JP2007155558A - Feeble light analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method capable of performing desired cell analysis even in the case of a feeble light sample, and an analysis method capable of analyzing a clear image in a short exposure time and in real time when an objective lens satisfies a specific condition. <P>SOLUTION: In this method, a sample including a cell into which the first imperfect reporter gene expressibly connected to a promoter domain of a gene manifested by a specific cell and the second imperfect reporter gene expressively connected to a promoter domain of a gene whose manifestation is induced by stimulus generated by contact of some material with the cell are introduced is arranged within an imaging visual field, and stimulus is performed by bringing the material into contact with the sample. The method has an optical processing process wherein optical imaging of a detectable signal based on interaction between the first reporter gene and the second reporter gene manifested in the cell subjected to the stimulus is performed by an imaging means, to thereby determine quantitatively the amount of a signal generated by the reporter genes. An image enabling image analysis of the feeble light is generated in the optical processing process. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a weak light analysis method for detecting a biological activity in a biological sample such as a cell or tissue for a long period or continuously without losing the activity as much as possible. The present invention can also be applied to an automation apparatus executed by the method and software associated with the apparatus.

  In research in the fields of biology and medicine, techniques for detecting biological activity of biological samples such as cells by reporter assays have been widely used. Reporter assays can be used to visualize biological activities that cannot be visually examined. In conventional clinical tests, only biological substances (nucleic acid, blood, hormones, proteins, etc.) to be examined from biological samples are isolated by various separation methods, and the amount and activity of the isolated biological substances are determined as reagents. And reacted. However, in living organisms, the interaction between various biological materials shows true biological activity. In recent years, when researching or developing medical drugs, drugs that most effectively act on biological activity in living biological samples have become critical conditions. In reporter assays for living biological samples, there is a growing need to image biological samples and biological substances to be examined and observe dynamic changes in and out of biological samples over time. .

Specifically, in research fields that utilize observation using luminescence (bioluminescence, chemiluminescence) or fluorescence as a reporter substance, time-lapse or video imaging is required to capture the dynamic functional expression of protein molecules in a sample. It has been. At present, dynamic changes are observed using an image picked up from a fluorescent sample (for example, observation of a moving image of one protein molecule using fluorescence). In the case of imaging a fluorescent sample, the amount of light emitted from the fluorescent sample decreases with the lapse of time by continuing to irradiate excitation light, so that a stable image that can be used for quantitative evaluation can be taken over time. However, it was possible to take a clear, high spatial resolution image with a short exposure time. On the other hand, in the time-dependent observation of dynamic changes by images for a luminescent sample, the luminescence from the luminescent sample is extremely small, so a CCD camera equipped with an image intensifier was used to observe the luminescent sample. It was done. In the case of imaging of a luminescent sample, it was not necessary to irradiate excitation light, so that stable images that could be used for quantitative evaluation could be taken over time.

Until now, in the observation of a luminescent sample, the amount of luminescence from the luminescent sample has been measured. For example, in the observation of a cell into which a luciferase gene has been introduced, the amount of luminescence from the cell due to the luciferase activity was measured in order to investigate the intensity of the luciferase gene expression (specifically, the expression level). . The amount of luminescence from the cells due to luciferase activity is measured by first reacting a cell lysate in which cells are lysed with a substrate solution containing luciferin, ATP, magnesium, etc., and then reacting with the substrate solution. The amount of luminescence from was quantified with a luminometer using a photomultiplier tube. That is, the amount of luminescence was measured after cell lysis. Thereby, the expression level of the luciferase gene at a certain time point could be measured as an average value of the whole cell. Here, methods for introducing a luminescent gene such as a luciferase gene into a cell as a reporter gene include, for example, a calcium phosphate method, a lipofectin method, an electroporation method, and the like, and each method is properly used depending on the purpose and the type of cell. Yes. In addition, when investigating the intensity of luciferase gene expression in cells transfected with a luciferase gene as a reporter gene using the amount of luminescence from the cell due to luciferase activity as an indicator, the target luciferase gene is introduced upstream or downstream of the luciferase gene introduced into the cell. By linking DNA fragments, it is possible to examine the effect of the DNA fragment on the transcription of the luciferase gene. In addition, genes such as transcription factors that are thought to affect the transcription of the luciferase gene to be introduced into cells are linked to the expression vector. By co-expressing with the luciferase gene, the effect of the gene product of the gene on the expression of the luciferase gene can be examined.

