WO2018216181A1 - Measurement method - Google Patents

Measurement method Download PDF

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Publication number
WO2018216181A1
WO2018216181A1 PCT/JP2017/019612 JP2017019612W WO2018216181A1 WO 2018216181 A1 WO2018216181 A1 WO 2018216181A1 JP 2017019612 W JP2017019612 W JP 2017019612W WO 2018216181 A1 WO2018216181 A1 WO 2018216181A1
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Prior art keywords
color
sample
luminescence
luciferase
amount
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PCT/JP2017/019612
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French (fr)
Japanese (ja)
Inventor
竜太郎 秋吉
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オリンパス株式会社
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Priority to PCT/JP2017/019612 priority Critical patent/WO2018216181A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

Definitions

  • the present invention relates to a measurement method.
  • a photoprotein is expressed in a cell and bioluminescence generated by the photoprotein is detected. Such detection of luminescence is used for analysis of the expression state of a gene or protein of interest. Moreover, visualization of intracellular molecular dynamics is also performed by photographing a cell in which a photoprotein is expressed and acquiring a luminescent image. Even based on such luminescent images, analysis of the expression state of the gene or protein of interest can be performed.
  • Japanese Patent Application Laid-Open No. 2004-333457 discloses a technique for obtaining a light emission amount for each color by using N-1 filters for N light having different wavelength characteristics that are generated simultaneously. Is disclosed. Using this technology, the transmission characteristics of each filter are obtained for N-color light, and a matrix operation is performed to obtain the light emission amount for each color based on the light intensity obtained when each filter is applied. Can be.
  • the luminescent properties may change even if the same photoprotein is used. Such a change leads to a decrease in the quantitativeness of the analysis.
  • the above-described technique is based on the premise that the state of cells and the like is stable and light emission is stable.
  • An object of the present invention is to provide a measurement method capable of highly quantitative analysis in a sample in which light having different wavelength characteristics is simultaneously emitted.
  • a measurement method includes using N or N-1 filters for a sample to be measured in which N photoproteins are mixed and luminescence of each color is mixed.
  • a method for obtaining a value related to the amount of luminescence wherein a plurality of first samples in which each of the N-color photoproteins is independently present are used, and none of the filters are transmitted for luminescence of each color. Calculating the transmittance of each of the filters for each color emission based on the emission intensity measured in the state and the emission intensity measured when passing through each of the filters; Based on the emission intensity measured when passing through each of the filters for the second sample prepared such that the respective amounts of protein are known, and the transmittance of the filters.
  • the present invention it is possible to provide a measurement method capable of analyzing with high quantitativeness in a sample in which light having different wavelength characteristics is simultaneously emitted.
  • FIG. 1 is a diagram showing an outline of an example of a measurement system used in an embodiment of the present invention.
  • FIG. 2 is a flowchart illustrating an outline of an example of a measurement method according to an embodiment.
  • FIG. 3A is a diagram showing an outline of a partial configuration example of a vector used for an example of measurement.
  • FIG. 3B is a diagram showing an outline of a configuration example of a part of a vector used for an example of measurement.
  • FIG. 4A is a diagram showing an outline of a partial configuration example of a vector used in an example of preparing a sample exhibiting monochromatic light emission.
  • FIG. 3A is a diagram showing an outline of a partial configuration example of a vector used in an example of preparing a sample exhibiting monochromatic light emission.
  • FIG. 4B is a diagram showing an outline of a part of a configuration example of a vector used in an example of preparing a sample exhibiting monochromatic light emission.
  • FIG. 5 is a diagram showing an outline of a partial configuration example of a vector used in an example of preparing a sample in which plural types of luciferases that emit light having different wavelengths from each other are expressed in the same cell in the same amount.
  • FIG. 6 is a diagram for explaining the relationship between the wavelength and the emission intensity.
  • FIG. 7A is a diagram showing an outline of a configuration example of a vector used for production of iPS cells.
  • FIG. 7B is a diagram showing an outline of a configuration example of a vector used for production of iPS cells.
  • FIG. 7A is a diagram showing an outline of a configuration example of a vector used for production of iPS cells.
  • FIG. 7B is a diagram showing an outline of a configuration example of a vector used for production of iPS cells.
  • FIG. 7C is a diagram showing an outline of a configuration example of a vector used for production of iPS cells.
  • FIG. 8 is a diagram showing an outline of a part of the structure of a vector used for preparing a sample in which MA-Luci2 luciferase and SfRE1 luciferase are expressed in equal amounts in the same cell in the examples.
  • FIG. 9A is a luminescence imaging image of the sample to be analyzed when no filter is used according to the example.
  • FIG. 9B is a luminescence imaging image of the sample to be analyzed when filter 1 is used according to the example.
  • FIG. 9C is a luminescence imaging image of the sample to be analyzed when filter 2 is used according to the example.
  • FIG. 9A is a luminescence imaging image of the sample to be analyzed when no filter is used according to the example.
  • FIG. 9B is a luminescence imaging image of the sample to be analyzed when filter 1 is used according to the example.
  • FIG. 10 is a diagram showing the luminescence intensity related to MA-Luci2 luciferase and SfRE1 luciferase obtained for each region of interest (ROI) of the luminescence imaging image of the sample to be analyzed according to the example.
  • FIG. 11 is a ratio of the vector A to the vector C related to each region of interest (ROI) obtained as an analysis result according to the example, before and after performing correction based on the luminescence characteristics of MA-Luci2 luciferase and SfRE1 luciferase It is a figure which shows a result.
  • the measurement method according to the present embodiment is used when there are a plurality of measurement objects.
  • this measurement method light having a different wavelength is associated with each measurement target, and the sample is prepared so that the light emission amount of the corresponding wavelength changes according to the change of the measurement target. More specifically, a photoprotein associated with the measurement target is used, and the amount of the photoprotein changes according to the change of the measurement target.
  • the characteristics of the measurement object are analyzed by measuring the emission intensity.
  • N filters are used to separate light for each wavelength.
  • N-1 filters N different emission intensities can be acquired for the case where no filter is used and the case where each filter is used. Therefore, based on these results, the emission intensity can be obtained for each of the N colors.
  • N filters having a characteristic of mainly transmitting each of N colors of light. If N is 4 or less, that is, 4 colors or less, light separation is relatively easy.
  • the measurement system 1 includes a measurement device 10 and a control device 40.
  • the measuring apparatus 10 is an apparatus that can quantify the intensity of light emission of a sample, such as a microscope equipped with an image sensor, a luminometer equipped with a photomultiplier tube, and the like.
  • the microscope may include a luminescence imaging system LV200 (Olympus Corporation).
  • the control device 40 is a device that controls the operation of the measurement device 10 such as a personal computer (PC) or analyzes data obtained by the measurement device 10.
  • PC personal computer
  • the measuring apparatus 10 includes a dark box 19.
  • the measuring device 10 detects the light emission of the sample 90 arranged in the dark box 19.
  • the measuring device 10 includes, for example, an objective lens 11, an imaging lens 12, a detector 13, and a filter unit 20.
  • the detector 13 detects light emitted from the sample 90 via the objective lens 11 and the imaging lens 12.
  • the detector 13 may be an imaging device including a CCD sensor, a photomultiplier tube, or the like.
  • the filter unit 20 includes a plurality of types of filters 21 and filter changing mechanisms 22 having different transmission characteristics.
  • the filter unit 20 inserts any one of the plurality of filters 21 in the optical path that reaches the detector 13 from the sample 90 or does not insert any filter 21.
  • a color CCD camera may be used as the detector 13 without switching the filter.
  • a color CCD camera since a red, green or blue color filter is provided for each pixel, red, green and blue data are acquired simultaneously.
  • the control device 40 operates the filter changing mechanism 22 to switch the filter 21 and acquire data from the detector 13.
  • the control device 40 analyzes the acquired data.
  • the control device 40 includes, for example, a processor 41, a random access memory (RAM) 42, a recording device 43, an input device 44, and a display device 45.
  • RAM random access memory
  • the processor 41 performs various calculations related to the operation of the measurement system 1.
  • the processor 41 may include, for example, a Central Processing Unit (CPU), an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA), or a Graphics Processing Unit (GPU).
  • the processor 41 may be configured by one integrated circuit or the like, or may be configured by combining a plurality of integrated circuits.
  • the RAM 42 functions as a main storage device for the processor 41.
  • the recording device 43 is, for example, a semiconductor memory or a hard disk.
  • the recording device 43 can record programs, parameters, and the like used by the processor 41.
  • the recording device 43 can record data obtained by using the measuring device 10, analysis results based on the data, and the like.
  • the input device 44 can include, for example, a keyboard, a mouse, a touch panel, and the like.
  • the display device 45 can include, for example, a liquid crystal display.
  • the measurement method mainly includes three processes.
  • the first process is a process for obtaining the transmittance at which the light emitted from the sample passes through the filter 21 for each emission color of the sample and for each filter 21 to be used.
  • the second process is a process for determining the relationship between the amount of photoprotein and the amount of luminescence using samples prepared so that photoproteins related to luminescence of different colors exist in equal amounts.
  • the third process is a measurement for the analysis target. The light emission intensity to be analyzed is measured, and the value for the object to be measured is calculated in consideration of the transmittance of the filter 21 and the relationship of the light emission amount to the amount of the photoprotein.
  • the outline of the measurement method according to this embodiment is shown in the flowchart of FIG.
  • the first process that is, the process for obtaining the transmittance of the filter is the process of steps S1 to S4 shown in FIG. This process will be described.
  • the filter whose transmittance is required is a filter used for the third process, that is, the measurement of the analysis target.
  • the sample used here is a sample to which monochromatic light is emitted.
  • a sample that emits this monochromatic light is prepared as a first sample (step S1).
  • the luminescence used here is luminescence used for the third process, that is, measurement of the analysis target.
  • Luminescence is related to bioluminescence by photoproteins.
  • luciferin as a substrate and luciferase as an enzyme as a photoprotein can be used.
  • this luciferin-luciferase system any system such as firefly luciferin, bacterial luciferin, dinoflagellate luciferin, vargulin, coelenterazine and the like may be used. Since a plurality of types of light having different wavelengths are used for the third process, that is, measurement of the analysis target, a plurality of types of luciferases having different emission wavelengths are used. Examples of luciferase include P.I.
  • luciferase Pyralis, Crick beetle, MA-Luci2, SfRE1, Oki_mut1, etc.
  • Beetle luciferase Renilla luciferase, Cypridina luciferase, Aequorin, Chiacyl luciferase, Luminous luciferase, Luminous luciferase, Dinoflagellate, etc.
  • luciferase can be used.
  • the sample prepared in step S1 is, for example, a cell into which an expression vector containing a luciferase gene has been introduced. Since a sample is prepared for each luminescent color, one type of luciferase gene among a plurality of types of luciferases having different emission wavelengths is introduced into a cell or the like in one sample. Samples relating to cells having such one type of luciferase gene are prepared for the number of types of luciferase.
  • the cells and the like can include various cells such as cell-lined cultured cells, isolated cells, and stem cells.
  • Stem cells can include iPS cells or ES cells.
  • the cell or the like may be a cell derived from a mammal, or may be a cell derived from another organism such as an insect or a fungus.
  • the isolated tissue etc. may be contained in the cell etc.
  • Various forms of the cell form can be taken. Colonies may be formed, cell clusters such as embryoid bodies, spheroids, and the like may be formed.
  • the sample prepared in step S1 is obtained by introducing, for example, one type of luciferase gene or the like used for measurement of the analysis target into a cell or the like used for measurement of the analysis target. For example, in a state where the introduced luciferase is expressed, luciferin is added to the medium to prepare a sample that generates luminescence.
  • step S2 For each sample prepared in step S1, the emission intensity is first measured without the filter 21 being inserted into the optical path (step S2). Subsequently, the emission intensity of the same sample is measured through the filter 21. The measurement of the emission intensity through the filter 21 is performed in order for all the filters used (step S3).
  • the detection result of the emission intensity when the filter is not inserted in the optical path and when each filter is inserted in the optical path can be obtained.
  • the transmittance for each filter is calculated for each emission color (step S4).
  • the subsequent processing for obtaining the relationship between the amount of the second photoprotein and the amount of luminescence is the processing of steps S5 to S7 shown in FIG. This process will be described.
  • the sample used here is a sample in which equal amounts of photoproteins related to light emission at each wavelength are contained. Such a multicolor equivalent light emission sample is prepared as a second sample (step S5).
  • a sample is prepared in which a plurality of types of luciferases that emit light having different wavelengths are expressed in equal amounts in the same cell.
  • a plurality of types of luciferase genes are linked in a polycistronic manner so that a plurality of types of luciferases are expressed in equal amounts in a cell.
  • multiple types of luciferase genes can be linked by a sequence related to the 2A peptide.
  • multiple types of luciferase genes introduced into cells can be linked by an IRES sequence.
  • a plurality of types of luciferase genes may be configured such that a plurality of types of luciferases are linked and expressed via a linker.
  • the emission intensity of the sample prepared in step S5 is measured through the filter 21.
  • the measurement of the light emission intensity is performed in order for each case through each filter (step S6).
  • the light emission detection result of the multicolor equivalent light emission sample through each filter is obtained.
  • Step S7 the light emission amount of each color when the substances related to light emission are contained in equal amounts is calculated. That is, the relationship between the amount of photoprotein and the amount of luminescence is acquired for each color.
  • the light emission amount for each wavelength can be calculated, for example, by matrix calculation. That is, each of the light emission amount of the N-color I 1, I 2, ... and I N.
  • the transmittance of each of the N light emission is T 11 , T 12 ,... T 1N for the first filter, and the transmittance of each of the N light emission is for the Nth filter.
  • T N1 , T N2 are defined as T N1 , T N2 .
  • Each of the N filters S 1, S 2 each detected value detected when inserted into the optical path, ... and S N. At this time, the following equation holds.
  • the light emission amounts of the N colors can be calculated by the following formula.
  • the present invention is not limited thereto. Even if the amount of photoprotein is not equal, if the quantitative ratio is known, the relationship between the amount of photoprotein and the amount of photoprotein is obtained.
  • luciferase A and luciferase B are used, two luciferase A genes and one luciferase B gene are linked by a 2A peptide, for example, a sequence such as luciferase A-2A-luciferase A-2A-luciferase B Can be built. In this case, the expression level ratio between luciferase A and luciferase B is 2: 1.
  • luciferase A when three or more kinds of luciferases are used, all of them may not be expressed in the same cell. If two or more of three or more luciferases are expressed in the same cell, and if one luciferase is expressed in two or more cells, the luciferase can be used for other cells. Comparison is possible. For example, when luciferase A, luciferase B, and luciferase C are used, if luciferase A and luciferase B are expressed in cell A and luciferase A and luciferase C are expressed in cell B, luciferase A is used as a reference. The relationship between the amount of photoprotein and the amount of luminescence among luciferase A, luciferase B, and luciferase C is obtained.
  • a sample to be analyzed is prepared as a third sample (step S8).
  • the emission intensity of this sample is measured through the filter 21.
  • the measurement of the emission intensity is performed in order for each case through each filter (step S9).
  • the luminescence detection result of the sample to be analyzed through each filter is obtained.
  • the transmittance for each filter calculated in step S4 is taken into consideration, whereby the light emission amount for each color emitted from the analysis target sample is calculated (step S10).
  • the amount of photoprotein in the sample to be analyzed can be calculated based on the relationship between the amount of photoprotein for each color and the amount of luminescence calculated in step S7. Since the amount of photoprotein is associated with the value related to the analysis target, the value related to the analysis target is calculated. As described above, the analysis target is measured based on the measurement of the emission intensity.
  • Example of measurement An application example of the above measurement method will be described.
