evaluate Cell Characteristics of Subject Cell
A method of evaluating the characteristics of a subject cell using the above-described reporter vector may comprise the following steps shown in
(A) preparing a reporter vector;
(B) introducing the reporter vector into the subject cell;
(C) replicating the reporter vector to express a first reporter gene and a second reporter gene;
(D) detecting a first signal from the first reporter gene and a second signal from the second reporter gene; and
(E) evaluating the cell characteristics of the subject cell based on the first signal and the second signal.
The steps will now be further described.
(A) Preparation of Reporter Vector
The reporter vector can be produced and prepared by integrating a first reporter gene expression unit, a second reporter gene expression unit and a replication initiation sequence on the same vector.
The vector may be any conventionally known vector in this technical field, for example, a plasmid, a cosmid or a phage. A plasmid vector is preferable.
The vector may be produced by any method known to a person skilled in the art. The vector may be prepared in the state of being contained in a carrier, which will be described later.
(B) Introduction of Reporter Vector to Subject Cell
The reporter vector obtained in the step (A) may be introduced to a subject cell and/or a cell used as a standard.
The subject cell may be extracted from a subject. The subject may be, for example, any of mammalians including primates such as apes and humans, rodents such as mice and rats, companion animals such as rabbits, dogs and cats and livestock including horses and cows.
The subject cell should sufficiently be a cell whose characteristics are to be evaluated. The subject cell may be a cell colony or a single cell, or may be contained in a sample. The sample may be, for example, blood, urine, feces, saliva, mucosa in the oral cavity, coelomic fluid, expectoration, or a tissues obtained by an biopsy or the like.
The cell characteristics to be evaluated as to the subject cell is canceration of the cell, and when the object of detection is a cancer cell, the subject cell may be, for example, an epithelial tumor, non-epithelial tumor, hemopoietic tissue tumor, fetal tumor, or any combination of these. The epithelial tumor may be, for example, a mammary tumor or a lung tumor.
The reporter vector may be introduced by any conventionally known method.
(C) Replication of Reporter Vector and Expression of Reporter Vector
After introducing the reporter gene into the subject cell, the subject cell may be maintained under the conditions that the vector can be self-replicated and the first and second reporter genes can be expressed. The conditions may be culture conditions conventionally known to a person skilled in the art, in which a subject cell is divided to be reproduced.
Under such conditions, the first and second reporter genes may be expressed. At the same time, the reporter vector can self-replicate as the replication initiation protein expressed by the activation of the promoter sequence exhibiting constitutive activity binds to the replication initiation sequence on the reporter vector. As the first and second reporter genes on the reporter vector cumulatively self-replicating are expressed, the first signal and the second signal can be amplified.
(D) Detection of Signals from Reporter Genes
The first and second signals may be detected, for example, by detecting or quantifying an optical signal obtained from the reporter proteins synthesized from the first and second reporter genes. The detection method may be selected appropriately according to the type of reporter protein encoded by the reporter gene. The method will be described below.
When the reporter gene is luciferase gene, a luciferase protein is extracted from the subject cell, and luciferin, which is a kind of the substrate, is added thereto, to cause a photogenesis reaction, and then the luminescence intensity of the solution may be detected as a signal using a luminance measuring device, for example, a luminometer.
When the reporter gene is a fluorescent protein gene, for example, an extract containing the subject cell or the fluorescent protein extracted from the subject cell is irradiated with the light of a specific wavelength, and the intensity of the fluorescence generated from the fluorescent protein in the subject cell may be detected as a signal using a fluorometry device.
When the reporter gene is a β-galactosidase gene, β-galactosidase protein is extracted and a substrate such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) or o-nitrophenyl-β-D-galactopyranoside (ONPG) is added to the solution, and then the absorbance thereof may be measured as a signal using a spectrometry device.
When the reporter gene is a nitric-monooxide-synthetase gene or xanthine-oxidase gene, the nitric-monooxide-synthetase or xanthine oxidase is extracted and the substrate is added to cause generation of active oxygen, and then the presence/absence or quantity thereof may be detected as a signal with an electron spin resonance device (ESR device) or the like. With the method using an ESR device, the quantity of active oxygen generated may be measured directly, or may be measured using a specific spin trap agent. When using a spin trap agent, the active oxygen generation enzyme is first trapped with the spin trap agent, and then the quantity of the active oxygen generated from the trapped active oxygen generation enzyme may be measured.