In addition, in order to capture the expression level of the luminescent gene over time, it is necessary to measure the amount of luminescence from living cells over time. In order to measure the amount of luminescence from living cells over time, first add the function of a luminometer to the incubator that cultivates the cells, and then quantitate the amount of luminescence from the whole cell population cultured at regular intervals with the luminometer. The procedure was to do. As a result, it was possible to measure an expression rhythm with a certain periodicity, and thus, it was possible to capture changes over time in the expression level of the luminescent gene in the entire cell. On the other hand, when the expression of the luminescent gene is transient, the expression level in individual cells varies greatly. For example, even in the case of cloned cultured cells such as HeLa cells, the response of drugs via receptors on the cell membrane surface may vary among individual cells. That is, several cells may be responding even if the response as a whole cell is not detected. For this reason, when the expression of the luminescent gene is transient, it is important to measure the amount of luminescence from individual cells over time rather than from the whole cell . The time-dependent measurement of the amount of light emitted from living individual cells using a microscope shows that the light emitted from each cell is extremely weak, so it can be exposed for a long time with a cooled CCD camera at a liquid nitrogen temperature level, or an image intensifier. Or a photon counting device. As a result, it was possible to capture changes over time in the expression level of the luminescent gene in individual living cells.

In the above description, for example, a method and an apparatus for analyzing gene expression using a fluorescent protein as a reporter gene are disclosed in Patent Document 1 (Japanese Translation of PCT International Publication No. 2004-5000576). A method and apparatus for analyzing gene expression by bioluminescence using a luminometer is disclosed in Patent Document 2 (Japanese Patent Laid-Open No. 2005-1118050).

Special table 2004-500576 JP 2005-1118050 A

However, when imaging a weakly luminescent sample, the amount of light emitted from the luminescent sample is extremely small, so it cannot be viewed with the naked eye, and the amount of light must be accumulated using a storage-type imaging means such as a CCD. Therefore, there is a restriction that an image cannot be generated. Moreover, in the cell group constituting a single cell or tissue, the weak light generated from each cell is too weak, so that the exposure time required for taking a clear image becomes long. there were. That is , since the imaging time interval is limited by the amount of light per unit time, when imaging a luminescent sample with weak light emission, a clear image can be taken over time at a long time interval, for example, at an interval of 60 minutes. Even if it was possible, there was a problem that imaging could not be performed in real time with a short exposure time of about 10 to 30 minutes , and thus with an exposure of 1 to 5 minutes . In particular, when living cells are exposed for a long time (for example, 50 minutes or longer), the cells themselves may move even on a support such as a culture vessel and a clear image may not be formed. Generally, in order to perform an analysis using an image, it is necessary to recognize an accurate contour. Therefore, the analysis result may be inaccurate when the image is unclear.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide an analysis method capable of performing desired cell analysis even with a faint light sample that generates faint light that cannot be seen with the naked eye. . It is another object of the present invention to provide an analysis method combined with a luminescent sample imaging method capable of taking a clear image with a short exposure time and in real time.

In order to solve the above-described problems and achieve the object, the method for analyzing weak light according to claim 1 according to the present invention is a method in which expression is linked to a promoter region of a gene expressed in a specific cell. Including cells into which an incomplete reporter gene of 1 and a second incomplete reporter gene operably linked to the promoter region of a gene whose expression is induced by a stimulus that contacts a substance with the cell are included A step of placing a sample in an imaging field of an imaging means, a step of bringing the substance into contact with the sample to perform stimulation, and the first reporter gene and the second reporter expressed in the cells subjected to the stimulation Detectable signals based on gene interactions are optically imaged by the imaging means, and the amount of signal generated by the reporter gene is quantified And an optical processing step of determining the further characterized by having a step of the optical processing step to generate an image analyzable image weak light.
Here, the promoter region of the first reporter gene is preferably a promoter region of a gene that is specifically expressed in nerve cells. The promoter region is preferably a promoter region of an early gene. In addition, it is preferable that the reporter gene is a photoprotein because a light amount corresponding to the expression level of the gene is stably generated for a long period of time in a mixed state with a certain amount of substrate solution. Moreover, it is preferable because accurate analysis can be performed by converting the intensity of the signal generated by the reporter gene into a luminance value. Moreover, it is preferable at the point which can identify the light-emitted cell correctly by superimposing the optical imaging image of the signal which the said reporter gene produces, and the bright field image of a cell.