  • a promoter assay using luciferase will be described as an example.
  • the activity of two types of promoters is measured.
  • two types of luciferases are used as reporters. That is, two types of expression vectors are introduced into one type of cell. These two types of expression vectors are referred to as a first vector and a second vector.
  • a luminescence microscope is used for the measurement. That is, luciferin is added to the cell culture medium into which the above-described expression vector has been introduced, and a microscopic image relating to luminescence generated at that time is obtained. In this microscope image, for example, a region of interest is set, and the luminance in the region of interest is analyzed to obtain the emission intensity.
  • FIG. 3A shows a configuration example of a part of the first vector
  • FIG. 3B shows a configuration example of a part of the second vector.
  • the first luciferase gene is arranged downstream of the first promoter, which is one of the promoters whose activity is to be measured.
  • a second luciferase gene is arranged downstream of a second promoter, which is another promoter whose activity is to be measured.
  • the first luciferase and the second luciferase are luciferases having different emission wavelengths.
  • MA-Luci2 related to green light emission can be used as the first luciferase
  • SfRE1 related to red light emission can be used as the second luciferase.
  • green light emission and red light emission are detected will be described as an example.
  • the following sample is manufactured as the first sample manufactured in step S1. That is, in order to prepare a cell that expresses the first luciferase alone, a third vector having the first luciferase gene is prepared. Similarly, in order to prepare a cell that expresses the second luciferase alone, a fourth vector having the gene for the second luciferase is prepared.
  • a configuration example of a part of the third vector is shown in FIG. 4A
  • a configuration example of a part of the fourth vector is shown in FIG. 4B.
  • a third promoter is arranged upstream of the first luciferase gene.
  • the third promoter is a promoter that can be expected to stably and highly express the first luciferase.
  • the third promoter is also arranged upstream of the second luciferase gene, as in the case of the third vector.
  • the third promoter for example, CAG promoter, CMV promoter, EF1a promoter, RSV promoter, SV40 promoter and the like can be used.
  • cells into which the third vector has been introduced and cells into which the fourth vector has been introduced are prepared.
  • green light is emitted from the cells into which the third vector has been introduced by the addition of luciferin
  • red light from the cells into which the fourth vector has been introduced by the addition of luciferin. Is emitted.
  • step S2 the luminescence intensity of luminescence related to the first luciferase in the cell into which the third vector has been introduced is measured without passing through the filter 21. That is, for example, without using the filter 21, a luminescent image related to the green luminescence of the first luciferase is acquired, and the luminance related to the luminescence of the image is analyzed.
  • the luminescence intensity of luminescence related to the first luciferase in the cell into which the third vector is introduced is measured through the green filter 21. That is, for example, a luminescent image related to green light emission related to the first luciferase is acquired via the green filter 21, and the luminance related to light emission of the image is analyzed.
  • the luminescence intensity of luminescence related to the first luciferase in the cell into which the third vector has been introduced is measured through the red filter 21. That is, for example, a light emission image related to green light emission related to the first luciferase is acquired via the red filter 21, and the luminance related to light emission of the image is analyzed.
  • the luminescence intensity of luminescence related to the second luciferase in the cell into which the fourth vector has been introduced is measured without passing through the filter 21. That is, for example, without passing through the filter 21, a luminescence image related to red light emission related to the second luciferase is acquired, and the luminance related to light emission of the image is analyzed.
  • the luminescence intensity of luminescence related to the second luciferase in the cell into which the fourth vector has been introduced is measured through the green filter 21. That is, for example, a luminescent image related to red light emission related to the second luciferase is acquired via the green filter 21, and the luminance related to light emission of the image is analyzed.
  • the luminescence intensity of luminescence related to the second luciferase in the cell into which the fourth vector has been introduced is measured through the red filter 21. That is, for example, a luminescence image related to red light emission related to the second luciferase is acquired via the red filter 21, and the luminance related to light emission of the image is analyzed.
  • step S4 with respect to the green light emission associated with the first luciferase, the transmittance with respect to the green filter and the transmittance with respect to the red filter are obtained. Similarly, with respect to the red light emission related to the second luciferase, the transmittance with respect to the green filter and the transmittance with respect to the red filter are obtained.
  • step S5 a sample in which the same amount of the first luciferase and the second luciferase are expressed is prepared. For this reason, the 5th vector which has the 1st luciferase and the 2nd luciferase is produced.
  • An example of the configuration of part of the fifth vector is shown in FIG.
  • the third promoter is used as in the third vector and the fourth vector. Downstream of the third promoter, a first luciferase gene and a second luciferase gene are arranged in a state of being bound by a sequence related to the 2A peptide.
  • the first luciferase and the second luciferase are continuously transcribed and translated, and the first luciferase and the second luciferase are expressed in equal amounts.
  • a second sample in which the fifth vector having such a configuration is introduced into the cell is prepared.
  • the luminescence intensity of luminescence related to the first luciferase and the second luciferase in the cell into which the fifth vector has been introduced is measured through the green filter 21. That is, for example, via the green filter 21, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired. Further, the luminescence intensity of luminescence related to the first luciferase and the second luciferase in the cell into which the fifth vector has been introduced is measured through the red filter 21. That is, for example, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired via the red filter 21.
  • step S7 based on the image obtained in step S6, the amount of luminescence when the same amount of the first luciferase and the second luciferase are expressed is calculated.
  • the transmittance obtained in step S4 is used. That is, the detection value obtained using the green filter is Sg, and the detection value obtained using the red filter is Sr. Further, the transmittance of the green light to the green filter obtained in step S4 is T gg , the transmittance of the green light to the red filter is T rg , the transmittance of the red light to the green filter is T gr , and the red light Let T rr be the transmittance of the red filter.
  • the green light emission amount is g and the red light emission amount is r
  • the ratio of the green light emission amount and the red light emission amount with respect to equal amounts of the first luciferase and the second luciferase is obtained as g: r.
  • step S8 a third sample to be analyzed is prepared. That is, a cell into which the first vector shown in FIG. 3A and the second vector shown in FIG. 3B have been introduced is prepared.
  • the luminescence intensity of the luminescence related to the first luciferase and the second luciferase in the cell into which the first vector and the second vector are introduced is measured through the green filter 21. That is, for example, via the green filter 21, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired.
  • the luminescence intensity of luminescence related to the first luciferase and the second luciferase in the cells into which the first vector and the second vector are introduced is measured through the red filter 21. That is, for example, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired via the red filter 21.
  • step S10 the green light emission amount g and the red light emission amount r are calculated using the above formulas (4) and (5).
  • step S11 the relationship between the luminescence amount obtained in step S7 and the substance amounts of the first luciferase and the second luciferase is used.
  • the amount of the first luciferase and the amount of the second luciferase in the cell are calculated from this relationship and the green light emission amount g and the red light emission amount r obtained in step S10.
  • This amount indicates the relationship between the activity of the first promoter and the activity of the second promoter.
  • the analysis on the activities of the first promoter and the second promoter is performed.
  • the relationship between the wavelength of luciferase and the emission intensity can be measured using, for example, purified protein.
  • An example of the relationship between the wavelength of luciferase and the luminescence intensity measured using the purified protein is shown in FIG. In FIG. 6, the solid line indicates the emission spectrum of MA-Luci2, and the broken line indicates the emission spectrum of SfRE1.
  • the emission spectrum in the cell shows different characteristics from those measured using a purified protein as shown in FIG.
  • This characteristic may vary depending on, for example, the environment inside and outside the cell, such as cell type, pH or temperature.
  • the relationship between the amount of protein and the amount of luminescence differs for each type.
  • the luminescence intensity for light caused by a certain luciferase A measured to measure the activity of a certain promoter A and the other luciferase B measured for measuring the activity of another promoter B Even if the luminescence intensity for the light to be obtained is obtained, the activity of promoter A and the activity of promoter B cannot generally be compared based on the result. For example, let us consider a case where the amount of luminescence caused by luciferase A is three times higher than the amount of luminescence caused by luciferase B. At this time, there is a possibility that the activity of promoter A is 3 times higher than that of promoter B.
  • the luminescence amount of luciferase A per unit protein amount may be three times the luminescence amount of luciferase B. In general, it is not possible to determine which of these possibilities is true or whether the two reductions are mixed.
  • the filter transmittance of each luciferase and the correction term for the intracellular luminescence amount of each luciferase are calculated. It becomes possible to calculate the expression level ratio of the promoter more accurately.
  • the ratio of the number of vectors introduced into each cell during reprogramming of iPS cells can be accurately calculated.
  • a vector including a transcription factor and having a configuration as shown in FIGS. 7A, 7B, and 7C is used for iPS reprogramming.
  • the ratio of the number of each introduced vector can be specified by measuring luminescence derived from luciferase introduced by each vector and expressed together with the transcription factor.
  • each filter is measured for the luminescence of cells expressing SfRE1 luciferase according to the vector shown in FIG. 7A. Similarly, the transmittance of each filter is measured for the luminescence of cells expressing Oki_mut1 luciferase according to the vector shown in FIG. 7B. Similarly, the transmittance of each filter is measured for the luminescence of cells expressing MA-Luci2 luciferase according to the vector shown in FIG. 7C.
  • an image using each filter is obtained for luminescence of cells in which SfRE1 luciferase, Oki_mut1 luciferase and MA-Luci2 luciferase are expressed in an equal amount using, for example, 2A peptide.
  • the above-mentioned filter transmittances, and the above equation (2) the amount of luminescence of each color is calculated, and each luciferase is expressed in an equal amount.
  • the light emission ratio can be calculated.
  • a luminescence image of a cell into which a vector having the structure shown in FIGS. 7A, 7B, and 7C is introduced is obtained, the detection value obtained from the image, each filter transmittance described above, and the above equation (2) Is used to calculate the light emission amount of each color in the cell.
  • the introduction number ratio of each vector shown in FIGS. 7A, 7B and 7C in the cell can be calculated.
  • transcription factors used for cell reprogramming include Oct3 / 4, Klf4, Sox2, c-myc, Lin28, and L-myc.
  • the technique of this embodiment when quantitatively analyzing the undifferentiation ability or differentiation state in the reprogramming stage for producing pluripotent cells such as iPS cells using a plurality of markers, luminescence having different wavelengths for each marker is used. Then, the technique of this embodiment can be used. In addition, when quantitatively analyzing the differentiation state using markers corresponding to various differentiation states in the differentiation stage of differentiating from pluripotent cells to various organs, etc., luminescence with different wavelengths is used for each marker. Then, the technique of this embodiment can be used.
  • the technology of the present embodiment can be used if light emission having a different wavelength is used for each tag. Can be. As a result, the ratio of the amounts of various proteins expressed in the cells can be accurately calculated.
  • Fluorescence may be used as a measurement method similar to the measurement according to the present embodiment.
  • the method of the present embodiment using light emission has the following advantages. That is, there is a fading phenomenon in fluorescence. That is, the measured light intensity changes due to the measurement. On the other hand, in light emission, there is no change in light intensity due to performing such measurement. Therefore, when using luminescence, the quantitativeness is higher than when using fluorescence.
  • the transmittance calculated in step S4 and the relationship between the expression level and the luminescence level calculated in step S7 are generally used when different cell types are used even when the same measurement system 1 is used. It needs to be acquired again. On the other hand, when the same cell type is used, the same value can be used.
  • the intracellular ratio of the vector used for iPS cell induction was calculated.
  • vector A a vector having the configuration shown in FIG. 7A
  • vector C a vector having the configuration shown in FIG. 7C
  • Vector A was prepared by digesting pCXLE-HOCT3 / 4-shp53 (Addgene) with Kpn I and Bgl II and incorporating SfRE1 luciferase downstream of hOCT3 / 4 via the 2A sequence.
  • Vector C was prepared by digesting pCXLE-hUL (Addgene) with KpnI and PacI and incorporating MA-Luci2 downstream of LIN28 via the 2A sequence.
  • PBMC Peripheral blood mononuclear cells
  • US CTL Peripheral blood mononuclear cells
  • PBMC medium AK02 medium (Ajinomoto) supplemented with IL3, IL6, SCF, TPO, Flt-3L, CSF.
  • the cell density was 2.5 ⁇ 10 6 cells / well, and culture was performed using a 24-well plate.
  • the PBMCs were seeded again in a 6-well plate coated with a coating agent (iMatrix (Nippi)).
  • the vector A and vectors pCE-mp53DD, pCE-hSK, pCE-hUL, and pCXB-EBNA1 were introduced into PBMC seeded in a 6-well plate using Amaxa (Lonza).
  • the vector C and each vector of pCE-hOCT3 / 4, pCE-mp53DD, pCE-hSK, and pCXB-EBNA1 were introduced into PBMC seeded in a 6-well plate using Amaxa (Lonza).
  • iPS cell culture medium (AK02 medium (Ajinomoto)
  • AK02 medium Ajinomoto
  • iPS cell culture medium AK02 medium (Ajinomoto)
  • vector A a vector having the configuration shown in FIG. 8
  • vector MA-Luci2-SfRE1 having the configuration shown in FIG. 7C
  • the vector MA-Luci2-SfRE1 was prepared by digesting pCXLE-hUL (Addgene) with EcoRI and incorporating a sequence in which SfRE1 luciferase was ligated downstream of MA-Luci2 luciferase via a 2A sequence.
  • PBMC Peripheral blood mononuclear cells
  • US CTL Peripheral blood mononuclear cells
  • PBMC medium AK02 medium (Ajinomoto) supplemented with IL3, IL6, SCF, TPO, Flt-3L, CSF.
  • the cell density was 2.5 ⁇ 10 6 cells / well, and culture was performed using a 24-well plate.
  • the PBMCs were seeded again in a 6-well plate coated with a coating agent (iMatrix (Nippi)).
  • the vector MA-Luci2-SfRE1 (Fig. 8) and pCE-hOCT3 / 4, pCE-mp53DD, pCE-hSK, pCE-hUL, and pCXB-EBNA1 using Amaxa (Lonza) on PBMC seeded in a 6-well plate Each vector was introduced.
  • iPS cell culture medium (AK02 medium (Ajinomoto)
  • AK02 medium Ajinomoto
  • all the medium was aspirated, and 2 ml of iPS cell medium was added. Thereafter, the medium was replaced every other day with 2 ml of iPS cell medium.
  • D-luciferin Promega was added to the medium to a final concentration of 1 mM, and luminescence imaging of iPS cell-like colonies was performed using a luminescence microscope system LV200 (Olympus).
  • T 2r is the transmittance of the light emission related to SfRE1 with respect to filter 2.
  • T 1g , T 2g , T 1r , and T 2r values obtained by measurement using the above-described monochromatic light emitting sample were used.
  • the light emission amount of MA-Luci2 is g_ratio
  • the light emission amount of SfRE1 is r_ratio. At this time, the following formula (6) holds.
  • PBMC peripheral blood mononuclear cell PBMC
  • PBMC medium AK02 medium (Ajinomoto) supplemented with IL3, IL6, SCF, TPO, Flt-3L, CSF
  • the cell density was 2.5 ⁇ 10 6 cells / well, and culture was performed using a 24-well plate.
  • the PBMCs were seeded again in a 6-well plate coated with a coating agent (iMatrix (Nippi)).
  • Vector A (FIG. 7A) and vector C (FIG. 7C), and pCE-mp53DD, pCE-hSK, and pCXB-EBNA1 (Addgene) were introduced into PBMC seeded in a 6-well plate using Amaxa (Lonza).
  • iPS cell culture medium (AK02 medium (Ajinomoto)
  • AK02 medium Ajinomoto
  • all the medium was aspirated, and 2 ml of iPS cell medium was added. Thereafter, the medium was replaced every other day with 2 ml of iPS cell medium.