When the reporter gene is a heavy-metal binding protein gene, the quantity of the protein bound to the heavy metal originated from the reporter protein may be detected as a signal with a magnetic resonance imaging device, a nuclear-medicine diagnostic device, an MRI imaging device, or computerized transverse axial tomography. The quantity of the heavy-metal binding protein may be detected, for example, as follows. First, a measurable heavy metal is added in advance to the culture medium of the subject cell. Then, after washing the subject cell as needed, the heavy-metal binding protein is extracted from the subject cell. Next, the extracted heavy metal binding protein is imaged with a diagnostic imaging device applicable to the heavy metal added to the medium, and the quantity of the heavy metal binding protein is measured. The heavy-metal binding protein may be expressed inside the subject cell or on an outer surface of the subject cell.
The heavy-metal binding protein gene may be, for example, a base sequence encoding a metallic compound binding peptide. For example, the metallic compound binding protein may just be a peptide, oligopeptide, polypeptide and/or protein binding specifically to a specific metallic compound.
For example, the sequence encoding a metallic compound binding peptide may be a base sequence of an antibody gene or a single-strand antibody gene known to bind a desired metallic compound, and may just be designed based on such a base sequence. Such a base sequence may be designed, for example, by modification and/or changing such as substitution, deletion and addition of one or some bases within a range that can maintain the binding with the metallic compound, or by such modification and/or changing according to the object to be applied.
The gene encoding a single-strand antibody peptide can be designed from an amino acid sequence of the antibody to which the metallic compound binds.
For example, in an MRI imaging, a gadolinium compound is used preferably because of its high contrasting effect. The gadolinium compound should just be, for example, a metallic compound of gadolinium, gadolinium ion, gadolinium complex, a salt or derivative of any of these, a derivative containing any of these, or an analogue of a gadolinium compound.
Moreover, the heavy-metal binding protein gene may be a reporter gene exhibiting a metallic compound binding capacity extracellularly. Such a reporter gene may be a base sequence which encodes a metallic compound binding peptide exhibited extracellularly. Such a base sequence should only be configured, for example, so that it is transcribed and translated in the cell to produce a metallic compound binding peptide and then the produced metallic compound binding peptide moves to the cell membrane to exhibit the metallic compound binding capacity extracellularly. An example of such a reporter gene is disclosed in JP 2012-200245 A, for example. As disclosed therein, such a reporter gene should only comprise a base sequence encoding a metallic compound binding peptide, a base sequence encoding a signal peptide which conveys the metallic compound binding peptide to a cell membrane, and a base sequence encoding an anchor peptide which immobilizes the metallic compound binding peptide conveyed to the cell membrane by the signal peptide thereto.
The result obtained by detection of a signal may be the presence/absence of the signal, may be the size of the signal, or both.
When detecting the first signal, the second signal may be detected after abolishing the first signal, or the order may be reversed. Or the first signal and the second signal may be separated from each other with an appropriate filter and then detected simultaneously.
(E) Evaluation of Characteristics of Subject Cell Based on First Signal and Second Signal
The value of the first signal acquired in the steps of (D) above may be divided by the second signal A to calculate out a relative value. The relative value may be compared between the subject cell and a cell used as a standard.
The comparison may be carried out by performing the steps of (A) to (E) on the subject cell and the standard cell simultaneously to calculate out the relative values to be compared. Or the relative value of a cell used as the standard may be computed in advance, to be compared therewith.
In the above-provided descriptions, a method including preparation of a reporter vector as step (A) is discussed, but the method may not necessarily include preparation of a reporter vector. The method may be carried out using a reporter vector prepared in advance.
With the above-described method of evaluating the characteristics of a subject cell, it is possible to evaluate the cell characteristics more precisely.
4. Device of Evaluation of Subject Cell
The above-described method of evaluating the characteristics of a subject cell can be carried out with a cell characteristics evaluation device. The cell characteristics evaluation device will be described with reference to
The cell characteristics evaluation device comprises the following structural elements:
(i) a gene introduction unit which introduces a reporter vector into a subject cell;
(ii) a thermo-regulating unit which receives a sample containing the subject cell obtained in the gene introduction unit and cultures the subject cell for a desired time;
(iii) a detection unit which receives the sample from the thermo-regulating unit and detects signals produced from the first reporter gene and the second reporter gene of the subject cell contained in the sample; and
(iv) an evaluation unit which evaluates cell characteristics of the subject cell based on a result of the detection.