  In the present invention, for optical imaging of a luminescent sample, by using an objective lens having a value of (NA ÷ β) 2 expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 or more, This is preferable in that an image of only light emission can be imaged. According to this optical condition, there is an advantage that imaging can be performed without using an expensive cryogenic cooling type imaging device.

  In particular, by using an objective lens whose numerical value (NA ÷ β) 2 expressed by numerical aperture (NA) and projection magnification (β) is 0.039 or more, imaging can be performed at a higher speed than in the past. preferable.

  Furthermore, by using the objective lens in a short time of 1 to 5 minutes when the value of (NA ÷ β) 2 expressed by the numerical aperture (NA) and the projection magnification (β) is 0.071 or more, This is preferable in that real-time imaging can be performed.

  Further, the present invention makes it possible to perform image analysis at short time intervals using an objective lens having a high numerical aperture (NA) for luminescence imaging, so that all stimulus responsiveness is not missed. This provides an excellent method in drug discovery and diagnosis. In addition, a luminescent sample (eg, a luminescent protein (eg, a luminescent protein expressed from an introduced gene (eg, luciferase gene)), a luminescent cell or a collection of luminescent cells, Even a tissue sample or a luminescent individual (such as an animal or an organ) can be analyzed with a clear exposure time and in real time.

According to the feeble light analysis method of the present invention, it is possible to provide an analysis method capable of performing desired cell analysis even with a feeble light sample that generates faint light that cannot be seen with the naked eye. In addition, when the objective lens satisfies a specific condition, it is possible to provide an analysis method capable of analyzing a clear image with a short exposure time and in real time.

  Embodiments of a faint light analysis method according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  A configuration of an apparatus for carrying out the method according to the present invention will be described with reference to FIG. FIG. 1 is a diagram showing an example of the configuration of an apparatus for carrying out the luminescent sample imaging method according to the present invention. As shown in FIG. 1, an apparatus for carrying out a luminescent sample imaging method according to the present invention is for imaging a sample 1 to be imaged with a short exposure time, and in real time. The condenser lens 3, the CCD camera 4, and the monitor 5 are included. The apparatus may further include a zoom lens 6 as shown.

  Sample 1 is a luminescent sample, for example, a luminescent protein (for example, a luminescent protein expressed from an introduced gene (such as a luciferase gene)), a luminescent cell, an aggregate of luminescent cells, or a luminescent property. Tissue samples, luminescent organs, and luminescent individuals (animals, etc.). Sample 1 may specifically be a luminescent cell into which a luciferase gene has been introduced. The objective lens 2 has a numerical value (NA ÷ β) 2 expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 or more. The condenser lens 3 collects light emitted from the sample 1 that has reached through the objective lens 2. The CCD camera 4 is a cooled CCD camera at about 0 ° C. and images the sample 1 through the objective lens 2 and the condenser lens 3. The monitor 5 outputs an image captured by the CCD camera 4.

  The value of (NA / β) 2 is written in the objective lens 2 and the packaging container (package) of the objective lens 2. The conventional objective lens indicates the lens type (eg “PlanApo”), magnification / NA oil immersion (eg “100 × / 1.40 oil”) and infinity / cover glass thickness (eg “∞ / 0.17”). It had been. However, the objective lens (objective lens 2) of the imaging means according to the method of the present invention includes a lens type (for example, “PlanApo”), magnification / NA oil immersion (for example, “100 × / 1.40 oil”), infinity / In addition to the cover glass thickness (for example, “∞ / 0.17”), an injection opening angle (for example, “(NA / β) 2: 0.05”) is also described.