  • D-luciferin Promega was added to the medium to a final concentration of 1 mM, and luminescence imaging of iPS cell-like colonies was performed using a luminescence microscope system LV200 (Olympus).
  • T 1g , T 2g , T 1r , and T 2r values obtained by measurement using the above-described monochromatic light-emitting sample were used.
  • the light emission amount of MA-Luci2 is g and the light emission amount of SfRE1 is r, the following equation (9) is established.
  • FIGS. 9A to 9C show the emission imaging images obtained for the analysis target sample.
  • 9A is a luminescence imaging image when no filter is used
  • FIG. 9B is a luminescence imaging image when filter 1 is used
  • FIG. 9C is a luminescence imaging image when filter 2 is used.
  • G and r were calculated using the detected values in each region of interest (ROI) shown in FIGS. 9A to 9C and the above equations (10) and (11). The obtained results are shown in the graph of FIG.
  • the corrected emission intensity ratio (g / r; MA-Luci2 / Sf-RE1) shown in FIG. 11 represents the quantitative ratio of vector introduced into each cell (vector C / vector A).
  • vector C the quantitative ratio of vector introduced into each cell
  • the method according to the present embodiment can provide a measurement method capable of highly quantitative analysis in a sample in which light having different wavelength characteristics is simultaneously emitted.

Abstract

This measurement method includes the calculation of filter transmittances and the calculation of ratios between photoprotein amounts and light emission amounts. Filter transmittances are determined using a plurality of first samples independently having photoproteins of N colors. The ratios between the amount of photoprotein and the amount of emitted light are determined using a second sample prepared such that the amounts of each of the photoproteins of N colors are known. The amounts of light emission for each color are determined on the basis of the light emission intensities measured when light from a third sample that is the object of measurement and is such that the photoproteins of N colors are mixed and emit mixed light passes through each of the filters, and the filter transmittances. Values for the amounts of the photoproteins of each color in the third sample are calculated on the basis of the obtained ratios and light emission amounts for each light emission color.

Description

測定方法Measuring method
　本発明は、測定方法に関する。 The present invention relates to a measurement method.
　細胞に発光タンパク質を発現させ、当該発光タンパク質による生物発光を検出することが行われている。このような発光の検出は、注目する遺伝子又はタンパク質の発現の状態の解析等に利用されている。また、発光タンパク質を発現させた細胞を撮影し、発光画像を取得することで、細胞内の分子動態を可視化することも行われている。このような発光画像に基づいても、注目する遺伝子又はタンパク質の発現の状態の解析等が行われ得る。 A photoprotein is expressed in a cell and bioluminescence generated by the photoprotein is detected. Such detection of luminescence is used for analysis of the expression state of a gene or protein of interest. Moreover, visualization of intracellular molecular dynamics is also performed by photographing a cell in which a photoprotein is expressed and acquiring a luminescent image. Even based on such luminescent images, analysis of the expression state of the gene or protein of interest can be performed.
　上述のような発光を利用した解析において、波長が異なる光を同時に定量的に検出することができれば、得られる情報量が多くなり、種々の解析が可能となる。試料から放射される光の波長にはある程度の幅があるので、異なる発光タンパク質に由来する光であっても、波長が互いに重なり合うことがある。例えば日本国特開２００４－３３３４５７号公報には、同時に生じている互いに波長特性が異なるＮ色の光について、Ｎ－１個のフィルターを用いることで、色毎に発光量を求めることに係る技術が開示されている。この技術を用いれば、Ｎ色の光について各フィルターの透過特性を求め、行列演算をすることで、各フィルターが適用されたときに得られた光強度に基づいて、色毎の発光量を求められ得る。 In the analysis using light emission as described above, if light having different wavelengths can be detected simultaneously and quantitatively, the amount of information obtained increases, and various analyzes are possible. Since the wavelength of the light emitted from the sample has a certain range, the wavelengths may overlap each other even if the light originates from different photoproteins. For example, Japanese Patent Application Laid-Open No. 2004-333457 discloses a technique for obtaining a light emission amount for each color by using N-1 filters for N light having different wavelength characteristics that are generated simultaneously. Is disclosed. Using this technology, the transmission characteristics of each filter are obtained for N-color light, and a matrix operation is performed to obtain the light emission amount for each color based on the light intensity obtained when each filter is applied. Can be.
　一方で、細胞等の状態（例えばｐＨ、温度等の細胞内外の環境等）が変化すれば、同じ発光タンパク質を用いても発光特性が変化することがある。このような変化は、解析の定量性を低下させることにつながる。上述の技術では、細胞等の状態が安定しており、発光が安定していることが前提となっている。 On the other hand, if the state of the cells (for example, the environment inside and outside the cells such as pH, temperature, etc.) changes, the luminescent properties may change even if the same photoprotein is used. Such a change leads to a decrease in the quantitativeness of the analysis. The above-described technique is based on the premise that the state of cells and the like is stable and light emission is stable.
　本発明は、異なる波長特性を有する光が同時に放射される試料において、定量性の高い解析が可能な測定方法を提供することを目的とする。 An object of the present invention is to provide a measurement method capable of highly quantitative analysis in a sample in which light having different wavelength characteristics is simultaneously emitted.
　本発明の一態様によれば、測定方法は、Ｎ色の発光タンパク質が混在し各色の発光が混在する測定対象の試料について、Ｎ個又はＮ－１個のフィルターを利用して各色の発光タンパク質の量に係る値を取得する測定方法であって、前記Ｎ色の発光タンパク質の各々が独立して存在する複数の第１の試料を用いて、各色の発光について前記フィルターの何れをも透過しない状態で測定される発光強度と各々の前記フィルターを透過したときに測定される発光強度とに基づいて、各色の発光についての各々の前記フィルターの透過率を算出することと、前記Ｎ色の発光タンパク質のそれぞれの量が既知となるように調製した第２の試料についての各々の前記フィルターを透過したときに測定される発光強度と、前記フィルターの透過率とに基づいて、前記発光タンパク質の量と発光量との比を算出することと、Ｎ色の発光タンパク質が混在し各色の発光が混在する前記測定対象である第３の試料についての各々の前記フィルターを透過したときに測定される発光強度と、各々の前記フィルターの透過率とに基づいて、各色の発光についての発光量を求めることと、得られた前記各色の発光についての発光量と前記比とに基づいて、前記第３の試料における前記各色の発光タンパク質の量に係る値を算出することとを含む。 According to one aspect of the present invention, a measurement method includes using N or N-1 filters for a sample to be measured in which N photoproteins are mixed and luminescence of each color is mixed. A method for obtaining a value related to the amount of luminescence, wherein a plurality of first samples in which each of the N-color photoproteins is independently present are used, and none of the filters are transmitted for luminescence of each color. Calculating the transmittance of each of the filters for each color emission based on the emission intensity measured in the state and the emission intensity measured when passing through each of the filters; Based on the emission intensity measured when passing through each of the filters for the second sample prepared such that the respective amounts of protein are known, and the transmittance of the filters. And calculating the ratio between the amount of the photoprotein and the amount of luminescence, and transmitting each of the filters for the third sample as the measurement object in which N-color photoproteins are mixed and luminescence of each color is mixed. The light emission amount for each color light emission is obtained based on the light emission intensity measured at that time and the transmittance of each filter, and the light emission amount and the ratio for the obtained light emission of each color are And calculating a value relating to the amount of the photoprotein of each color in the third sample.
　本発明によれば、異なる波長特性を有する光が同時に放射される試料において、定量性の高い解析が可能な測定方法を提供できる。 According to the present invention, it is possible to provide a measurement method capable of analyzing with high quantitativeness in a sample in which light having different wavelength characteristics is simultaneously emitted.
図１は、本発明の一実施形態で用いられる測定システムの一例の概略を示す図である。FIG. 1 is a diagram showing an outline of an example of a measurement system used in an embodiment of the present invention. 図２は、一実施形態に係る測定方法の一例の概略を示すフローチャートである。FIG. 2 is a flowchart illustrating an outline of an example of a measurement method according to an embodiment. 図３Ａは、測定の一例に用いられるベクターの一部の構成例の概略を示す図である。FIG. 3A is a diagram showing an outline of a partial configuration example of a vector used for an example of measurement. 図３Ｂは、測定の一例に用いられるベクターの一部の構成例の概略を示す図である。FIG. 3B is a diagram showing an outline of a configuration example of a part of a vector used for an example of measurement. 図４Ａは、単色の発光を示す試料の調製の一例に用いられるベクターの一部の構成例の概略を示す図である。FIG. 4A is a diagram showing an outline of a partial configuration example of a vector used in an example of preparing a sample exhibiting monochromatic light emission. 図４Ｂは、単色の発光を示す試料の調製の一例に用いられるベクターの一部の構成例の概略を示す図である。FIG. 4B is a diagram showing an outline of a part of a configuration example of a vector used in an example of preparing a sample exhibiting monochromatic light emission. 図５は、互いに波長が異なる発光を起こす複数種類のルシフェラーゼが同一の細胞内で互いに等量発現する試料の調製の一例に用いられるベクターの一部の構成例の概略を示す図である。FIG. 5 is a diagram showing an outline of a partial configuration example of a vector used in an example of preparing a sample in which plural types of luciferases that emit light having different wavelengths from each other are expressed in the same cell in the same amount. 図６は、波長と発光強度との関係について説明するための図である。FIG. 6 is a diagram for explaining the relationship between the wavelength and the emission intensity. 図７Ａは、ｉＰＳ細胞の作製に用いられるベクターの構成例の概略を示す図である。FIG. 7A is a diagram showing an outline of a configuration example of a vector used for production of iPS cells. 図７Ｂは、ｉＰＳ細胞の作製に用いられるベクターの構成例の概略を示す図である。FIG. 7B is a diagram showing an outline of a configuration example of a vector used for production of iPS cells. 図７Ｃは、ｉＰＳ細胞の作製に用いられるベクターの構成例の概略を示す図である。FIG. 7C is a diagram showing an outline of a configuration example of a vector used for production of iPS cells. 図８は、実施例でＭＡ－Ｌｕｃｉ２ルシフェラーゼとＳｆＲＥ１ルシフェラーゼとが同一の細胞内で互いに等量発現する試料の調製に用いたベクターの一部の構成の概略を示す図である。FIG. 8 is a diagram showing an outline of a part of the structure of a vector used for preparing a sample in which MA-Luci2 luciferase and SfRE1 luciferase are expressed in equal amounts in the same cell in the examples. 図９Ａは、実施例に係る、フィルターを用いなかった場合の解析対象試料の発光イメージング像である。FIG. 9A is a luminescence imaging image of the sample to be analyzed when no filter is used according to the example. 図９Ｂは、実施例に係る、filter 1を用いた場合の解析対象試料の発光イメージング像である。FIG. 9B is a luminescence imaging image of the sample to be analyzed when filter 1 is used according to the example. 図９Ｃは、実施例に係る、filter 2を用いた場合の解析対象試料の発光イメージング像である。FIG. 9C is a luminescence imaging image of the sample to be analyzed when filter 2 is used according to the example. 図１０は、実施例に係る、解析対象試料の発光イメージング像の各関心領域（ＲＯＩ）について得られた、ＭＡ－Ｌｕｃｉ２ルシフェラーゼとＳｆＲＥ１ルシフェラーゼとに係る発光強度を示す図である。FIG. 10 is a diagram showing the luminescence intensity related to MA-Luci2 luciferase and SfRE1 luciferase obtained for each region of interest (ROI) of the luminescence imaging image of the sample to be analyzed according to the example. 図１１は、実施例に係る、解析結果として得られた各関心領域（ＲＯＩ）に係るベクターＣに対するベクターＡの比であり、ＭＡ－Ｌｕｃｉ２ルシフェラーゼとＳｆＲＥ１ルシフェラーゼとの発光特性による補正を行う前後の結果を示す図である。FIG. 11 is a ratio of the vector A to the vector C related to each region of interest (ROI) obtained as an analysis result according to the example, before and after performing correction based on the luminescence characteristics of MA-Luci2 luciferase and SfRE1 luciferase It is a figure which shows a result.
　本発明の一実施形態について図面を参照して説明する。本実施形態に係る測定方法は、測定対象が複数ある場合に用いられる。この測定方法では、測定対象毎に波長が異なる光が関連付けられており、測定対象の変化に応じて対応する波長の光の発光量が変化するように、試料が調製されている。より具体的には、測定対象と関連付けられた発光タンパク質が用いられており、測定対象の変化に応じて発光タンパク質の量が変化する。本測定方法では、発光強度が計測されることで、測定対象の特性が解析される。 An embodiment of the present invention will be described with reference to the drawings. The measurement method according to the present embodiment is used when there are a plurality of measurement objects. In this measurement method, light having a different wavelength is associated with each measurement target, and the sample is prepared so that the light emission amount of the corresponding wavelength changes according to the change of the measurement target. More specifically, a photoprotein associated with the measurement target is used, and the amount of the photoprotein changes according to the change of the measurement target. In this measurement method, the characteristics of the measurement object are analyzed by measuring the emission intensity.
　この測定方法では、異なる波長を含む光は、同時に同一試料から放射される。そこで、波長毎に光を分離するために、フィルターが用いられる。例えば、Ｎ色の光が混在する場合には、Ｎ個又はＮ－１個のフィルターが用いられる。Ｎ－１個のフィルターが用いられれば、フィルターが用いられない場合と、各々のフィルターが用いられた場合とを合わせてＮ通りの発光強度が取得され得る。したがって、これらの結果に基づけば、Ｎ色の色毎に発光強度を求めることができる。ただし、一般的には、Ｎ色の光の各々を主に透過させる特性を有するＮ個のフィルターが用いられることが好ましい。なお、Ｎが４以下、すなわち４色以下であれば、光の分離は比較的容易である。 In this measurement method, light having different wavelengths is simultaneously emitted from the same sample. Therefore, a filter is used to separate light for each wavelength. For example, when N colors of light are mixed, N or N-1 filters are used. If N-1 filters are used, N different emission intensities can be acquired for the case where no filter is used and the case where each filter is used. Therefore, based on these results, the emission intensity can be obtained for each of the N colors. However, in general, it is preferable to use N filters having a characteristic of mainly transmitting each of N colors of light. If N is 4 or less, that is, 4 colors or less, light separation is relatively easy.
　［測定システムの構成］
　本実施形態で用いられる測定システム１の構成例の概略を図１に示す。図１に示すように、測定システム１は、測定装置１０と制御装置４０とを備える。測定装置１０は、例えば撮像素子を備えた顕微鏡、光電子増倍管を備えたルミノメーター等といった、試料の発光の強度を定量できる装置である。顕微鏡には、発光イメージングシステムＬＶ２００（オリンパス株式会社）が含まれ得る。制御装置４０は、例えばパーソナルコンピュータ（ＰＣ）等の測定装置１０の動作を制御したり、測定装置１０で得られたデータの解析を行ったりする装置である。 [Configuration of measurement system]
An outline of a configuration example of the measurement system 1 used in the present embodiment is shown in FIG. As shown in FIG. 1, the measurement system 1 includes a measurement device 10 and a control device 40. The measuring apparatus 10 is an apparatus that can quantify the intensity of light emission of a sample, such as a microscope equipped with an image sensor, a luminometer equipped with a photomultiplier tube, and the like. The microscope may include a luminescence imaging system LV200 (Olympus Corporation). The control device 40 is a device that controls the operation of the measurement device 10 such as a personal computer (PC) or analyzes data obtained by the measurement device 10.