In the gene introduction unit, the reporter vector is introduced into a nucleus of the subject cell. The reporter vector may be any reporter vector discussed above.
In the thermo-regulating unit, the self-replicating vector self-replicates within the nucleus of the subject cell.
In the detection unit, the signals produced from the first reporter gene and the second reporter gene are detected. In the detection unit, the subject cell is subjected to, for example, photogenesis measurement, fluorometry, absorbance measurement, X-ray measurement, electron spin resonance, nuclear medicine diagnosis and/or magnetic resonance imaging, etc. The measuring devices for these measurements may be selected according to the first reporter gene and the second reporter gene. The information acquired by the detection unit is sent to the evaluation unit.
The evaluation unit may comprise a processor, a display unit, an entry unit, etc. In the evaluation unit, the information acquired by the detection unit may be subjected to data processing by the processor and displayed on the display unit as needed. That is, the result obtained by the detection unit may be sent to the processor of the evaluation unit, where it may be analyzed, calculated and processed according to the predetermined procedures, and may be indicated on the display unit.
The insides of the gene introduction unit, the thermo regulating unit and the detection unit may be communicated to each other via connection windows so that processing can be continuously carried out. After the subject cell is passed to one unit to the following unit, the respective connection window can be closed by a closure shield.
The cell characteristics evaluation device may further include a cell separation unit (not shown) configured to separate a subject cell from a sample and send the subject cell to the gene introduction unit.
The procedure of the method of evaluating cell characteristics with the cell characteristics evaluation device will be described with reference to
First, an experimenter may place a container containing a subject cell and a reporter vector into the gene introduction unit (S1). Then, the instruction to start the analysis may be entered from the entry unit. The instruction from the entry unit may be sent to the processor and with an instruction from the processor, a treatment to enabling the introduction of the reporter vector to the subject cell may be performed in the gene introduction unit (S2). After a predetermined time elapses, the introduction process may be finished and the subject cell accommodated in the container may be sent to the thermo-regulating unit. In the thermo-regulating unit, the subject cell may be maintained under predetermined conditions for a predetermined time. Thus, the reporter vector may self-replicate (S3). After the predetermined time elapses, the subject cell accommodated in the container may be sent to the detection unit from the thermo-regulating unit. In the detection unit, the signals produced from the first reporter gene and the second reporter gene of the subject cell may be detected (S4). The detected information may be sent to the evaluation unit to be subjected to data processing by the processor, and the cell characteristics may be evaluated based on the processed data (S5). The information thus obtained may be shown in the display unit. The container may be moved to the gene introduction unit, the thermo-regulating unit and the detection unit by a transportation means such as a conveyor belt, a movable arm and/or a movable tray, etc. All of these movements may be performed under control of the processor according to the directions of the processor based on a preset program. The gene introduction unit has a configuration required for the gene introduction by a selected method. The thermo-regulating unit has a configuration required in order to fulfill conditions required for the reporter vector to self-replicate. The detection unit has a configuration required for detection the signal according to the signal detected.
A cell characteristics evaluation device 50 shown in
The cell separation unit 51 may include a separator 55 for separating a cell to be examined, i.e., a subject cell, from a specimen obtained from an object. The separator 55 may comprise a receptor 56a for receiving the specimen obtained from the object, a treatment unit 56b which, for example, digest the specimen received with the receptor 56a, and a separation portion 57 for separating the subject cell from the specimen treated with the treatment unit 56b. The receptor 56a, the treatment unit 56b, and the separation portion 57 may be stacked one on another in this order as one liquid-tight integral body while the three hollow cylindrical portions having different internal diameters are connected together from the top to the bottom. The respective boundaries between the receptor 56a, the treatment unit 56b, and the separation portion 57 may be joined in a liquid junction manner with valves to be openable/closable. The receptor 56a may be a tapered cylindrical member with an aperture in its upper part and may comprise an openable/closable cover. The boundary between the receptor 56a and the treatment unit 56b is closed with a valve to be liquid-tight. When this valve is closed, the valve serves as a bottom of the receptor 56a. The treatment unit 56b is a cylindrical member continuing from the lower end of the receptor 56a. When the valve in the boundary to the receptor 56a is closed liquid-tightly, the treatment unit 56b is provided with a cover. When the valve in the boundary to the separation portion 57 is closed, the treatment unit 56b is provided with a bottom. The separation portion 57 is a cylindrical member having an inner wall which is liquid-lightly continuous from the inner wall of the treatment unit 56b and the valve is provided at its lower end. When this valve is closed liquid-tightly, the separation portion 57 is provided with a bottom. A separating member is fixed to an inside of the separation portion 57. When using the device 50, the container 58 is placed on the stage 54 beneath the lower part of the bottom of the separation portion 57. Here, the container 58 receives the specimen containing the separated subject cell 59 from the separation portion 57 and accommodates it therein. The treatment unit 56b may comprise a heating and/or thermo-regulating mechanism for a desired process such as digestion, a biochemical reaction and the like on the specimen contained therein. The separating member may be a component which separates and extracts a sample containing the subject cell from the specimen dropped from the treatment unit 56b, or may be a component which removes a contaminant from the specimen dropped from the treatment unit 56b. The separating member may spread along the surface intersecting the central axis of the separation portion 57 to be fixed over a circumference of the inner wall of the separation portion 57. The separating member may be, for example, a filter, a membrane and/or a magnet.