  As described above, in the apparatus for carrying out the luminescent sample imaging method according to the present invention, the objective lens 2 is represented by (NA ÷ β) 2 represented by the numerical aperture (NA) and the projection magnification (β). The value is 0.01 or more. As a result, a luminescent sample (for example, a luminescent protein (for example, a luminescent protein expressed from an introduced gene (for example, luciferase gene)), a luminescent cell, or a collection of luminescent cells, A clear image can be taken with a short exposure time and in real time even with a sex tissue sample or a luminescent individual (such as an animal or an organ). Specifically, a luminescent cell into which a luciferase gene has been introduced can be used as an imaging target, and a clear image can be taken with a short exposure time and in real time. In addition, since the objective lens 2 has a larger numerical aperture and a lower magnification as compared with a conventional objective lens, a wide range can be imaged with high resolution by using the objective lens 2. Thereby, for example, a moving luminescent sample, a moving luminescent sample, or a luminescent sample distributed over a wide range can be set as an imaging target. The objective lens 2 is expressed by the numerical aperture (NA) and the projection magnification (β) (NA ÷ β) 2 on the objective lens 2 and / or a packaging container (package) that wraps the objective lens 2. Value (for example, 0.01 or more) was expressed. Thus, for example, a person who observes a luminescent image can easily find an objective lens suitable for imaging a luminescent sample with a short exposure time and in real time if the value of (NA ÷ β) 2 is confirmed. You can choose.

  Conventional reporter assays using the luciferase gene measure the amount of luminescence after lysing the cells, so only the expression level at a certain point in time can be captured, and the average value of the entire cell is measured. . Further, in measurement while culturing, changes in the expression level of cell colonies over time can be caught, but changes in the expression level in individual cells cannot be caught. In order to observe the luminescence of individual cells with a microscope, the amount of luminescence from living cells is extremely weak, so it can be exposed for a long time with a cooled CCD camera at the liquid nitrogen temperature level, or an image intensifier is attached. You have to do photon counting with a CCD camera. Therefore, the camera for detecting light emission is expensive and large. However, when observing the luminescence of individual cells exhibiting luciferase activity as a reporter gene product with a microscope, an image intensifier is attached if the apparatus for carrying out the luminescent sample imaging method according to the present invention is used. The quantitative image can be acquired using a cooled CCD camera at about 0 ° C. That is, if the apparatus for carrying out the luminescent sample imaging method according to the present invention is used, the luminescence of individual cells can be observed with a cooled CCD camera at about 0 ° C. in an alive state. No fire or photon counting device is required. That is, the luminescent sample can be imaged at low cost. In addition, if the apparatus for performing the luminescent sample imaging method according to the present invention is used, the luminescence of individual living cells can be observed over time while being cultured, and can also be observed in real time. . Moreover, if the apparatus for performing the luminescent sample imaging method according to the present invention is used, it is possible to monitor the response of drugs and stimuli under different conditions for the same cell.

Here, in order to facilitate understanding of the luminescent sample imaging method, the luminescent cell imaging method and the objective lens according to the present invention, a conventional objective lens and luminescent image observation using the objective lens will be briefly described.
In general, the spatial resolution ε in microscopic observation is expressed by the following mathematical formula 1.
ε = 0.61 × λ ÷ NA (Formula 1)
(In Equation 1, λ is the wavelength of light and NA is the numerical aperture.)
Further, the diameter d of the observation range is expressed by the following mathematical formula 2.
d = D ÷ M (Formula 2)
(In Equation 2, D is the number of fields of view and M is the magnification. The number of fields of view is generally 22 to 26.)
Conventionally, the focal length of an objective lens for a microscope has been set to 45 mm according to international standards. Recently, an objective lens having a focal length of 60 mm has been used. If a lens with a large NA, that is, a high spatial resolution is designed on the assumption of this focal length, the working distance (WD) is generally about 0.5 mm, and even a long WD design is about 8 mm. When such an objective lens is used, the observation range is about 0.5 mm in diameter.