　例えば、測定装置１０は、暗箱１９を備える。測定装置１０は、暗箱１９の中に配置された試料９０の発光を検出する。測定装置１０は、例えば、対物レンズ１１と結像レンズ１２と検出器１３とフィルターユニット２０とを有する。検出器１３は、試料９０から放射された光を対物レンズ１１及び結像レンズ１２を介して検出する。検出器１３は、ＣＣＤセンサーを含む撮像素子等であってもよいし、光電子増倍管等であってもよい。 For example, the measuring apparatus 10 includes a dark box 19. The measuring device 10 detects the light emission of the sample 90 arranged in the dark box 19. The measuring device 10 includes, for example, an objective lens 11, an imaging lens 12, a detector 13, and a filter unit 20. The detector 13 detects light emitted from the sample 90 via the objective lens 11 and the imaging lens 12. The detector 13 may be an imaging device including a CCD sensor, a photomultiplier tube, or the like.
　フィルターユニット２０は、互いに透過特性が異なる複数種類のフィルター２１とフィルター変更機構２２とを有する。フィルターユニット２０は、試料９０から検出器１３へと到達する光路に複数のフィルター２１のうち何れか１つを挿入したり、何れのフィルター２１も挿入しなかったりする。 The filter unit 20 includes a plurality of types of filters 21 and filter changing mechanisms 22 having different transmission characteristics. The filter unit 20 inserts any one of the plurality of filters 21 in the optical path that reaches the detector 13 from the sample 90 or does not insert any filter 21.
　フィルターが切替えられず、例えば検出器１３にカラーＣＣＤカメラが用いられてもよい。カラーＣＣＤカメラでは、画素毎に赤色、緑色又は青色のカラーフィルターが設けられているので、赤色、緑色、青色のデータが同時に取得されることになる。 For example, a color CCD camera may be used as the detector 13 without switching the filter. In a color CCD camera, since a red, green or blue color filter is provided for each pixel, red, green and blue data are acquired simultaneously.
　制御装置４０は、フィルター変更機構２２を動作させてフィルター２１を切替えたり、検出器１３からデータを取得したりする。また、制御装置４０は、取得したデータの解析を行う。制御装置４０は、例えばプロセッサー４１と、random access memory（ＲＡＭ）４２と、記録装置４３と、入力装置４４と、表示装置４５とを備える。 The control device 40 operates the filter changing mechanism 22 to switch the filter 21 and acquire data from the detector 13. The control device 40 analyzes the acquired data. The control device 40 includes, for example, a processor 41, a random access memory (RAM) 42, a recording device 43, an input device 44, and a display device 45.
　プロセッサー４１は、測定システム１の動作に係る各種演算を行う。プロセッサー４１は、例えば、Central Processing Unit（ＣＰＵ）、Application Specific Integrated Circuit（ＡＳＩＣ）、Field Programmable Gate Array（ＦＰＧＡ）、又はGraphics Processing Unit（ＧＰＵ）等を含み得る。プロセッサー４１は、１つの集積回路等で構成されてもよいし、複数の集積回路等が組み合わされて構成されてもよい。ＲＡＭ４２は、プロセッサー４１の主記憶装置として機能する。記録装置４３は、例えば半導体メモリ、ハードディスク等である。記録装置４３には、プロセッサー４１で用いられるプログラム、パラメーター等が記録され得る。また、記録装置４３には、測定装置１０を用いて得られたデータ、それに基づく解析結果等が記録され得る。入力装置４４は、例えばキーボート、マウス、タッチパネル等を含み得る。表示装置４５は、例えば液晶ディスプレイ等を含み得る。 The processor 41 performs various calculations related to the operation of the measurement system 1. The processor 41 may include, for example, a Central Processing Unit (CPU), an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA), or a Graphics Processing Unit (GPU). The processor 41 may be configured by one integrated circuit or the like, or may be configured by combining a plurality of integrated circuits. The RAM 42 functions as a main storage device for the processor 41. The recording device 43 is, for example, a semiconductor memory or a hard disk. The recording device 43 can record programs, parameters, and the like used by the processor 41. The recording device 43 can record data obtained by using the measuring device 10, analysis results based on the data, and the like. The input device 44 can include, for example, a keyboard, a mouse, a touch panel, and the like. The display device 45 can include, for example, a liquid crystal display.
　［測定方法］
　一般に測定系の条件が異なると、発光タンパク質の量に対する発光強度の検出結果も異なり得る。そこで、本実施形態に係る測定方法では、測定系毎に発光タンパク質の量と発光強度の検出結果との関係を求める処理が行われる。本測定方法は、大きくは３つの処理を含んでいる。すなわち、第１の処理は、試料の発光色毎に、また、用いるフィルター２１毎に、試料から放射された光がフィルター２１を透過する透過率を求める処理である。第２の処理は、異なる色の発光に係る発光タンパク質が互いに等量存在するように調製された試料を用いて、発光タンパク質の量と発光量との関係を求める処理である。第３の処理は、解析対象についての測定である。解析対象の発光強度が測定され、上述のフィルター２１の透過率と発光タンパク質の量に対する発光量の関係とを考慮して、被測定物についての値が算出される。 [Measuring method]
In general, when the conditions of the measurement system are different, the detection result of the luminescence intensity with respect to the amount of photoprotein can also be different. Therefore, in the measurement method according to the present embodiment, processing for obtaining the relationship between the amount of photoprotein and the detection result of the luminescence intensity is performed for each measurement system. This measurement method mainly includes three processes. In other words, the first process is a process for obtaining the transmittance at which the light emitted from the sample passes through the filter 21 for each emission color of the sample and for each filter 21 to be used. The second process is a process for determining the relationship between the amount of photoprotein and the amount of luminescence using samples prepared so that photoproteins related to luminescence of different colors exist in equal amounts. The third process is a measurement for the analysis target. The light emission intensity to be analyzed is measured, and the value for the object to be measured is calculated in consideration of the transmittance of the filter 21 and the relationship of the light emission amount to the amount of the photoprotein.
　本実施形態に係る測定方法の概略を図２のフローチャートに示す。第１の処理、すなわちフィルターの透過率を求める処理が、図２に示すステップＳ１乃至ステップＳ４の処理である。この処理について説明する。ここで透過率が求められるフィルターは、第３の処理、すなわち解析対象の測定に用いられるフィルターである。 The outline of the measurement method according to this embodiment is shown in the flowchart of FIG. The first process, that is, the process for obtaining the transmittance of the filter is the process of steps S1 to S4 shown in FIG. This process will be described. Here, the filter whose transmittance is required is a filter used for the third process, that is, the measurement of the analysis target.
　発光色毎に以下の処理が行われる。ここで用いられる試料は、単色の光が放射される試料である。この単色の光を放射する試料が第１の試料として調製される（ステップＳ１）。ここで用いられる発光は、第３の処理、すなわち解析対象の測定に用いられる発光である。 The following processing is performed for each emission color. The sample used here is a sample to which monochromatic light is emitted. A sample that emits this monochromatic light is prepared as a first sample (step S1). The luminescence used here is luminescence used for the third process, that is, measurement of the analysis target.
　発光は、発光タンパク質による生物発光に係るものである。生物発光には、基質であるルシフェリンと発光タンパク質としての酵素であるルシフェラーゼとが用いられ得る。このルシフェリン－ルシフェラーゼの系としては、ホタルルシフェリン、バクテリアルシフェリン、渦鞭毛藻類ルシフェリン、ヴァルグリン、セレンテラジン等、何れの系が用いられてもよい。第３の処理、すなわち、解析対象の測定には、波長が異なる複数種類の光が用いられるので、発光波長が互いに異なる複数種類のルシフェラーゼが用いられることになる。ルシフェラーゼとしては、例えば、Ｐ．ｐｙｒａｌｉｓ、クリックビートル、ＭＡ－Ｌｕｃｉ２、ＳｆＲＥ１、Ｏｋｉ＿ｍｕｔ１等を含む甲虫ルシフェラーゼ、ウミシイタケルシフェラーゼ、ウミホタルルシフェラーゼ、エクオリン、カイアシルシフェラーゼ、発光エビルシフェラーゼ等を含む海洋性ルシフェラーゼ、バクテリアルシフェラーゼ、渦鞭毛藻ルシフェラーゼなど、各種のルシフェラーゼが用いられ得る。 Luminescence is related to bioluminescence by photoproteins. For bioluminescence, luciferin as a substrate and luciferase as an enzyme as a photoprotein can be used. As this luciferin-luciferase system, any system such as firefly luciferin, bacterial luciferin, dinoflagellate luciferin, vargulin, coelenterazine and the like may be used. Since a plurality of types of light having different wavelengths are used for the third process, that is, measurement of the analysis target, a plurality of types of luciferases having different emission wavelengths are used. Examples of luciferase include P.I. Pyralis, Crick beetle, MA-Luci2, SfRE1, Oki_mut1, etc. Beetle luciferase, Renilla luciferase, Cypridina luciferase, Aequorin, Chiacyl luciferase, Luminous luciferase, Luminous luciferase, Dinoflagellate, etc. Of luciferase can be used.
　ステップＳ１で調製される試料は、例えばルシフェラーゼの遺伝子を含む発現ベクターが導入された細胞等である。発光色毎に試料が調製されるので、１つの試料において、発光波長が異なる複数種類のルシフェラーゼのうち１種類のルシフェラーゼの遺伝子が細胞等に導入される。このような１種類のルシフェラーゼの遺伝子を持つ細胞に係る試料が、ルシフェラーゼの種類数だけ調製される。 The sample prepared in step S1 is, for example, a cell into which an expression vector containing a luciferase gene has been introduced. Since a sample is prepared for each luminescent color, one type of luciferase gene among a plurality of types of luciferases having different emission wavelengths is introduced into a cell or the like in one sample. Samples relating to cells having such one type of luciferase gene are prepared for the number of types of luciferase.
　細胞等には、セルライン化された培養細胞、単離された細胞、幹細胞等の各種細胞が含まれ得る。幹細胞には、ｉＰＳ細胞又はＥＳ細胞等が含まれ得る。また、細胞等は、哺乳類由来の細胞であってもよいし、昆虫、菌等、その他の生物に由来する細胞であってもよい。また、細胞等には、単離された組織等が含まれてもよい。細胞の形態についても種々の形態が取られ得る。コロニーが形成されていてもよいし、胚様体等の細胞塊、スフェロイド等が形成されていてもよい。 The cells and the like can include various cells such as cell-lined cultured cells, isolated cells, and stem cells. Stem cells can include iPS cells or ES cells. Further, the cell or the like may be a cell derived from a mammal, or may be a cell derived from another organism such as an insect or a fungus. Moreover, the isolated tissue etc. may be contained in the cell etc. Various forms of the cell form can be taken. Colonies may be formed, cell clusters such as embryoid bodies, spheroids, and the like may be formed.
　ステップＳ１で調製される試料は、例えば、解析対象の測定に用いられる細胞等に、解析対象の測定に用いられるルシフェラーゼの遺伝子等が１種類ずつ導入されたものとなる。例えば導入されたルシフェラーゼが発現している状態で培地にルシフェリンが添加され、発光が生じる試料が調製される。 The sample prepared in step S1 is obtained by introducing, for example, one type of luciferase gene or the like used for measurement of the analysis target into a cell or the like used for measurement of the analysis target. For example, in a state where the introduced luciferase is expressed, luciferin is added to the medium to prepare a sample that generates luminescence.
　ステップＳ１で調製された試料の各々について、まず、フィルター２１を光路に挿入していない状態で発光強度が測定される（ステップＳ２）。続いて同一の試料について、フィルター２１を介して発光強度が測定される。フィルター２１を介した発光強度の測定は、用いる全てのフィルターについて順に行われる（ステップＳ３）。 For each sample prepared in step S1, the emission intensity is first measured without the filter 21 being inserted into the optical path (step S2). Subsequently, the emission intensity of the same sample is measured through the filter 21. The measurement of the emission intensity through the filter 21 is performed in order for all the filters used (step S3).
　以上により、各色の発光について、光路にフィルターを挿入していないときと光路に各々のフィルターを挿入したときの発光強度の検出結果が得られる。光路にフィルターを挿入していないときに検出された発光強度と挿入したときに検出された発光強度との比に基づいて、発光色毎に、フィルター毎の透過率が算出される（ステップＳ４）。 As described above, for the emission of each color, the detection result of the emission intensity when the filter is not inserted in the optical path and when each filter is inserted in the optical path can be obtained. Based on the ratio between the emission intensity detected when no filter is inserted in the optical path and the emission intensity detected when the filter is inserted, the transmittance for each filter is calculated for each emission color (step S4). .
　続いて行われる２つ目の発光タンパク質の量と発光量との関係を求める処理が、図２に示すステップＳ５乃至ステップＳ７の処理である。この処理について説明する。ここで用いられる試料は、各々の波長の発光に係る発光タンパク質が互いに等量含まれている試料である。このような多色等量発光試料が第２の試料として調製される（ステップＳ５）。 The subsequent processing for obtaining the relationship between the amount of the second photoprotein and the amount of luminescence is the processing of steps S5 to S7 shown in FIG. This process will be described. The sample used here is a sample in which equal amounts of photoproteins related to light emission at each wavelength are contained. Such a multicolor equivalent light emission sample is prepared as a second sample (step S5).
　ステップＳ５では、例えば互いに波長が異なる発光を起こす複数種類のルシフェラーゼが同一の細胞内で互いに等量発現する試料が調製される。複数種類のルシフェラーゼが細胞内で等量発現するように、例えば複数種類のルシフェラーゼの遺伝子がポリシストロニックに連結される。例えば、細胞に導入される発現ベクターにおいて、複数種類のルシフェラーゼの遺伝子は、２Ａペプチドに係る配列によって連結され得る。また、細胞に導入される複数種類のルシフェラーゼの遺伝子は、ＩＲＥＳ配列によって連結され得る。なお、切断効率が高いため、２Ａペプチドが利用されることが好ましい。また、複数種類のルシフェラーゼがリンカーを介して連結して発現するように、複数種類のルシフェラーゼの遺伝子が構成されてもよい。 In step S5, for example, a sample is prepared in which a plurality of types of luciferases that emit light having different wavelengths are expressed in equal amounts in the same cell. For example, a plurality of types of luciferase genes are linked in a polycistronic manner so that a plurality of types of luciferases are expressed in equal amounts in a cell. For example, in an expression vector introduced into a cell, multiple types of luciferase genes can be linked by a sequence related to the 2A peptide. In addition, multiple types of luciferase genes introduced into cells can be linked by an IRES sequence. In addition, since cutting efficiency is high, it is preferable that 2A peptide is utilized. Further, a plurality of types of luciferase genes may be configured such that a plurality of types of luciferases are linked and expressed via a linker.
　ステップＳ５で調製された試料について、フィルター２１を介して発光強度が測定される。発光強度の測定は、各々のフィルターを介した場合について順に行われる（ステップＳ６）。このようにして、各フィルターを介した多色等量発光試料の発光検出結果が得られる。ここで得られた検出結果について、ステップＳ４で算出されたフィルターごとの透過率が考慮されることで、発光に係る物質が互いに等量含まれている場合の各色の発光量が算出される（ステップＳ７）。すなわち、色毎に、発光タンパク質の量と発光量との関係が取得される。 The emission intensity of the sample prepared in step S5 is measured through the filter 21. The measurement of the light emission intensity is performed in order for each case through each filter (step S6). In this way, the light emission detection result of the multicolor equivalent light emission sample through each filter is obtained. With respect to the detection result obtained here, by considering the transmittance for each filter calculated in step S4, the light emission amount of each color when the substances related to light emission are contained in equal amounts is calculated ( Step S7). That is, the relationship between the amount of photoprotein and the amount of luminescence is acquired for each color.