The sample containing the subject cell 59 separated with the separator 55 is accommodated in the container 58 placed on the stage 54. The container 58 which accommodates the subject cell is sent to the DNA introduction unit 52.
The DNA introduction unit 52 comprises a controller 60 and the introduction probe 61, which introduce a reporter vector to the subject cell accommodated in the container 58. The reporter vector may be introduced to the subject cell by a conventionally known method by itself. The controller 60 and the introduction probe 61 may only be devices which employ any introduction mechanism to realize the selected introduction method. For example, the DNA introduction unit 52 may be a device which performs electroporation. In that case, the controller 60 may be the DNA introducer and the introduction probe 61 may be an electrode probe, by which the electropolator may be constituted. Further, the DNA introduction unit 52 may comprise a nozzle (not shown) for supplying a reagent containing a reporter vector to the container 58. Furthermore, the DNA introduction unit 52 may comprise a liquid transfer system (not shown) for sending a reagent to the nozzle, and a reagent storage container (not shown) for supplying a reagent to the liquid transfer system.
Moreover, the DNA introduction unit 52 may function as an incubator which cultures the subject cell 59 in the container 58 under a predetermined culture condition over a predetermined time. The culture of the DNA introduction unit 52 may just be controlled by the controller 60.
In the DNA introduction unit 52, the container 58 containing the subject cell into which the reporter vector is introduced is, next, sent to the measurement unit 53.
The measurement unit 53 may comprise a black box 62 which covers a part of stage 54 from above and below, a light source 63 provided inside the black box 62, a window opened in a stage surface below the light source 63, an image pick-up unit 68 with an objective lens 67, installed under the window, a controller 65 communicating to the light source 63 and the image pick-up unit 68, and a display unit 64 and a storage unit 66, which are both connected to the controller 65. For example, the light source 63, the objective lens 67 and the image pick-up unit 68 of the measurement unit 53 may be installed to be contained in a microscope equipped with a CCD camera, a CMOS camera or the like. Moreover, the controller 65, the display unit 64 and the storage unit 66 may be a computer comprising these members.
An example of the evaluation of the cell characteristics by the device 50 shown in
The reaction stop reagent may be accommodated in the separation portion 57 in advance, or such a reagent may be added through a closable reagent inlet opening made in a location of a wall surface of the separator 55.
With the sample containing the subject cell 59 accommodated therein, the container 58 is sent to the DNA introduction unit 52. A reagent containing a reporter vector is added to the container 58 through the nozzle of the DNA introduction unit 52. The introduction probe 61 is brought into contact with the sample in the container 58 and the reporter vector is introduced into the subject cell 59 under the control of the controller 60. Under the control of the controller 60, the subject cell 59 is cultured under a predetermined culture condition over a predetermined time.
Then, the container 58 is sent to the measurement unit 53. In the measurement unit 53, the quantity of the signal produced from the first reporter gene and the second reporter gene in the subject cell 59 accommodated in the container 58 is measured. All the procedures in the measurement unit 53 are controlled by the controller 65 according to a table in which the programs and a plurality of information pre-stored in the storage unit 66 are organized. For example, the light irradiation from the light source 63, the irradiation conditions such as the irradiation time and irradiation interval, the imaging with the image pick-up unit 68 and the image pick-up conditions, the image processing of images picked up, image processing, etc. are controlled by the measurement unit 53. Further, the evaluation of the cell characteristics is carried out by the controller 65 according to the program pre-stored in the storage unit 66 with reference to a table including the information which associate images and cell characteristics with each other.