  However, when observing a cell group, tissue, or individual dispersed in a dish or glass bottom dish, the observation range may range from 1 to several centimeters. When it is desired to observe such a range with a high resolution, the NA must be maintained at a large value while the magnification is low. In other words, since NA is the ratio of the lens radius to the focal length, an objective lens that can observe a wide range with a large NA needs to be low magnification. As a result, such an objective lens has a large aperture. In manufacturing a large-diameter objective lens, generally high accuracy is required in terms of uniformity of physical properties and coating uniformity of an optical material and lens shape.

In the case of microscopic observation, the light transmittance of the optical system, the numerical aperture of the objective lens, the projection magnification on the chip surface of the CCD camera, the performance of the CCD camera, etc. greatly affect the brightness of the image. The brightness of the image is evaluated by the square of the value obtained by dividing the numerical aperture (NA) by the projection magnification (β), that is, (NA / β) 2. Here, the objective lens generally has a relationship of the following Equation 3 between the incident aperture angle NA and the exit aperture angle NA ′, and NA ′ 2 indicates the brightness that reaches the observer's eyes, a CCD camera, or the like. Value.
NA ′ = NA ÷ β (Formula 3)
(In Equation 3, NA is the incident aperture angle (numerical aperture), NA ′ is the exit aperture angle, and β is the projection magnification.)
In a general objective lens, NA ′ is at most 0.04, and NA ′ 2 is 0.0016. Further, when the value of image brightness (NA / β) 2 in a general microscope objective lens currently on the market was examined, it was in the range of 0.0005 to 0.002.

However, using a microscope equipped with a commercially available objective lens as described above, for example, even if a cell expressing a luciferase gene and emitting light is observed, the light emitted from the cell is visually observed. In addition, even if a light emission image taken using a CCD camera cooled to about 0 ° C. is observed, light emission from the cells cannot be confirmed. When observing a luminescent sample, it is not necessary to project excitation light necessary for fluorescence observation. For example, in epifluorescence observation, the objective lens satisfies both functions of an excitation light projection lens and a lens that collects fluorescence to form an image.
Therefore, in order to observe light emission with a small amount of light with an image, an objective lens having large NA and small β characteristics is required. As a result, the objective lens tends to have a large aperture. Note that such an objective lens is required to be easily designed and manufactured by simplifying the function without considering the function of projecting the excitation light.

In the research field using luminescence and fluorescence observation, time-lapse and moving image capturing are required to capture the dynamic functional expression of protein molecules in a sample. Recently, video observation of one molecule of protein using fluorescence has been performed. In these imaging operations, the exposure time per image frame becomes shorter as the number of imaging frames per unit time increases. Such observation requires a bright optical system, particularly a bright objective lens. However, since the amount of photoprotein is less than that of fluorescence, it often takes an exposure time of, for example, 20 minutes to image one frame. In order to perform time- lapse observation with such an exposure time, a dynamic change is limited to a very slow sample. For example, in cells that divide once every hour, changes within that cycle cannot be observed. Therefore, it is important to improve the brightness of the optical system in order to efficiently image a small amount of light while maintaining a high signal-to-noise ratio.

  The objective lens of the present invention manufactured based on the above circumstances has characteristics of a large NA and a small β as compared with the above-described objective lenses generally on the market. Therefore, NA′2 of the objective lens of the present invention is a large value. That is, it can be said that the objective lens of the present invention is a bright objective lens. Thereby, if a bright objective lens like the objective lens of this invention is used, the light emission from the light emission sample with little light quantity can be observed with an image. In order to observe a darker image, a CCD camera cooled to about 0 ° C. without mounting an image intensifier by mounting the objective lens of the present invention having a large numerical aperture on a stereomicroscope, Cell luminescence can be observed with images. In addition, there is a method of increasing the sensitivity with a CCD camera using liquid nitrogen cooling. In this case, the CCD camera becomes very expensive and large-scale. However, if the objective lens of the present invention is used, the light emission of the cells can be observed as an image even with a CCD camera using Peltier cooling. Moreover, the objective lens of the present invention has a large diameter of several to about 10 cm. Thereby, a moving luminescent sample that could not be an imaging target in the past or a luminescent sample distributed over a wide range can be set as an imaging target.