　波長毎の発光量は、例えば行列演算によって算出され得る。すなわち、Ｎ色のそれぞれの発光量をＩ1、Ｉ2、…ＩNとする。Ｎ個のフィルターのうち１個目のフィルターについて、Ｎ色の発光のそれぞれの透過率をＴ11、Ｔ12、…Ｔ1Nとし、Ｎ個目のフィルターについて、Ｎ色の発光のそれぞれの透過率をＴN1、ＴN2、…ＴNNとする。Ｎ個のフィルターのそれぞれが光路に挿入されたときに検出されるそれぞれの検出値をＳ1、Ｓ2、…ＳNとする。このとき、下記式が成り立つ。 The light emission amount for each wavelength can be calculated, for example, by matrix calculation. That is, each of the light emission amount of the N-color I 1, I 2, ... and I N. Of the N filters, the transmittance of each of the N light emission is T 11 , T 12 ,... T 1N for the first filter, and the transmittance of each of the N light emission is for the Nth filter. Are defined as T N1 , T N2 ,. Each of the N filters S 1, S 2 each detected value detected when inserted into the optical path, ... and S N. At this time, the following equation holds.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
したがって、下記式によりＮ色のそれぞれの発光量が算出され得る。 Therefore, the light emission amounts of the N colors can be calculated by the following formula.
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-M000002
　なお、ここでは、複数種類のルシフェラーゼが同一の細胞内で互いに等量発現する例を挙げたがこれに限らない。発光タンパク質の量は等量でなくても量比が既知であれば同様に発光量と発光タンパク質の量との関係が取得される。例えば、ルシフェラーゼＡとルシフェラーゼＢとが用いられるとき、２つのルシフェラーゼＡの遺伝子と１つのルシフェラーゼＢの遺伝子とが２Ａペプチドで連結され、例えば、ルシフェラーゼＡ－２Ａ－ルシフェラーゼＡ－２Ａ－ルシフェラーゼＢといった配列が構築され得る。この場合、ルシフェラーゼＡとルシフェラーゼＢとの発現量比が２：１となる。 In addition, although an example in which multiple types of luciferases are expressed in the same amount in the same cell is given here, the present invention is not limited thereto. Even if the amount of photoprotein is not equal, if the quantitative ratio is known, the relationship between the amount of photoprotein and the amount of photoprotein is obtained. For example, when luciferase A and luciferase B are used, two luciferase A genes and one luciferase B gene are linked by a 2A peptide, for example, a sequence such as luciferase A-2A-luciferase A-2A-luciferase B Can be built. In this case, the expression level ratio between luciferase A and luciferase B is 2: 1.
　また、３種類以上のルシフェラーゼ等が用いられるときには、それら全てが同一の細胞内に発現していなくてもよい。３種類以上のルシフェラーゼ等のうち２種類以上が同一の細胞に発現しており、さらに１つのルシフェラーゼが２種類以上の細胞に発現していれば、そのルシフェラーゼを利用して、他の細胞についての比較が可能となる。例えば、ルシフェラーゼＡとルシフェラーゼＢとルシフェラーゼＣとが用いられるとき、細胞ＡにルシフェラーゼＡとルシフェラーゼＢとが発現し、細胞ＢにルシフェラーゼＡとルシフェラーゼＣとが発現していれば、ルシフェラーゼＡを基準として、ルシフェラーゼＡとルシフェラーゼＢとルシフェラーゼＣとの間の発光タンパク質の量と発光量との関係が求まる。 In addition, when three or more kinds of luciferases are used, all of them may not be expressed in the same cell. If two or more of three or more luciferases are expressed in the same cell, and if one luciferase is expressed in two or more cells, the luciferase can be used for other cells. Comparison is possible. For example, when luciferase A, luciferase B, and luciferase C are used, if luciferase A and luciferase B are expressed in cell A and luciferase A and luciferase C are expressed in cell B, luciferase A is used as a reference. The relationship between the amount of photoprotein and the amount of luminescence among luciferase A, luciferase B, and luciferase C is obtained.
　続いて行われる３つ目の解析対象についての測定が、図２に示すステップＳ８乃至ステップＳ１１である。この処理について説明する。まず、解析対象の試料が第３の試料として調製される（ステップＳ８）。この試料について、フィルター２１を介して発光強度が測定される。発光強度の測定は、各々のフィルターを介した場合について順に行われる（ステップＳ９）。このようにして、各フィルターを介した解析対象試料の発光検出結果が得られる。ここで得られた検出結果について、ステップＳ４で算出されたフィルターごとの透過率が考慮されることで、解析対象試料が放射した各色についての発光量が算出される（ステップＳ１０）。さらに、ステップＳ７で算出された色毎の発光タンパク質の量と発光量との関係に基づいて、解析対象の試料における発光タンパク質の量が算出され得る。発光タンパク質の量は、解析対象に係る値と関連付けられているので、解析対象に係る値が算出される。以上によって、発光強度の測定に基づく解析対象の測定がなされる。 Subsequently, the measurement performed on the third analysis target is steps S8 to S11 shown in FIG. This process will be described. First, a sample to be analyzed is prepared as a third sample (step S8). The emission intensity of this sample is measured through the filter 21. The measurement of the emission intensity is performed in order for each case through each filter (step S9). In this way, the luminescence detection result of the sample to be analyzed through each filter is obtained. With respect to the detection result obtained here, the transmittance for each filter calculated in step S4 is taken into consideration, whereby the light emission amount for each color emitted from the analysis target sample is calculated (step S10). Furthermore, the amount of photoprotein in the sample to be analyzed can be calculated based on the relationship between the amount of photoprotein for each color and the amount of luminescence calculated in step S7. Since the amount of photoprotein is associated with the value related to the analysis target, the value related to the analysis target is calculated. As described above, the analysis target is measured based on the measurement of the emission intensity.
　［測定の例］
　上述の測定方法の適用例について説明する。ここでは、ルシフェラーゼを用いたプロモーターアッセイを例として説明する。この例では、２種類のプロモーターの活性が測定される。測定のために、レポーターとして２種類のルシフェラーゼが用いられるものとする。すなわち、１種類の細胞に２種類の発現ベクターが導入されるものとする。この２種類の発現ベクターを第１のベクター及び第２のベクターとする。 [Example of measurement]
An application example of the above measurement method will be described. Here, a promoter assay using luciferase will be described as an example. In this example, the activity of two types of promoters is measured. For the measurement, two types of luciferases are used as reporters. That is, two types of expression vectors are introduced into one type of cell. These two types of expression vectors are referred to as a first vector and a second vector.
　また、測定には、発光顕微鏡が用いられるものとする。すなわち、上述の発現ベクターが導入された細胞の培地にルシフェリンが添加され、その際に生じる発光に係る顕微鏡画像が取得される。この顕微鏡画像において、例えば関心領域が設定され、関心領域内の輝度が解析されることで発光強度が得られる。 In addition, a luminescence microscope is used for the measurement. That is, luciferin is added to the cell culture medium into which the above-described expression vector has been introduced, and a microscopic image relating to luminescence generated at that time is obtained. In this microscope image, for example, a region of interest is set, and the luminance in the region of interest is analyzed to obtain the emission intensity.
　第１のベクターの一部の構成例を図３Ａに示し、第２のベクターの一部の構成例を図３Ｂに示す。図３Ａに示すように、活性を測定したいプロモーターの１つである第１のプロモーターの下流に第１のルシフェラーゼの遺伝子が配置されている。図３Ｂに示すように、活性を測定したいもう１つのプロモーターである第２のプロモーターの下流に第２のルシフェラーゼの遺伝子が配置されている。第１のルシフェラーゼと第２のルシフェラーゼとは、互いに発光の波長が異なるルシフェラーゼである。 FIG. 3A shows a configuration example of a part of the first vector, and FIG. 3B shows a configuration example of a part of the second vector. As shown in FIG. 3A, the first luciferase gene is arranged downstream of the first promoter, which is one of the promoters whose activity is to be measured. As shown in FIG. 3B, a second luciferase gene is arranged downstream of a second promoter, which is another promoter whose activity is to be measured. The first luciferase and the second luciferase are luciferases having different emission wavelengths.
　例えば、第１のルシフェラーゼとして、緑色の発光に係るＭＡ－Ｌｕｃｉ２が用いられ、第２のルシフェラーゼとして、赤色の発光に係るＳｆＲＥ１が用いられ得る。ここでは、緑色の発光と赤色の発光とが検出される場合を例に挙げて説明する。 For example, MA-Luci2 related to green light emission can be used as the first luciferase, and SfRE1 related to red light emission can be used as the second luciferase. Here, a case where green light emission and red light emission are detected will be described as an example.
　この例では、ステップＳ１で作製される第１の試料として、以下の試料が作製される。すなわち、第１のルシフェラーゼを単独で発現する細胞を用意するために、第１のルシフェラーゼの遺伝子を有する第３のベクターが作製される。同様に、第２のルシフェラーゼを単独で発現する細胞を用意するために、第２のルシフェラーゼの遺伝子を有する第４のベクターが作製される。第３のベクターの一部の構成例を図４Ａに示し、第４のベクターの一部の構成例を図４Ｂに示す。図４Ａに示すように、第３のベクターにおいて、第１のルシフェラーゼの遺伝子の上流には、第３のプロモーターが配置されている。ここで、第３のプロモーターは、第１のルシフェラーゼの安定した高い発現が期待できるプロモーターである。図４Ｂに示すように、第４のベクターにおいて、第２のルシフェラーゼの遺伝子の上流にも、第３のベクターの場合と同様に、第３のプロモーターが配置されている。第３のプロモーターとしては、例えば、ＣＡＧプロモーター、ＣＭＶプロモーター、ＥＦ１ａプロモーター、ＲＳＶプロモーター、ＳＶ４０プロモーター等が用いられ得る。 In this example, the following sample is manufactured as the first sample manufactured in step S1. That is, in order to prepare a cell that expresses the first luciferase alone, a third vector having the first luciferase gene is prepared. Similarly, in order to prepare a cell that expresses the second luciferase alone, a fourth vector having the gene for the second luciferase is prepared. A configuration example of a part of the third vector is shown in FIG. 4A, and a configuration example of a part of the fourth vector is shown in FIG. 4B. As shown in FIG. 4A, in the third vector, a third promoter is arranged upstream of the first luciferase gene. Here, the third promoter is a promoter that can be expected to stably and highly express the first luciferase. As shown in FIG. 4B, in the fourth vector, the third promoter is also arranged upstream of the second luciferase gene, as in the case of the third vector. As the third promoter, for example, CAG promoter, CMV promoter, EF1a promoter, RSV promoter, SV40 promoter and the like can be used.
　ステップＳ１の試料の作製では、第３のベクターが導入された細胞と、第４のベクターが導入された細胞とがそれぞれ調製される。ここで、上述の例では、第３のベクターが導入された細胞からは、ルシフェリンの添加によって緑色の光が放射され、第４のベクターが導入された細胞からは、ルシフェリンの添加によって赤色の光が放射される。 In the preparation of the sample in step S1, cells into which the third vector has been introduced and cells into which the fourth vector has been introduced are prepared. Here, in the above example, green light is emitted from the cells into which the third vector has been introduced by the addition of luciferin, and red light from the cells into which the fourth vector has been introduced by the addition of luciferin. Is emitted.
　ステップＳ２の測定では、第３のベクターが導入された細胞における第１のルシフェラーゼに係る発光の発光強度がフィルター２１を介さずに測定される。すなわち、例えばフィルター２１を介さずに、第１のルシフェラーゼの緑色の発光に係る発光画像が取得され、当該画像の発光に係る輝度が解析される。 In the measurement of step S2, the luminescence intensity of luminescence related to the first luciferase in the cell into which the third vector has been introduced is measured without passing through the filter 21. That is, for example, without using the filter 21, a luminescent image related to the green luminescence of the first luciferase is acquired, and the luminance related to the luminescence of the image is analyzed.
　ステップＳ３の測定では、第３のベクターが導入された細胞における第１のルシフェラーゼに係る発光の発光強度が緑色のフィルター２１を介して測定される。すなわち、例えば緑色のフィルター２１を介して、第１のルシフェラーゼに係る緑色の発光に係る発光画像が取得され、当該画像の発光に係る輝度が解析される。また、第３のベクターが導入された細胞における第１のルシフェラーゼに係る発光の発光強度が赤色のフィルター２１を介して測定される。すなわち、例えば赤色のフィルター２１を介して、第１のルシフェラーゼに係る緑色の発光に係る発光画像が取得され、当該画像の発光に係る輝度が解析される。 In the measurement of step S3, the luminescence intensity of luminescence related to the first luciferase in the cell into which the third vector is introduced is measured through the green filter 21. That is, for example, a luminescent image related to green light emission related to the first luciferase is acquired via the green filter 21, and the luminance related to light emission of the image is analyzed. In addition, the luminescence intensity of luminescence related to the first luciferase in the cell into which the third vector has been introduced is measured through the red filter 21. That is, for example, a light emission image related to green light emission related to the first luciferase is acquired via the red filter 21, and the luminance related to light emission of the image is analyzed.
　同様に、ステップＳ２の測定では、第４のベクターが導入された細胞における第２のルシフェラーゼに係る発光の発光強度がフィルター２１を介さずに測定される。すなわち、例えばフィルター２１を介さずに、第２のルシフェラーゼに係る赤色の発光に係る発光画像が取得され、当該画像の発光に係る輝度が解析される。 Similarly, in the measurement in step S2, the luminescence intensity of luminescence related to the second luciferase in the cell into which the fourth vector has been introduced is measured without passing through the filter 21. That is, for example, without passing through the filter 21, a luminescence image related to red light emission related to the second luciferase is acquired, and the luminance related to light emission of the image is analyzed.
　同様に、ステップＳ３の測定では、第４のベクターが導入された細胞における第２のルシフェラーゼに係る発光の発光強度が緑色のフィルター２１を介して測定される。すなわち、例えば緑色のフィルター２１を介して、第２のルシフェラーゼに係る赤色の発光に係る発光画像が取得され、当該画像の発光に係る輝度が解析される。また、第４のベクターが導入された細胞における第２のルシフェラーゼに係る発光の発光強度が赤色のフィルター２１を介して測定される。すなわち、例えば赤色のフィルター２１を介して、第２のルシフェラーゼに係る赤色の発光に係る発光画像が取得され、当該画像の発光に係る輝度が解析される。 Similarly, in the measurement of step S3, the luminescence intensity of luminescence related to the second luciferase in the cell into which the fourth vector has been introduced is measured through the green filter 21. That is, for example, a luminescent image related to red light emission related to the second luciferase is acquired via the green filter 21, and the luminance related to light emission of the image is analyzed. In addition, the luminescence intensity of luminescence related to the second luciferase in the cell into which the fourth vector has been introduced is measured through the red filter 21. That is, for example, a luminescence image related to red light emission related to the second luciferase is acquired via the red filter 21, and the luminance related to light emission of the image is analyzed.
　ステップＳ４では、第１のルシフェラーゼに係る緑色の発光について、緑色のフィルターに対する透過率と、赤色のフィルターに対する透過率とが求められる。同様に、第２のルシフェラーゼに係る赤色の発光について、緑色のフィルターに対する透過率と、赤色のフィルターに対する透過率とが求められる。 In step S4, with respect to the green light emission associated with the first luciferase, the transmittance with respect to the green filter and the transmittance with respect to the red filter are obtained. Similarly, with respect to the red light emission related to the second luciferase, the transmittance with respect to the green filter and the transmittance with respect to the red filter are obtained.