After culturing the subject cell in the DNA introduction unit 52, the container 58 is sent to the measurement unit 53. Then, the light from the light source 63 is irradiated into the container 58. The light passing through the container 58 is concentrated by the objective lens 67, and with the light, a bright field image can be arbitrarily obtained with the image pick-up unit 68. Then, in the black box 62, the signal (for example, light emission) produced from the first reporter gene and the second reporter gene introduced to the subject cell is imaged by the image pick-up unit 68 through the objective lens 67. The light irradiation of the light source 63 is stopped and the light emission from the subject cell is collected with the objective lens 67, by which an image of the light emission is obtained with the image pick-up unit 68. Arbitrarily bright field images and light-emission images obtained by the image pick-up unit 68 are subjected to image processing by the controller 65, to be superimposed (merged) one on another and displayed on the display unit 64. At the same time or approximately the time of display of an image on the display unit 64, the controller 65 evaluates the characteristics of the subject cell based on the bright field image and light emission image with reference to the table stored in the storage unit 66. The result of the evaluation is displayed on the display unit 64 together with the merged image.
The above-provided example is directed to the case where light is emitted as a signal produced from the first reporter gene and the second reporter gene, but a signal other than light emission can be measured similarly by exchanging the sensing device which constitutes the image pick-up unit 68 as needed.
Moreover, the measurement unit 53 may further comprise a processor for performing computational operation, image processing, etc. or an analyzer for evaluation of characteristics. The processor and analyzer may perform the computational operation, image processing, and evaluation of characteristics carried out by the controller 65 in the above-discussed case.
The above-described embodiment discusses the example in which the subject cell is cultured by the DNA introduction unit 52, but the subject cell may be cultured in the measurement unit 53, in which case, the device 50 should only comprise a component which unable cell culture. Or a subject cell may be cultured in both of the DNA introduction unit 52 and the measurement unit 53.
In order to control the motions of the units of the device 50, each unit may comprise a controller, or all the units included in the device 50 may be controlled by one controller, for example, the controller 65. Further, the container 58 placed on the stage 54 may be moved between units by a container transfer mechanism mounted in the stage 54. Such a mechanism may be, for example, the robot arm provided in the device 50 and controlled by the controller as to its movement. Or the conveyor belt may be attached to the stage 54.
Moreover, the reporter vector may be added to the container 58 at any stage before the container 58 is sent to a predetermined position of the DNA introduction unit 52. In that case, the device 50 should only further comprise a reporter vector addition mechanism at any location upstream the the DNA introduction unit 52.
The characteristics of a subject cell are evaluated by the above-described device. With this device, it is possible to evaluate the characteristics of the subject cell at higher precision. Further, it is possible to perform the test at a high throughput, the testing procedure is simple, thus making it possible to prevent mix-up of samples or contamination.
Moreover, the morphological feature of the subject cell may be acquired based on the bright field image, and the characteristics of the subject cell may be evaluated thereby together with the data from the reporter vector. The morphological feature may be, for example, the area of the subject cell, the ratio of the area of the nucleus to the area of the subject cell, the ratio of the minor axis to the major axis of the subject cell, the number of nuclei in the subject cell, or the feature of the marginal part of the subject cell, that is, for example, the contrast ratio of the marginal part, or the like. These features can be obtained by a noninvasive optical image pick-up method. For example, a hyper-spectrum imaging method, a phase-contrast microscope method, or a differential interferometry may be used to acquire these data.
For example, when performing morphologic observation by a bright field, it is preferable to irradiate light having a wavelength of a near-infrared region. For example, the light having a wavelength of a near-infrared region should preferably be near-infrared light having a wavelength of 700 nm to 750 nm or multiple-spectrum light having a wavelength of 350 nm to 1050 nm. For example, the light of a wavelength of the near-infrared region may be irradiated onto the subject cell to acquire the morphological data on the subject cell based on reflection or absorption of the light, for example, the reflectance or absorbance as an index.