  The measurement principle for implementing the method of the present invention, the configuration of the optical system, and the like have already been described with reference to FIG. 1, but refer to the applications by the present applicant (Japanese Patent Application Nos. 2005-267531 and 2005-44737). May be. As described in the above-mentioned application, the applicant, through optical experiment, has a square value of (NA ÷ β) represented by a numerical aperture (NA) of the objective lens and a projection magnification (β) of 0. By taking an image with an optical system of 0.01 or more, proof data that can be imaged only by light emission generated from a single cell was obtained. Furthermore, the present applicant has proceeded with studies and found that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish. Surprisingly, the above imaging conditions can also be applied to weak luminescent images handled in the application examples implemented in the present invention, and weak such as bioluminescence in a short time (for example, 1 to 20 minutes). Cell images can be captured with various luminescent components. Further, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) is 0.071 or more, the objective lens of the imaging device can be imaged within 1 to 5 minutes. It was also found that cell images that can be analyzed can be provided.

  As shown in FIG. 1 described above, the apparatus for carrying out the imaging method according to the present invention is for imaging a sample 1 to be imaged with a short exposure time and in real time, and has been conventionally employed. A high numerical aperture (NA) objective lens 2, a CCD camera 4 and a monitor 5 are basically configured. In the present invention, an apparatus having these basic configurations is referred to as a light emission microscope. In order to perform imaging in a dark field, the light emission microscope is preferably accommodated by a light shielding lid or housing as appropriate. Moreover, imaging can be automatically performed over a long period of time by combining a culture apparatus capable of maintaining appropriate culture conditions integrally with a light emission microscope. In addition, if it is a structure which has an imaging mechanism, it does not need to be in the form of a microscope, and may be in the form of a measuring instrument such as a microplate reader.

Next, the configuration, form, and operation of the light emission microscope used in the present invention will be described. The basic feature of the luminescence microscope is that light emitted from the photoprotein expressed in the cells in the microscope field can be monitored in a short time by imaging with a digital camera through the objective lens (NA0.75) and optical filter. That is. Since a gene expression pattern can be obtained in real time as a luminescence image, it is a technology that is expected to be applicable to a wide range of research subjects in gene expression assay systems using cells. The basic measurement system consists of a sample observation unit that is an optical system adjacent to the culture apparatus unit, and the light emitted from the optical system is captured by a digital camera through an objective lens (NA 0.75, preferably NA 0.75 or more) and an optical filter. Later, data recording and analysis are performed on a digital image capturing personal computer. The culture apparatus part can be kept warm and moisturized by a heat plate and a chamber. The temperature in the culture apparatus is set to 25-37 ° C and the sample is observed. The sample is preferably observed at 37 ° C. The humidity is set to 0 to 100% and observed. Preferably, it is set to 60 to 100%. Furthermore, by acquiring a bright-field image in the same field as when optical luminescence imaging was performed, and superimposing the light-emission image and the bright-field image with image analysis software or the like, it is possible to identify the cells that emit light. .

The method and apparatus of the present invention are useful for application to the embodiments described below.

TECHNICAL FIELD This aspect relates to a method for analyzing gene expression in specific cells in the present invention.

Background Art In the conventional phlomotor assay method, it was impossible to see that the change in gene expression level with respect to external stimuli was different for each cell. However, the response to stimuli is more complex in heterogeneous cell populations, as it is known that the response of individual cells to stimuli is different even when using cell lines with relatively uniform properties. Is expected. Furthermore, conventionally, since an experimental system in which uniform cells are gathered was used, it was not necessary to identify individual cells, and the average value of all cells was observed. However, in experimental systems using heterogeneous cell populations such as primary cultures and slice cultures, signals from the target cells overlap with those of surrounding cells using the conventional phlomotor assay, so that the cells are accurately identified. I can't. In addition, early genes (such as c-fos) whose expression is induced by stimulation from outside the cell are known to be expressed in many cell types. It is possible to observe the reactivity of cells to the real time. However, in the presence of multiple types of cells, it is difficult to identify cells that have been expressed. Therefore, it is necessary to construct an experimental system in which changes in the expression level of the initial gene can be seen in real time only in specific cells.