　ステップＳ５において、第１のルシフェラーゼと第２のルシフェラーゼとが等量発現する試料が調製される。このため、第１のルシフェラーゼと第２のルシフェラーゼとを有する第５のベクターが作製される。第５のベクターの一部の構成例を図５に示す。図５に示すように、第５のベクターでは、第３のベクター及び第４のベクターと同様に、第３のプロモーターが用いられる。第３のプロモーターの下流には、第１のルシフェラーゼの遺伝子と第２のルシフェラーゼの遺伝子とが２Ａペプチドに係る配列で結合された状態で配置される。２Ａペプチドで結合されているので、第１のルシフェラーゼと第２のルシフェラーゼとは一続きに転写され、それぞれ翻訳され、第１のルシフェラーゼと第２のルシフェラーゼとが等量発現することになる。このような構成を有する第５のベクターが細胞に導入された第２の試料が調製される。 In step S5, a sample in which the same amount of the first luciferase and the second luciferase are expressed is prepared. For this reason, the 5th vector which has the 1st luciferase and the 2nd luciferase is produced. An example of the configuration of part of the fifth vector is shown in FIG. As shown in FIG. 5, in the fifth vector, the third promoter is used as in the third vector and the fourth vector. Downstream of the third promoter, a first luciferase gene and a second luciferase gene are arranged in a state of being bound by a sequence related to the 2A peptide. Since they are linked by the 2A peptide, the first luciferase and the second luciferase are continuously transcribed and translated, and the first luciferase and the second luciferase are expressed in equal amounts. A second sample in which the fifth vector having such a configuration is introduced into the cell is prepared.
　ステップＳ６の測定では、第５のベクターが導入された細胞における第１のルシフェラーゼ及び第２のルシフェラーゼに係る発光の発光強度が緑色のフィルター２１を介して測定される。すなわち、例えば緑色のフィルター２１を介して、第１のルシフェラーゼに係る緑色の発光と第２のルシフェラーゼに係る赤色の発光とに係る発光画像が取得される。また、第５のベクターが導入された細胞における第１のルシフェラーゼ及び第２のルシフェラーゼに係る発光の発光強度が赤色のフィルター２１を介して測定される。すなわち、例えば赤色のフィルター２１を介して、第１のルシフェラーゼに係る緑色の発光と第２のルシフェラーゼに係る赤色の発光とに係る発光画像が取得される。 In the measurement of step S6, the luminescence intensity of luminescence related to the first luciferase and the second luciferase in the cell into which the fifth vector has been introduced is measured through the green filter 21. That is, for example, via the green filter 21, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired. Further, the luminescence intensity of luminescence related to the first luciferase and the second luciferase in the cell into which the fifth vector has been introduced is measured through the red filter 21. That is, for example, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired via the red filter 21.
　ステップＳ７では、ステップＳ６で得られた画像に基づいて、第１のルシフェラーゼと第２のルシフェラーゼとが等量発現している場合の発光量が算出される。この際、ステップＳ４で求められた透過率が用いられる。すなわち、緑色のフィルターを用いて得られた検出値をＳｇとし、赤色のフィルターを用いて得られた検出値をＳｒとする。また、ステップＳ４で得られた、緑色光の緑色フィルターに対する透過率をＴｇｇとし、緑色光の赤色フィルターに対する透過率をＴｒｇとし、赤色光の緑色フィルターに対する透過率をＴｇｒとし、赤色光の赤色フィルターに対する透過率をＴｒｒとする。ここで、緑色の発光量をｇとし、赤色の発光量をｒとすると下記式が成り立つ。 In step S7, based on the image obtained in step S6, the amount of luminescence when the same amount of the first luciferase and the second luciferase are expressed is calculated. At this time, the transmittance obtained in step S4 is used. That is, the detection value obtained using the green filter is Sg, and the detection value obtained using the red filter is Sr. Further, the transmittance of the green light to the green filter obtained in step S4 is T gg , the transmittance of the green light to the red filter is T rg , the transmittance of the red light to the green filter is T gr , and the red light Let T rr be the transmittance of the red filter. Here, when the green light emission amount is g and the red light emission amount is r, the following equation holds.
Figure JPOXMLDOC01-appb-M000003
Figure JPOXMLDOC01-appb-M000003
この式の解を得ることで、以下が成り立つ。 By obtaining the solution of this equation, the following holds.
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000005
Figure JPOXMLDOC01-appb-M000005
このようにして、等量の第１のルシフェラーゼと第２のルシフェラーゼに対する緑色光の発光量と赤色光の発光量との比はｇ：ｒと求まる。 In this manner, the ratio of the green light emission amount and the red light emission amount with respect to equal amounts of the first luciferase and the second luciferase is obtained as g: r.
　ステップＳ８では、解析対象の第３の試料が調製される。すなわち、図３Ａに示した第１のベクターと図３Ｂに示した第２のベクターとが導入された細胞が用意される。 In step S8, a third sample to be analyzed is prepared. That is, a cell into which the first vector shown in FIG. 3A and the second vector shown in FIG. 3B have been introduced is prepared.
　ステップＳ９の測定では、第１のベクター及び第２のベクターが導入された細胞における第１のルシフェラーゼ及び第２のルシフェラーゼに係る発光の発光強度が緑色のフィルター２１を介して測定される。すなわち、例えば緑色のフィルター２１を介して、第１のルシフェラーゼに係る緑色の発光と第２のルシフェラーゼに係る赤色の発光とに係る発光画像が取得される。また、第１のベクター及び第２のベクターが導入された細胞における第１のルシフェラーゼ及び第２のルシフェラーゼに係る発光の発光強度が赤色のフィルター２１を介して測定される。すなわち、例えば赤色のフィルター２１を介して、第１のルシフェラーゼに係る緑色の発光と第２のルシフェラーゼに係る赤色の発光とに係る発光画像が取得される。 In the measurement of step S9, the luminescence intensity of the luminescence related to the first luciferase and the second luciferase in the cell into which the first vector and the second vector are introduced is measured through the green filter 21. That is, for example, via the green filter 21, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired. In addition, the luminescence intensity of luminescence related to the first luciferase and the second luciferase in the cells into which the first vector and the second vector are introduced is measured through the red filter 21. That is, for example, a luminescence image related to the green luminescence related to the first luciferase and the red luminescence related to the second luciferase is acquired via the red filter 21.
　ステップＳ１０の処理では、上述の式（４）及び（５）を用いて、緑色の発光量ｇと赤色の発光量ｒとが算出される。 In the process of step S10, the green light emission amount g and the red light emission amount r are calculated using the above formulas (4) and (5).
　ステップＳ１１の処理では、ステップＳ７で求められた発光量と第１のルシフェラーゼ及び第２のルシフェラーゼの物質量との関係が用いられる。この関係と、ステップＳ１０で求められた緑色の発光量ｇと赤色の発光量ｒとから、細胞における第１のルシフェラーゼの量と第２のルシフェラーゼの量とが算出される。この量は、第１のプロモーターの活性と第２のプロモーターの活性との関係を示す。以上のように、第１のプロモーター及び第２のプロモーターの活性に関する解析が行われる。 In the process of step S11, the relationship between the luminescence amount obtained in step S7 and the substance amounts of the first luciferase and the second luciferase is used. The amount of the first luciferase and the amount of the second luciferase in the cell are calculated from this relationship and the green light emission amount g and the red light emission amount r obtained in step S10. This amount indicates the relationship between the activity of the first promoter and the activity of the second promoter. As described above, the analysis on the activities of the first promoter and the second promoter is performed.
　［本測定方法の特長］
　一般に、ルシフェラーゼの波長と発光強度との関係は、例えば精製タンパク質を用いて測定され得る。精製タンパク質を用いて測定されたルシフェラーゼの波長と発光強度との関係の一例を図６に示す。図６において、実線は、ＭＡ－Ｌｕｃｉ２の発光スペクトルを示し、破線は、ＳｆＲＥ１の発光スペクトルを示す。 [Features of this measurement method]
In general, the relationship between the wavelength of luciferase and the emission intensity can be measured using, for example, purified protein. An example of the relationship between the wavelength of luciferase and the luminescence intensity measured using the purified protein is shown in FIG. In FIG. 6, the solid line indicates the emission spectrum of MA-Luci2, and the broken line indicates the emission spectrum of SfRE1.
　一方で、細胞内の発光スペクトルは、図６に示すような精製タンパク質を用いて計測される場合とは異なる特性を示す。この特性は、例えば細胞の種類、ｐＨ又は温度等の細胞内外の環境等に応じて異なり得る。また、複数種類のルシフェラーゼが用いられる場合、種類ごとにタンパク質量と発光量との関係が異なることが知られている。 On the other hand, the emission spectrum in the cell shows different characteristics from those measured using a purified protein as shown in FIG. This characteristic may vary depending on, for example, the environment inside and outside the cell, such as cell type, pH or temperature. In addition, when multiple types of luciferases are used, it is known that the relationship between the amount of protein and the amount of luminescence differs for each type.
　したがって、例えば、あるプロモーターＡの活性を測定するために計測されたあるルシフェラーゼＡに起因する光についての発光強度と、他のプロモーターＢの活性を測定するために計測された他のルシフェラーゼＢに起因する光についての発光強度とが求まっても、一般にはその結果に基づいてプロモーターＡの活性とプロモーターＢの活性とを比較することができない。例えば、ルシフェラーゼＡに起因する発光量が、ルシフェラーゼＢに起因する発光量よりも３倍高いときを考える。このとき、プロモーターＡの活性がプロモーターＢの活性と比較して３倍高い活性を示している可能性がある。また、プロモーターＡの活性とプロモーターＢの活性とが同じであるが、単位タンパク質量当たりのルシフェラーゼＡの発光量がルシフェラーゼＢの発光量の３倍である可能性がある。一般的には、これらの可能性の何れが真実なのかあるいは２つの減少が混在しているのかを判断することはできない。 Therefore, for example, the luminescence intensity for light caused by a certain luciferase A measured to measure the activity of a certain promoter A and the other luciferase B measured for measuring the activity of another promoter B Even if the luminescence intensity for the light to be obtained is obtained, the activity of promoter A and the activity of promoter B cannot generally be compared based on the result. For example, let us consider a case where the amount of luminescence caused by luciferase A is three times higher than the amount of luminescence caused by luciferase B. At this time, there is a possibility that the activity of promoter A is 3 times higher than that of promoter B. Further, although the activity of promoter A and the activity of promoter B are the same, the luminescence amount of luciferase A per unit protein amount may be three times the luminescence amount of luciferase B. In general, it is not possible to determine which of these possibilities is true or whether the two reductions are mixed.
　これに対して本実施形態に係る測定方法によれば、多色プロモーターアッセイを行う際に、各ルシフェラーゼのフィルター透過率と、各ルシフェラーゼの細胞内発光量の補正項とが算出されるので、異なるプロモーターの発現量比をより正確に算出することが可能になる。 On the other hand, according to the measurement method according to the present embodiment, when performing a multicolor promoter assay, the filter transmittance of each luciferase and the correction term for the intracellular luminescence amount of each luciferase are calculated. It becomes possible to calculate the expression level ratio of the promoter more accurately.
　この技術は、上述のプロモーターアッセイに限らず種々の解析に適用され得る。例えば、ｉＰＳ細胞のリプログラミング時に各細胞に導入されたベクター数の比が正確に算出され得る。例えば、転写因子を含む図７Ａ、図７Ｂ及び図７Ｃに示すような構成を有するベクターがｉＰＳのリプログラミングに用いられたとする。このとき、導入された各ベクター数の比は、各ベクターによって導入され転写因子とともに発現するルシフェラーゼに由来する発光が計測されることで特定され得る。 This technique is not limited to the promoter assay described above, and can be applied to various analyses. For example, the ratio of the number of vectors introduced into each cell during reprogramming of iPS cells can be accurately calculated. For example, it is assumed that a vector including a transcription factor and having a configuration as shown in FIGS. 7A, 7B, and 7C is used for iPS reprogramming. At this time, the ratio of the number of each introduced vector can be specified by measuring luminescence derived from luciferase introduced by each vector and expressed together with the transcription factor.
　より具体的には、図７Ａに示されるベクターに係るＳｆＲＥ１ルシフェラーゼを発現させた細胞の発光について、各フィルター透過率が測定される。同様に、図７Ｂに示されるベクターに係るＯｋｉ＿ｍｕｔ１ルシフェラーゼを発現させた細胞の発光について、各フィルター透過率が測定される。同様に、図７Ｃに示されるベクターに係るＭＡ－Ｌｕｃｉ２ルシフェラーゼを発現させた細胞の発光について、各フィルター透過率が測定される。さらに、ＳｆＲＥ１ルシフェラーゼとＯｋｉ＿ｍｕｔ１ルシフェラーゼとＭＡ－Ｌｕｃｉ２ルシフェラーゼとを、例えば２Ａペプチドを利用して、等量発現させた細胞の発光について、各フィルターを用いた画像を取得する。これらの画像から得られた発光の検出値と、上述の各フィルター透過率と、上記式（２）とを用いて、各色の発光量が算出され、各ルシフェラーゼが等量発現している場合の発光量比が算出され得る。 More specifically, the transmittance of each filter is measured for the luminescence of cells expressing SfRE1 luciferase according to the vector shown in FIG. 7A. Similarly, the transmittance of each filter is measured for the luminescence of cells expressing Oki_mut1 luciferase according to the vector shown in FIG. 7B. Similarly, the transmittance of each filter is measured for the luminescence of cells expressing MA-Luci2 luciferase according to the vector shown in FIG. 7C. Furthermore, an image using each filter is obtained for luminescence of cells in which SfRE1 luciferase, Oki_mut1 luciferase and MA-Luci2 luciferase are expressed in an equal amount using, for example, 2A peptide. Using the detected luminescence values obtained from these images, the above-mentioned filter transmittances, and the above equation (2), the amount of luminescence of each color is calculated, and each luciferase is expressed in an equal amount. The light emission ratio can be calculated.
　図７Ａ、図７Ｂ及び図７Ｃに示すような構成を有するベクターが導入された細胞の発光画像を取得し、画像から得られる検出値と、上述の各フィルター透過率と、上記式（２）とを用いて、当該細胞における各色の発光量が算出される。この発光量と、上述の等量発現の場合の発光量比を用いて、当該細胞における図７Ａ、図７Ｂ及び図７Ｃに示す各ベクターの導入数比が算出され得る。なお、細胞のリプログラミングに用いられる転写因子としては、Ｏｃｔ３／４、Ｋｌｆ４、Ｓｏｘ２、ｃ－ｍｙｃ、Ｌｉｎ２８、Ｌ－ｍｙｃ等が挙げられる。 A luminescence image of a cell into which a vector having the structure shown in FIGS. 7A, 7B, and 7C is introduced is obtained, the detection value obtained from the image, each filter transmittance described above, and the above equation (2) Is used to calculate the light emission amount of each color in the cell. Using this luminescence amount and the luminescence amount ratio in the case of the above-described equivalent expression, the introduction number ratio of each vector shown in FIGS. 7A, 7B and 7C in the cell can be calculated. Examples of transcription factors used for cell reprogramming include Oct3 / 4, Klf4, Sox2, c-myc, Lin28, and L-myc.
　また、例えばｉＰＳ細胞といった多能性細胞を作製するリプログラミングの段階における未分化能又は分化状態を複数のマーカーを用いて定量的に解析する場合にも、マーカー毎に波長が異なる発光が利用されれば本実施形態の技術が用いられ得る。また、多能性細胞から各種臓器等へ分化させる分化段階における多様な分化状態に対応するマーカー等を用いて定量的に分化状態を解析する場合にも、マーカー毎に波長が異なる発光が利用されれば本実施形態の技術が用いられ得る。 In addition, when quantitatively analyzing the undifferentiation ability or differentiation state in the reprogramming stage for producing pluripotent cells such as iPS cells using a plurality of markers, luminescence having different wavelengths for each marker is used. Then, the technique of this embodiment can be used. In addition, when quantitatively analyzing the differentiation state using markers corresponding to various differentiation states in the differentiation stage of differentiating from pluripotent cells to various organs, etc., luminescence with different wavelengths is used for each marker. Then, the technique of this embodiment can be used.