For example, when the malignancy of a subject cell is high, there is a tendency that the cell form has a high aspect ratio, such as of a spindle shape. For example, the morphological data on such a subject cell as well may only be acquired in the measurement unit 53. In that case, the measurement unit 53 should just be controlled by the controller 65 according to the table in which the programs and information pre-stored in the storage unit 66 are organized. The table should just include information on the relationship between detectable signals from reporter vectors and cell characteristics and further information indicating the relationship between the morphological characteristics of the subject cell and the cell characteristics and the relationship between the detectable signals, cell characteristics and morphological characteristics. Moreover, for example, the light irradiation from the light source 63, the irradiation conditions such as the irradiation time and irradiation interval, the imaging with the image pick-up unit 68 and the image pick-up conditions, the image processing of images picked up, image processing, etc. are controlled by the measurement unit 53. Further, the evaluation of the cell characteristics may just be carried out by the controller 65 according to the program pre-stored in the storage unit 66 with reference to the table including the information which associate the morphological characteristics, information from a reporter vector, and cell characteristics with each other. Thus, by evaluating the cell characteristics of a subject cell from a plurality of sides including the promoter activity and the morphological characteristics, it is possible to obtain more accurate results.
(a) Production of Dual Reporter Vector
A reporter vector to which a promoter region (−2426 to +276 bp, SEQ ID NO: 1) of a nicotinamidephosphoribosyl transferases (NAMPT) gene has been integrated was produced. The promoter region of the NAMPT gene amplified and adjusted by PCR using the human genome as the template. The PCR was performed using PrimeStar GXLDNA polymerase (TaKaRaBIO). The primer sequences used for the PCR are as follow.
The promoter sequence (NAMPT promoter) of the NAMPT gene thus adjusted was integrated to an upstream of an Oplophorus gracilirostris-origin NanoLuc (registered trademark) gene, to produce a reporter gene expression unit comprising a NAMPT promoter sequence, a NanoLuc (Nluc) (registered trademark) gene and an SV40 transcription termination sequence, a reporter gene expression unit comprising a cytomegalovirus (CMV) promoter, a firefly-origin luciferase (Fluc, Firefly Luciferase) gene and a transcription termination sequence of a bovine growth hormone gene, and a self-replicating dual reporter vector containing SV40 replication initiation sequence on the same nucleic acid. As to the self-replicating dual reporter vector, two kinds were produced, in which the directions of reporter gene expression units differ from each other (that is, pS-NAMPT-D in
(b) Lipofection (Introduction of Reporter Vector to Cell)
0.5 μg of the dual reporter vector was added to 100 μL of Opti-MEM (Life Technologies). Then, 0.5 μg of an enhancer reagent (Plus reagent of Life Technologies) was added and the resultant was incubated for 5 minutes at room temperature. To the solution, 1.5 μL of Lipofectamine LTX (Life Technologies) was added and well suspended therein, and the resultant was incubated for 25 minutes at room temperature. 40 μL of the solution was moved to a 48 well plate, and 200 μL of a suspension of human breast cancer cell strain (high metastatic malignant tumor cell strain: MDA-MB-231) was added thereto, followed by gently mixing. Then, the cells were cultured in an incubator at 37° C. in a 5%-CO2 atmosphere.
(c) Reporter Gene Assay
96 hours after the lipofection, the medium was removed from the 48 well plate and the cells were washed by PBS. Then, 150 μL of a cytolysis liquid (Glo-Lysis Buffer, Promega) was added thereto and the resultant was let stand still for 30 minutes or more at −80° C. to be frozen. When to be used, the cytolysis liquid was thawed at room temperature, and the solution was collected in a centrifuge tube and the cell residue was precipitated by centrifuge. The supernatant was collected in a new sample tube and 25 μL of the supernatant was dispensed to a 96 well plate (Nunc). An equivalent amount of a Luciferase substrate solution (One-Glo Luciferase Assay System, Promega) or NanoLuc (registered trademark) substrate solution (Nano-Glo Luciferase Assay System, Promega) was added to the well, and the luminescent intensity was measured with a luminometer (Mithras LB940, Berthold). Thus, the reporter activity of Nluc (the activation of the NAMPT promoter) for evaluation of the malignancy of the oncocyte, and the reporter activation of Fluc (the activation of the CMV promoter) for the correction of amount of reporter DNA introduced for each cell were measured. The relative reporter activation (RLU) used for evaluation of the malignancy of the oncocyte was defined as a value obtained by dividing the activation value (luminescent intensity) of Nluc by the activation value (luminescent intensity) of Fluc.