Problems arising from the above background The conventional method does not consider observing gene expression only in specific cells among a group of cells consisting of a plurality of cell types, and does not specify a detectable signal in which a luciferase gene is generated. We could not cope with the point of optical imaging only from cells. The present invention pays attention to this point, and provides a method for analyzing gene expression in specific cells by emitting a signal in which a luciferase gene is generated only in a specific cell and optically imaging the signal with a light emission microscope. And

Means and effects for solving the above problems
One of the Spherispheres fused to the MyoD gene or Id gene is expressed only in specific cells using the cell-specific promoter region, and the other uses the promoter region of the gene (such as the first-stage gene) whose expression level is to be analyzed. To express in cells. By observing this cell sample with a light emission microscope, it is possible to optically image specific cells in which both fusion genes are expressed and the luciferase enzyme activity is observed.

  In this embodiment, in a cell using a mammalian two-hybrid system as described in a patent (US Pat. No. 5,670,356) and a non-patent paper (PNAS (2002) vol.99 No.24 pp3105-3110). Reference can be made to methods for detecting interactions between tanks and fluoromotor assays. These methods include fusion protein expression vectors of genes known to cause interactions (MyoD and Id genes) and yeast GAL4 DNA binding domain or VP16 activation domain of herpes simplex virus, and GAL4 binding region. A step of introducing a luciferase expression vector containing 5 into a cell. Here, using the principle that the expression level of luciferase increases when the target genes (MyoD gene and Id gene) interact with each other, the interaction between tanks can be detected and the fluoromotor assay can be performed. .

  In addition, in a non-patent paper (PNAS (2002) vol.99 No.24 pp15608-15613), it is also possible to refer to a method for detecting an interaction between tanks in a cell and a phlomotor assay method using a spheripher phase. This method includes the steps of dividing the luciferase gene into the N-terminal side and the C-terminal side, creating a fusion tank with genes (MyoD gene and Id gene) that are known to cause interaction and expressing them in cells. It is out. Here, luciferase has no enzyme activity in the state of N-terminal or C-terminal only, so no luminescence is detected. However, if there is a bond between fused genes (MyoD gene and Id gene), The interaction between tanks can be detected and the fluoromotor assay can be performed using the principle that the C-terminal side also binds to have enzyme activity. The following embodiment is an example that is appropriately assembled so as to be applicable to this aspect, with reference to the above method, and does not limit the present invention.

Application examples
A. Definition of phrase <Promoter region of a gene expressed in a specific cell>
The promoter region of a gene expressed in a specific cell in the present invention includes, for example, a neuron-specific gene or a glial cell-specific gene promoter.
Examples of the promoter of a neuron-specific gene used in the present invention include a promoter region of an NSE (neuron-specific enolase) gene. Examples of the glial cell-specific gene promoter used in the present invention include the promoter region of a GFAP (glial fibrillary acidic protein) gene. The promoter used in the present invention is any of the above promoters. The counterparts of these animal species are also included.
Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 4000 bases upstream from the transcription site of the gene can be used.

<Promoter region of a gene whose expression is induced by stimulation>
Examples of the gene promoter region whose expression is induced by stimulation in the present invention include a promoter of an early gene.
Examples of the promoter of the early gene used in the present invention include the c-fos promoter region. In addition, the promoter used in the present invention includes a counterpart of any animal species of the promoter.
Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Reporter gene>
The reporter gene in the present invention means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example.

<Substance>
The substance used in the present invention means a natural substance existing in nature or any substance prepared artificially.
Specifically, for example, any compound synthesized chemically can be mentioned. The type and molecular weight of the compound are not particularly limited.
When the substance is a protein or peptide, those isolated from living tissues and cells and those prepared by genetic recombination or chemical synthesis are also included. Furthermore, those chemical modifications are also included.