　また、各種タンパク質とルシフェラーゼとの融合タンパク質を細胞内で発現させて、ルシフェラーゼを各種タンパク質のタグとして使用する場合にも、タグ毎に波長が異なる発光が利用されれば本実施形態の技術が用いられ得る。その結果、細胞内で発現した各種タンパク質量の比を正確に算出し得る。 In addition, when a fusion protein of various proteins and luciferase is expressed in a cell and luciferase is used as a tag for various proteins, the technology of the present embodiment can be used if light emission having a different wavelength is used for each tag. Can be. As a result, the ratio of the amounts of various proteins expressed in the cells can be accurately calculated.
　以上のように、本実施形態に係る測定方法を用いれば、波長が異なる光の定量的な解析が高い精度で可能になる。 As described above, if the measurement method according to the present embodiment is used, quantitative analysis of light having different wavelengths becomes possible with high accuracy.
　本実施形態に係る測定と類似の測定方法として、蛍光が利用されることがある。蛍光を利用する場合と比較して、発光を用いる本実施形態の方法は、以下のような利点がある。すなわち、蛍光では、退色現象が存在する。すなわち、計測を行うことに起因して、計測される光強度が変化する。これに対して、発光では、このような計測を行うことに起因する光強度の変化がない。したがって、発光を利用する場合、蛍光を利用する場合に比較して定量性が高い。 Fluorescence may be used as a measurement method similar to the measurement according to the present embodiment. Compared with the case of using fluorescence, the method of the present embodiment using light emission has the following advantages. That is, there is a fading phenomenon in fluorescence. That is, the measured light intensity changes due to the measurement. On the other hand, in light emission, there is no change in light intensity due to performing such measurement. Therefore, when using luminescence, the quantitativeness is higher than when using fluorescence.
　また、蛍光を利用する測定では、励起光を必要とするため、励起光の強度に係る光源の出力、励起光の光路に設けられたフィルターの特性、励起光と蛍光とを分離するフィルターの特性等、発光を利用する測定と比べて、検出される光強度に影響を与える要素が多い。したがって、蛍光を利用する測定では、調整すべき要素が多い。これに対して、発光を利用する測定では、上述の調整が行われれば、十分に高い定量性が得られる。 In addition, since measurement using fluorescence requires excitation light, the output of the light source related to the intensity of the excitation light, the characteristics of the filter provided in the optical path of the excitation light, and the characteristics of the filter that separates the excitation light and fluorescence Thus, there are many factors that affect the detected light intensity as compared with the measurement using luminescence. Therefore, there are many elements to be adjusted in the measurement using fluorescence. On the other hand, in the measurement using luminescence, sufficiently high quantitativeness can be obtained if the above-described adjustment is performed.
　なお、ステップＳ４で算出される透過率、及びステップＳ７で算出される発現量と発光量との関係は、同一の測定システム１が用いられる場合であっても、一般に異なる細胞種が用いられるときには再度取得される必要がある。一方で、同一の細胞種が用いられるときには、同一の値が援用され得る。 Note that the transmittance calculated in step S4 and the relationship between the expression level and the luminescence level calculated in step S7 are generally used when different cell types are used even when the same measurement system 1 is used. It needs to be acquired again. On the other hand, when the same cell type is used, the same value can be used.
　上述の実施形態の手法を用いて、ｉＰＳ細胞の誘導時に使用されるベクターの細胞内の量比を算出した。 Using the method of the above-described embodiment, the intracellular ratio of the vector used for iPS cell induction was calculated.
　［実験方法］
（１）単色発光試料の調製
　本実施例では、図７Ａに示した構成を有するベクター（以降、ベクターＡと称する）と、図７Ｃに示した構成を有するベクター（以降、ベクターＣと称する）とを用いた。ベクターＡは、pCXLE-HOCT3/4-shp53（Addgene）をKpn I及びBgl IIで消化し、hOCT3/4の下流に2A配列を介してSfRE1 luciferaseを組み込み作製した。ベクターＣは、pCXLE-hUL（Addgene）をKpnI及びPacIで消化し、LIN28の下流に2A配列を介してMA-Luci2を組み込み作製した。 [experimental method]
(1) Preparation of Monochromatic Luminescent Sample In this example, a vector having the configuration shown in FIG. 7A (hereinafter referred to as vector A) and a vector having the configuration shown in FIG. 7C (hereinafter referred to as vector C) Was used. Vector A was prepared by digesting pCXLE-HOCT3 / 4-shp53 (Addgene) with Kpn I and Bgl II and incorporating SfRE1 luciferase downstream of hOCT3 / 4 via the 2A sequence. Vector C was prepared by digesting pCXLE-hUL (Addgene) with KpnI and PacI and incorporating MA-Luci2 downstream of LIN28 via the 2A sequence.
　末梢血単核球PBMC（米国CTL）を解凍し、PBMC用培地（IL3, IL6, SCF, TPO, Flt-3L, CSFを添加したAK02培地（味の素））で培養した。細胞密度を2.5×106 cells/wellとし、24ウェルプレートを用いて培養を行った。培地交換を行わず、37℃、5% CO2で7日間培養後、PBMCをコーティング剤（iMatrix（ニッピ））でコートした6ウェルプレートに再度播種した。 Peripheral blood mononuclear cells PBMC (US CTL) were thawed and cultured in PBMC medium (AK02 medium (Ajinomoto) supplemented with IL3, IL6, SCF, TPO, Flt-3L, CSF). The cell density was 2.5 × 10 6 cells / well, and culture was performed using a 24-well plate. After exchanging the medium for 7 days at 37 ° C. and 5% CO 2 , the PBMCs were seeded again in a 6-well plate coated with a coating agent (iMatrix (Nippi)).
　６ウェルプレートに播種されたPBMCに、Amaxa（Lonza）を用いて、ベクターＡと、pCE-mp53DD、pCE-hSK、pCE-hUL、pCXB-EBNA1（それぞれAddgene）の各ベクターを導入した。また、６ウェルプレートに播種されたPBMCに、Amaxa（Lonza）を用いて、ベクターＣと、pCE-hOCT3/4、pCE-mp53DD、pCE-hSK、pCXB-EBNA1の各ベクターを導入した。 The vector A and vectors pCE-mp53DD, pCE-hSK, pCE-hUL, and pCXB-EBNA1 (each Addgene) were introduced into PBMC seeded in a 6-well plate using Amaxa (Lonza). Moreover, the vector C and each vector of pCE-hOCT3 / 4, pCE-mp53DD, pCE-hSK, and pCXB-EBNA1 were introduced into PBMC seeded in a 6-well plate using Amaxa (Lonza).
　ベクターＡを導入した細胞とベクターＣを導入した細胞とのそれぞれについて、導入日の翌翌日以降、1日おきに1.5 mlのiPS細胞用培地（AK02培地（味の素））を加えた。ベクター導入後8日目に、培地をすべて吸引し、2 mlのiPS細胞用培地を加えた。以後、1日おきに2 mlのiPS細胞用培地で培地交換した。iPS細胞様コロニーが形成された後、終濃度1 mMとなるようにD-luciferin（Promega）を培地に加え、iPS細胞様コロニーにおける発光イメージングを発光顕微鏡システムLV200（オリンパス）を用いて行った。 For each of the cells into which vector A was introduced and the cells into which vector C was introduced, 1.5 μml of iPS cell culture medium (AK02 medium (Ajinomoto)) was added every other day from the day after the day after the introduction. On the 8th day after the introduction of the vector, all the medium was aspirated, and 2 ml of iPS cell medium was added. Thereafter, the medium was replaced every other day with 2 ml of iPS cell medium. After iPS cell-like colonies were formed, D-luciferin (Promega) was added to the medium to a final concentration of 1 mM, and luminescence imaging of iPS cell-like colonies was performed using a luminescence microscope system LV200 (Olympus).
（２）発光イメージング像を用いた単色発光試料のフィルター透過率の算出
　発光イメージングにおける測定用フィルターとして、BP495-540（Filter 1）及び610ALP （Filter 2）を使用した。まず、ベクターＡ（SfRE1ルシフェラーゼ）を導入した細胞を用いて、(1) フィルターを用いない測定（全光）、(2) Filter 1を透過した透過光の測定、(3) Filter 2を透過した透過光の測定を実施した。画像取得時の露出時間はいずれの測定においても180秒に設定した。続いて、ベクターＣ（MA-Luci2ルシフェラーゼ）を導入した細胞を用いて、(1) フィルターを用いない測定（全光）、(2) Filter 1を透過した透過光の測定、(3) Filter 2を透過した透過光の測定を、ベクターＡの場合と同様の条件で実施した。発光イメージング像に設定した関心領域（Region of Interest: ROI）における検出値に基づいて、下記表１に示す係数を算出した。 (2) Calculation of filter transmittance of monochromatic luminescent sample using luminescence imaging image BP495-540 (Filter 1) and 610ALP (Filter 2) were used as measurement filters in luminescence imaging. First, using cells into which vector A (SfRE1 luciferase) was introduced, (1) measurement without filter (all light), (2) measurement of transmitted light that passed through Filter 1, and (3) transmission through Filter 2 Measurement of transmitted light was performed. The exposure time at the time of image acquisition was set to 180 seconds in all measurements. Subsequently, using cells into which vector C (MA-Luci2 luciferase) was introduced, (1) measurement without using a filter (total light), (2) measurement of transmitted light that passed through Filter 1, (3) Filter 2 Measurement of the transmitted light transmitted through was carried out under the same conditions as in Vector A. Based on the detection value in the region of interest (ROI) set in the emission imaging image, the coefficients shown in Table 1 below were calculated.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（３）多色等量発光試料の調製
　図８に示した構成を有するベクター（以降、ベクターＡと称する）と、図７Ｃに示した構成を有するベクターMA-Luci2-SfRE1を用いた。ベクターMA-Luci2-SfRE1は、pCXLE-hUL（Addgene）をEcoRIで消化し、MA-Luci2 luciferaseの下流に2A配列を介してSfRE1 luciferaseを連結させた配列を組み込み作製した。 (3) Preparation of Multicolor Equivalent Luminescent Sample A vector having the configuration shown in FIG. 8 (hereinafter referred to as vector A) and a vector MA-Luci2-SfRE1 having the configuration shown in FIG. 7C were used. The vector MA-Luci2-SfRE1 was prepared by digesting pCXLE-hUL (Addgene) with EcoRI and incorporating a sequence in which SfRE1 luciferase was ligated downstream of MA-Luci2 luciferase via a 2A sequence.
　末梢血単核球PBMC（米国CTL）を解凍し、PBMC用培地（IL3, IL6, SCF, TPO, Flt-3L, CSFを添加したAK02培地（味の素））で培養した。細胞密度を2.5×106 cells/wellとし、24ウェルプレートを用いて培養を行った。培地交換を行わず、37℃、5% CO2で7日間培養後、PBMCをコーティング剤（iMatrix（ニッピ））でコートした6ウェルプレートに再度播種した。 Peripheral blood mononuclear cells PBMC (US CTL) were thawed and cultured in PBMC medium (AK02 medium (Ajinomoto) supplemented with IL3, IL6, SCF, TPO, Flt-3L, CSF). The cell density was 2.5 × 10 6 cells / well, and culture was performed using a 24-well plate. After exchanging the medium for 7 days at 37 ° C. and 5% CO 2 , the PBMCs were seeded again in a 6-well plate coated with a coating agent (iMatrix (Nippi)).
　６ウェルプレートに播種されたPBMCにAmaxa（Lonza）を用いてベクターMA-Luci2-SfRE1（図８）と、pCE-hOCT3/4、pCE-mp53DD、pCE-hSK、pCE-hUL、pCXB-EBNA1の各ベクターとを導入した。 The vector MA-Luci2-SfRE1 (Fig. 8) and pCE-hOCT3 / 4, pCE-mp53DD, pCE-hSK, pCE-hUL, and pCXB-EBNA1 using Amaxa (Lonza) on PBMC seeded in a 6-well plate Each vector was introduced.
　導入日の翌翌日以降、1日おきに1.5 mlのiPS細胞用培地（AK02培地（味の素））を加えた。ベクター導入後8日目に、培地をすべて吸引し、2 mlのiPS細胞用培地を加えた。以後、1日おきに2 mlのiPS細胞用培地で培地交換した。iPS細胞様コロニーが形成された後、終濃度1 mMとなるようにD-luciferin（Promega）を培地に加え、iPS細胞様コロニーにおける発光イメージングを発光顕微鏡システムLV200（オリンパス）を用いて行った。 From the next day after the introduction day, 1.5 μml of iPS cell culture medium (AK02 medium (Ajinomoto)) was added every other day. On the 8th day after the introduction of the vector, all the medium was aspirated, and 2 ml of iPS cell medium was added. Thereafter, the medium was replaced every other day with 2 ml of iPS cell medium. After iPS cell-like colonies were formed, D-luciferin (Promega) was added to the medium to a final concentration of 1 mM, and luminescence imaging of iPS cell-like colonies was performed using a luminescence microscope system LV200 (Olympus).
（４）発光イメージング像を用いた多色等量発光試料の発光強度比の算出
　発光イメージング像に設定した関心領域（Region of Interest: ROI）におけるMA-Luci2ルシフェラーゼとSfRE1ルシフェラーゼの発光量を算出した。Filter 1を用いて得られた検出値をSgRatioとし、Filter 2を用いて得られた検出値をSrRatioとする。また、MA-Luci2に係る発光のfilter 1に対する透過率をT1ｇとし、MA-Luci2に係る発光のfilter 2に対する透過率をT2gとし、SfRE1に係る発光のfilter 1に対する透過率をT1rとし、SfRE1に係る発光のfilter 2に対する透過率をT2rとする。ここで、T1g、T2g、T1r、T2rは、上述の単色発光試料を用いた測定で得られた値を用いた。MA-Luci2の発光量をg_ratioとし、SfRE1の発光量をr_ratioとする。このとき、下記式（６）が成り立つ。 (4) Calculation of luminescence intensity ratio of multicolor equivalent luminescence sample using luminescence imaging image Calculated luminescence amount of MA-Luci2 luciferase and SfRE1 luciferase in the region of interest (ROI) set in luminescence imaging image . The detection value obtained using Filter 1 is S gRatio, and the detection value obtained using Filter 2 is S rRatio . In addition, the transmittance of the luminescence filter 1 according to MA-Luci2 is T 1g , the transmittance of the luminescence filter 2 according to MA-Luci2 is T 2g, and the transmittance of the luminescence filter 1 according to SfRE1 is T 1r. , T 2r is the transmittance of the light emission related to SfRE1 with respect to filter 2. Here, as T 1g , T 2g , T 1r , and T 2r , values obtained by measurement using the above-described monochromatic light emitting sample were used. The light emission amount of MA-Luci2 is g_ratio, and the light emission amount of SfRE1 is r_ratio. At this time, the following formula (6) holds.
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000007
　この式の解を得ることで、以下が成り立つ。 The following is established by obtaining the solution of this equation.
