WO2018216181A1 - Procédé de mesure - Google Patents

Procédé de mesure 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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English (en)
Japanese (ja)
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竜太郎 秋吉
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オリンパス株式会社
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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.

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Abstract

La présente invention concerne un procédé de mesure qui comprend le calcul de transmittances de filtre et le calcul de rapports entre des quantités de photoprotéine et des quantités d'émission de lumière. Des transmittances de filtre sont déterminées au moyen d'une pluralité de premiers échantillons comportant indépendamment des photoprotéines de N couleurs. Les rapports entre la quantité de photoprotéine et la quantité de lumière émise sont déterminés au moyen d'un deuxième échantillon préparé de sorte que les quantités de chacune des photoprotéines de N couleurs soient connues. Les quantités d'émission de lumière pour chaque couleur sont déterminées sur la base des intensités d'émission de lumière mesurées lorsqu'une lumière provenant d'un troisième échantillon qui est l'objet de mesure et est telle que les photoprotéines de N couleurs soient mélangées et émettent une lumière mixte traverse chacun des filtres, et des transmittances de filtre. Les valeurs pour les quantités des photoprotéines de chaque couleur dans le troisième échantillon sont calculées sur la base des rapports obtenus et des quantités d'émission de lumière pour chaque couleur d'émission de lumière.
PCT/JP2017/019612 2017-05-25 2017-05-25 Procédé de mesure WO2018216181A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
JP2006296294A (ja) * 2005-04-20 2006-11-02 Olympus Corp 発現量定量方法
JP2007330185A (ja) * 2006-06-16 2007-12-27 Toyo B-Net Co Ltd 多検体試料中における複数のルシフェラーゼを検出する方法
JP2012500005A (ja) * 2008-08-12 2012-01-05 セルラー ダイナミクス インターナショナル， インコーポレイテッド ｉＰＳ細胞を生成するための方法
JP2014014352A (ja) * 2012-06-11 2014-01-30 Olympus Corp 幹細胞の状態を同定する方法
WO2016203617A1 (fr) * 2015-06-18 2016-12-22 オリンパス株式会社 Procédé d'observation d'émission de lumière

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JP2006296294A (ja) * 2005-04-20 2006-11-02 Olympus Corp 発現量定量方法
JP2007330185A (ja) * 2006-06-16 2007-12-27 Toyo B-Net Co Ltd 多検体試料中における複数のルシフェラーゼを検出する方法
JP2012500005A (ja) * 2008-08-12 2012-01-05 セルラー ダイナミクス インターナショナル， インコーポレイテッド ｉＰＳ細胞を生成するための方法
JP2014014352A (ja) * 2012-06-11 2014-01-30 Olympus Corp 幹細胞の状態を同定する方法
WO2016203617A1 (fr) * 2015-06-18 2016-12-22 オリンパス株式会社 Procédé d'observation d'émission de lumière

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PAPAPETROU, E. P. ET AL.: "Stoichiometric and temporal requirements of Oct4, Sox2, Kil4, and c-Myc expression for efficient human iPSC induction and differentiation", PNAS, vol. 106, no. 31, 2009, pages 12759 - 12764, XP002560045 *

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