The results are shown in
By the method discussed in Example 1 (2), pS-NAMPT-D was introduced into a normal human mammary epithelial cell (HMEC) and three kinds of breast cancer cell strains (a high metastatic malignant oncocyte strain: MDA-MB-231 and low metastatic malignant oncocyte strains: T-47D and BT-474), and they were cultured. After 72 hours, a reporter gene assay was performed by the method discussed in Example 1 (3), and the relative reporter activity (RLU) was calculated. The results are shown in
(a) Construction of Reporter Vector
A self-replicating dual reporter vector (pAmp-NamNL-CMVLLT) was produced by insert a replication initiation protein (LT) gene of simian virus 40 (SV40) between a CMV promoter::luciferase gene, and a poly(A) addition signal of bovine growth hormone gene in pS-NAMPT-D (
(b) Electroporation (Introduction of Reporter Vector to Cell)
To ten kinds of malignant mammary gland-originated oncocyte strains (T-47D, MCF7, BT-474, UACC-812, SK-BR-3, HCC70, MDA-MB-468, BT-549, MDA-MB-231 and BT-20), one kind of benign mammary gland-originated tumor cell strain (MCF10A) and a normal human mammary epithelial cell (HMEC), the two kinds of self-replicating dual reporter vectors described in Section (a) above were integrated by electroporation. After pre-cultured cells were washed by PBS once, the cells were removed from the culture container using trypsin-EDTA, and a culture medium containing serum was added thereto to suspend the cells. Then, 50 mL of the cell suspension was collected in a plastic centrifugal tube and the cells were precipitated by centrifuge. After that, the supernatant was removed, and Opti-MEM (Life Technologies) was added to suspend the cells. After precipitating the cells by centrifuge and removing the supernatant, the cell suspension was adjusted by adding Opti-MEM thereto to have a concentration of 1×107 cell/mL. 100 μL of the cell suspension was dispensed to a 1.5 mL tube and 5.0 μg of a reporter vector (pAmp-NamNL-CMVLLT or pAmp-NamNLLT-CMVL) was added thereto. After that, electroporation was performed using an electropolator (CUY EDIT II, Bex) and a cuvette electrode (2-mm width, Bex). Then, 1 mL of a culture medium was added and gently mixed. After that, the cuvette electrode was set still in a 5%-CO2 incubator. 5 minutes after, the cuvette electrode was removed from the incubator and the cell suspension was moved to a 24 well plate to which the culture medium was added in advance. Then, the resultant was cultured at 37° C. in a 5%-CO2 atmosphere.
(c) Reporter Gene Assay
72 hours after the electroporation, a reporter gene assay was performed by the method discussed in Example 1 (3) to calculate the relative reporter activity (RLU). The results are shown in
When pAmp-NamNL-CMVLLT was introduced, all the malignant oncocyte strains exhibited RLUs of 20 times that of the normal human mammary epithelial cell (HMEC). Further, even if compared with the RLUs of nonmalignant oncocyte strain MCF10A and malignant oncocyte strain, a RLU higher than that of MCF10A was exhibited in all the malignant breast cancer cells. On the other hand, when pAmp-NamNLLT-CMVL was integrated, a RLU lower than that of pAmp-NamNL-CMVLLT was exhibited. Further, with the same reporter vector, two kinds of malignant breast cancer cells exhibited RLUs comparable as that of HMEC, and three kinds of malignant breast cancer cells exhibited RLUs comparable with or lower than that of MCF10A. From this result, it was clarified that pAmp-NamNL-CMVLLT in which IRES sequence::replication initiation protein gene was inserted to a reporter gene expression unit including a reporter sequence exhibiting constitutive activity, which includes a CMV promoter exhibited a high signal value, and therefore has a reporter vector structure appropriate for evaluation of malignancy of a tumor with higher accuracy.
T-47D and HMEC were mixed at 1:1 or 1:3. To the thus mixed cells, pAmp-NamNL-CMVLLT was introduced by electroporation, the cells were cultured at 37° C. in a 5%-CO2 atmosphere. 72 hours after, photographs of the luminescent cells were taken using a photogenesis imaging system, LuminoView (LV200, Olympus). The results are shown in
It was clarified from the above-described experiment that the self-replicating dual reporter vectors of the embodiments are appropriate for evaluation of the characteristics of a subject cell with higher accuracy.
While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-008125 | Jan 2016 | JP | national |