Figure JPOXMLDOC01-appb-M000008
Figure JPOXMLDOC01-appb-M000008
Figure JPOXMLDOC01-appb-M000009
Figure JPOXMLDOC01-appb-M000009
（５）解析対象試料の調製
　末梢血単核球PBMC（米国CTL）を解凍し、PBMC用培地（IL3, IL6, SCF, TPO, Flt-3L, CSFを添加したAK02培地（味の素））で培養した。細胞密度を2.5×106 cells/wellとし、24ウェルプレートを用いて培養を行った。培地交換を行わず、37℃、5% CO2で7日間培養後、PBMCをコーティング剤（iMatrix（ニッピ））でコートした6ウェルプレートに再度播種した。 (5) Preparation of sample to be analyzed Thaw peripheral blood mononuclear cell PBMC (US CTL) and culture in PBMC medium (AK02 medium (Ajinomoto) supplemented with IL3, IL6, SCF, TPO, Flt-3L, CSF) did. The cell density was 2.5 × 10 6 cells / well, and culture was performed using a 24-well plate. After exchanging the medium for 7 days at 37 ° C. and 5% CO 2 , the PBMCs were seeded again in a 6-well plate coated with a coating agent (iMatrix (Nippi)).
　６ウェルプレートに播種されたPBMCにAmaxa（Lonza）を用いてベクターＡ（図７Ａ）及びベクターＣ（図７Ｃ）と、pCE-mp53DD、pCE-hSK、pCXB-EBNA1（Addgene）を導入した。 Vector A (FIG. 7A) and vector C (FIG. 7C), and pCE-mp53DD, pCE-hSK, and pCXB-EBNA1 (Addgene) were introduced into PBMC seeded in a 6-well plate using Amaxa (Lonza).
　導入日の翌翌日以降、1日おきに1.5 mlのiPS細胞用培地（AK02培地（味の素））を加えた。ベクター導入後8日目に、培地をすべて吸引し、2 mlのiPS細胞用培地を加えた。以後、1日おきに2 mlのiPS細胞用培地で培地交換した。iPS細胞様コロニーが形成された後、終濃度1 mMとなるようにD-luciferin（Promega）を培地に加え、iPS細胞様コロニーにおける発光イメージングを発光顕微鏡システムLV200（オリンパス）を用いて行った。 From the next day after the introduction day, 1.5 μml of iPS cell culture medium (AK02 medium (Ajinomoto)) was added every other day. On the 8th day after the introduction of the vector, all the medium was aspirated, and 2 ml of iPS cell medium was added. Thereafter, the medium was replaced every other day with 2 ml of iPS cell medium. After iPS cell-like colonies were formed, D-luciferin (Promega) was added to the medium to a final concentration of 1 mM, and luminescence imaging of iPS cell-like colonies was performed using a luminescence microscope system LV200 (Olympus).
（６）発光イメージング像を用いた対象試料の解析
　発光イメージング像に設定したROIにおけるMA-Luci2ルシフェラーゼ及びSfRE1ルシフェラーゼの発光量を算出した。Filter 1を用いて得られた検出値をSgとし、filter 2を用いて得られた検出値をSrとする。また、MA-Luci2に係る発光のfilter 1に対する透過率をT1gとし、MA-Luci2に係る発光のfilter 2に対する透過率をT2gとし、SfRE1に係る発光のfilter 1に対する透過率をT1rとし、SfRE1に係る発光のfilter 2に対する透過率をT2rとする。ここで、T1g、T2g、T1r、T2rは、上述の単色発光試料を用いた測定で得られた値を用いた。MA-Luci2の発光量をgとし、SfRE1の発光量をrとすると下記式（９）が成り立つ。 (6) Analysis of target sample using luminescence imaging image The amount of luminescence of MA-Luci2 luciferase and SfRE1 luciferase in ROI set in the luminescence imaging image was calculated. The detection value obtained using Filter 1 is S g, and the detection value obtained using filter 2 is S r . In addition, the transmittance of the luminescence filter 1 according to MA-Luci2 is T 1g , the transmittance of the luminescence filter 2 according to MA-Luci2 is T 2g, and the transmittance of the luminescence filter 1 according to SfRE1 is T 1r. , T 2r is the transmittance of the light emission related to SfRE1 with respect to filter 2. Here, as T 1g , T 2g , T 1r , and T 2r , values obtained by measurement using the above-described monochromatic light-emitting sample were used. When the light emission amount of MA-Luci2 is g and the light emission amount of SfRE1 is r, the following equation (9) is established.
Figure JPOXMLDOC01-appb-M000010
Figure JPOXMLDOC01-appb-M000010
　この式の解を得ることで、以下が成り立つ。 The following is established by obtaining the solution of this equation.
Figure JPOXMLDOC01-appb-M000011
Figure JPOXMLDOC01-appb-M000011
Figure JPOXMLDOC01-appb-M000012
Figure JPOXMLDOC01-appb-M000012
　［結果］
（１）発光イメージング像を用いた単色発光試料のフィルター透過率の算出
　単色発光試料の各々の発光イメージング像上に関心領域（ROI）を設定し、フィルター透過率を算出した結果、下記表２の結果が得られた。 [result]
(1) Calculation of filter transmittance of monochromatic luminescent sample using luminescent imaging image Region of interest (ROI) was set on each luminescent imaging image of monochromatic luminescent sample, and the filter transmittance was calculated. Results were obtained.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
（２）発光イメージング像を用いた多色等量発光試料の発光強度比の算出
　多色等量発光試料の発光イメージング像上に関心領域（ROI）を設定し、検出値を得た。得られた検出値と、上記式（７）及び（８）を用いて、等量のMA-Luci2ルシフェラーゼとSfRE1ルシフェラーゼの発光強度を算出すると、下記の結果が得られた（N = 5）。
　g_ratio = 14307.7 ± 578
　r_ratio = 12580.5 ± 568 (2) Calculation of emission intensity ratio of multicolor equivalent luminescence sample using luminescence imaging image Region of interest (ROI) was set on the luminescence imaging image of multicolor equivalent luminescence sample, and the detection value was obtained. Using the obtained detection values and the above formulas (7) and (8), the emission intensity of equal amounts of MA-Luci2 luciferase and SfRE1 luciferase was calculated, and the following results were obtained (N = 5).
g_ratio = 14307.7 ± 578
r_ratio = 12580.5 ± 568
　この結果より、MA-Luci2とSfRE1が等量細胞内で発現しているときの発光強度の比はg_ratio : r_ratio ＝ 1.14 : 1であると求まった。 From this result, the ratio of luminescence intensity when MA-Luci2 and SfRE1 are expressed in the same amount of cells was determined to be g_ratio: r_ratio = 1.14: 1.
（３）発光イメージング像を用いた対象試料の解析
　解析対象試料についての得られた発光イメージング像を図９Ａ乃至図９Ｃに示す。図９Ａは、フィルターを用いない場合の発光イメージング像であり、図９Ｂは、filter 1を用いた場合の発光イメージング像であり、図９Ｃは、filter 2を用いた場合の発光イメージング像である。図９Ａ乃至図９Ｃに示した各関心領域（ROI）における検出値と、上記式（１０）及び（１１）とを用いてg及びrを算出した。得られた結果を図１０のグラフに示す。 (3) Analysis of target sample using emission imaging image FIGS. 9A to 9C show the emission imaging images obtained for the analysis target sample. 9A is a luminescence imaging image when no filter is used, FIG. 9B is a luminescence imaging image when filter 1 is used, and FIG. 9C is a luminescence imaging image when filter 2 is used. G and r were calculated using the detected values in each region of interest (ROI) shown in FIGS. 9A to 9C and the above equations (10) and (11). The obtained results are shown in the graph of FIG.
　図１０に示した発光強度の比（g/r; MA-Luci2/Sf-RE1）を図１１に補正無しとして示す。さらに、図１０に示した発光強度を多色等量発現サンプルで得られた発光強度比（g_ratio : r_ratio ＝ 1.14 : 1）を用いて補正した結果に基づいて得られた発光強度の比（g/r; MA-Luci2/Sf-RE1）を図１１に補正後として示す。 The emission intensity ratio (g / r; MA-Luci2 / Sf-RE1) shown in FIG. 10 is shown as no correction in FIG. Furthermore, the emission intensity ratio (g) obtained based on the result of correcting the emission intensity shown in FIG. 10 using the emission intensity ratio (g_ratio: r_ratio = 1.14:） 1) obtained with the multicolor equivalent expression sample. / r; MA-Luci2 / Sf-RE1) is shown in FIG.
　［考察］
　図１１に示した補正後の発光強度の比（g/r; MA-Luci2/Sf-RE1）は、各細胞に導入されたベクターの量比（ベクターＣ／ベクターＡ）を表す。このように、各細胞に導入されたベクターＡ及びベクターＣの正確な量比を求めることができた。 [Discussion]
The corrected emission intensity ratio (g / r; MA-Luci2 / Sf-RE1) shown in FIG. 11 represents the quantitative ratio of vector introduced into each cell (vector C / vector A). Thus, the exact quantitative ratio of the vector A and the vector C introduced into each cell could be obtained.
　本実施形態に係る方法によれば、異なる波長特性を有する光が同時に放射される試料において定量性の高い解析が可能な測定方法を提供できることが明らかになった。 It has been clarified that the method according to the present embodiment can provide a measurement method capable of highly quantitative analysis in a sample in which light having different wavelength characteristics is simultaneously emitted.

Claims (13)

  1. 　Ｎ色の発光タンパク質が混在し各色の発光が混在する測定対象の試料について、Ｎ個又はＮ－１個のフィルターを利用して各色の発光タンパク質の量に係る値を取得する測定方法であって、
    　前記Ｎ色の発光タンパク質の各々が独立して存在する複数の第１の試料を用いて、各色の発光について前記フィルターの何れをも透過しない状態で測定される発光強度と各々の前記フィルターを透過したときに測定される発光強度とに基づいて、各色の発光についての各々の前記フィルターの透過率を算出することと、
    　前記Ｎ色の発光タンパク質のそれぞれの量が既知となるように調製した第２の試料についての各々の前記フィルターを透過したときに測定される発光強度と、前記フィルターの透過率とに基づいて、前記発光タンパク質の量と発光量との比を算出することと、
    　Ｎ色の発光タンパク質が混在し各色の発光が混在する前記測定対象である第３の試料についての各々の前記フィルターを透過したときに測定される発光強度と、各々の前記フィルターの透過率とに基づいて、各色の発光についての発光量を求めることと、
    　得られた前記各色の発光についての発光量と前記比とに基づいて、前記第３の試料における前記各色の発光タンパク質の量に係る値を算出することと
    　を含む測定方法。     A measurement method for obtaining a value related to the amount of photoproteins of each color using N or N-1 filters for a sample to be measured in which N color photoproteins are mixed and luminescence of each color is mixed. ,
    Using a plurality of first samples in which each of the N-color photoproteins is independently present, the luminescence intensity measured in a state in which none of the filters is transmitted for each color luminescence and the respective filters are transmitted. Calculating the transmittance of each filter for each color emission based on the emission intensity measured when
    Based on the luminescence intensity measured when passing through each of the filters for the second sample prepared so that the respective amounts of the N-color photoproteins are known, and the transmittance of the filters, Calculating the ratio of the amount of photoprotein and the amount of luminescence;
    The luminescence intensity measured when passing through each of the filters of the third sample, which is the measurement target, in which N photoproteins are mixed and the light emission of each color is mixed, and the transmittance of each of the filters Based on the amount of light emission for each color emission,
    Calculating a value related to the amount of the photoprotein of each color in the third sample based on the obtained light emission amount and the ratio of the light emission of each color.
  2. 　前記第１の試料、前記第２の試料及び前記第３の試料は、それぞれ前記発光タンパク質を発現する細胞を含む、請求項１に記載の測定方法。 The measurement method according to claim 1, wherein each of the first sample, the second sample, and the third sample includes cells that express the photoprotein.
  3. 　前記発光タンパク質はルシフェラーゼである、請求項１又は２に記載の測定方法。 The measuring method according to claim 1 or 2, wherein the photoprotein is luciferase.
  4. 　前記第２の試料は、前記Ｎ色の発光タンパク質の各々の遺伝子がポリシストロニックに連結された構成を有する発現ベクターが導入された細胞を含む、請求項１乃至３のうち何れか１項に記載の測定方法。 4. The method according to claim 1, wherein the second sample includes a cell into which an expression vector having a configuration in which genes of the N-color photoproteins are linked polycistronically is introduced. 5. The measuring method described.
  5. 　前記第２の試料は、前記Ｎ色の発光タンパク質の各々の遺伝子がリンカーを介して連結された構成を有する発現ベクターが導入された細胞を含む、請求項１乃至３のうち何れか１項に記載の測定方法。 4. The method according to claim 1, wherein the second sample includes cells into which an expression vector having a configuration in which genes of the N-color photoproteins are linked via a linker is introduced. 5. The measuring method described.
  6. 　前記第２の試料が含む前記Ｎ色の発光タンパク質の量は、互いに等量である、請求項１乃至５のうち何れか１項に記載の測定方法。 The measurement method according to any one of claims 1 to 5, wherein the amount of the N-color photoprotein included in the second sample is equal to each other.
  7. 　前記フィルターの個数はＮ個である、請求項１乃至６のうち何れか１項に記載の測定方法。 The measurement method according to any one of claims 1 to 6, wherein the number of the filters is N.
  8. 　前記発光量の測定は、発光イメージングを用いて行われる、請求項１乃至７のうち何れか１項に記載の測定方法。 The measurement method according to any one of claims 1 to 7, wherein the measurement of the light emission amount is performed using light emission imaging.
  9. 　前記発光量の測定は、ルミノメーターを用いて行われる、請求項１乃至７のうち何れか１項に記載の測定方法。 The measurement method according to any one of claims 1 to 7, wherein the light emission amount is measured using a luminometer.
  10. 　前記第３の試料における各色の発光についての発光量を求めるときに、行列演算が用いられる、請求項１乃至９のうち何れか１項に記載の測定方法。 The measurement method according to any one of claims 1 to 9, wherein a matrix operation is used when obtaining a light emission amount for each color light emission in the third sample.
  11. 　前記第３の試料において、前記Ｎ色の発光タンパク質は、細胞のリプログラミングに関連する複数の転写因子と共に発現するように構成されており、
    　前記発光タンパク質の量に係る値は、前記転写因子の遺伝子が導入された量を示す、
    　請求項１乃至１０のうち何れか１項に記載の測定方法。     In the third sample, the N-color photoprotein is configured to be expressed together with a plurality of transcription factors related to cell reprogramming,
    The value relating to the amount of the photoprotein indicates the amount of the transcription factor gene introduced,
    The measurement method according to any one of claims 1 to 10.
  12. 　前記第１の試料は、複数の発現ベクターのうち何れか１つが導入された細胞を含み、
    　前記複数の発現ベクターの各々は、前記Ｎ色の発光タンパク質の遺伝子のうち何れか１つと前記複数の転写因子の遺伝子のうち少なくとも何れか１つとがポリシストロニックに連結された構成を有し、
    　前記第３の試料は、前記複数の発現ベクターの全てが導入された細胞を含む、
    　請求項１１に記載の測定方法。     The first sample includes cells into which any one of a plurality of expression vectors has been introduced,
    Each of the plurality of expression vectors has a configuration in which any one of the N-color photoprotein genes and at least one of the plurality of transcription factor genes are linked in a polycistronic manner,
    The third sample includes cells into which all of the plurality of expression vectors have been introduced.
    The measurement method according to claim 11.
  13. 　前記複数の転写因子は、Ｏｃｔ３／４、Ｋｌｆ４、Ｓｏｘ２、ｃ－ｍｙｃ、Ｌｉｎ２８、Ｌ－ｍｙｃからなる群より選択される少なくとも２つである、請求項１１又は１２に記載の測定方法。 The measurement method according to claim 11 or 12, wherein the plurality of transcription factors are at least two selected from the group consisting of Oct3 / 4, Klf4, Sox2, c-myc, Lin28, and L-myc.
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