<Optical imaging>
Optical imaging in the present invention means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted by a reporter gene in a cell into which the reporter gene has been introduced. In order to achieve optical imaging, the intensity of the signal emitted by the reporter gene must be high enough so that the signal can be analyzed from outside the cell. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously. Note that the technology for processing image information two-dimensionally or three-dimensionally at an arbitrary position of an imaged sample image can also be referred to Japanese Patent Application Nos. 2004-172156 and 2004-178254 by the present applicant. Good. The difference between time-series image acquisition in fluorescence observation and light emission observation is that there is no influence of extra light because light emission observation does not require optical scanning (laser scan) with excitation light. According to the imaging technique performed in the present invention, a luminescent image can be captured in real time.

B. Optical imaging method of gene expression <Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The gene-introduced cells used in the present invention include both transiently expressing cells and stable expressing cells.

<Optical imaging of gene expression by stimulation>
The present invention can be implemented, for example, as follows.
N-terminal luciferase (NLuc) gene (amino acid number 1-416) and mouse Id gene (amino acid numbers 29-148) fusion gene (NLuc-Id gene), and C-terminal luciferase (CLuc) gene (amino acid number) 398-550) and a mouse MyoD gene (amino acid numbers 1-318) are prepared (MyoD-CLuc gene). As shown in FIG. 2, when the Id gene site in the NLuc-Id gene expressed in the cell interacts with the MyoD gene site in the MyoD-CLuc gene, the NLuc gene and the CLuc gene are bound and detected. Emits possible signals.

  In addition, cell-specific expression is expressed together with the NLuc-Id gene operably linked to the promoter region of the early gene (for example, the promoter region of the c-fos gene) whose expression is induced by stimulation with the substance contacting the cell. If you want to observe neurons, use the promoter region of the NSE (neuron-specific enolase) gene, which is expressed specifically in nerve cells when you want to observe nerve cells. A cell group consisting of a plurality of cell types obtained by mixing and culturing a MyoD-CLuc gene operably linked to a GFAP (glial fibrillary acidic protein) gene expressed using a promoter region) Introduced into a mixed culture of neurons and glia cells). Alternatively, the promoter region of a gene that expresses cell-specifically together with the MyoD-CLuc gene operably linked to the promoter region of the initial gene (eg, the promoter region of the c-fos gene) In some cases, the NSE (neuron-specific enolase) gene promoter region expressed specifically in nerve cells is used. To observe glial cells, the GFAP (glial fibrillary acidic protein) gene promoter region expressed specifically in the glial cells The NLuc-Id gene operably linked to a cell group (for example, a mixed culture of nerve cells and glia cells) composed of a plurality of cell types mixed and cultured using a conventional gene transfer method.

Use a device (for example, petri dish, multi-plate having many wells, etc.) capable of culturing a desired cell for the constant of the introduced cells (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ^{3 to} 1 × 10 ⁶ cells) And cultured in a desired nutrient medium (eg, D-MEM medium). A sample of this constant number of cells is preliminarily kept at a temperature optimum for the cells (for example, 25 to 37 ° C., preferably 35 to 37 ° C.) and water is injected to prevent the sample from being dried. A luminescent image is recorded by a digital camera through an objective lens in a sample observation section of the luminescence microscope that is installed in the culture apparatus. A substance (for example, a compound) for stimulating by contacting cells is added to the sample at a desired concentration (for example, 1 pM to 1 M, preferably 100 nM to 1 mM), and a desired time interval (for example, 5 minutes) is added. -5 hours, preferably 10 minutes to 1 hour). As shown in FIG. 3, in a specific cell B in which the NLuc-Id gene (or MyoD-CLuc gene) is expressed, the expression of the MyoD-CLuc gene (or NLuc-Id gene) by stimulating the substance to contact the cell When induced, the enzyme activity of luciferase is enhanced and a detectable signal is emitted. On the other hand, no detectable signal is emitted in the cell A in which the expression of only the MyoD-CLuc gene (or NLuc-Id gene) is induced by the stimulation of contacting the substance with the cell. The recorded image uses a commercially available image analysis software (for example, MetaMorph; manufactured by Universal Imaging Co., Ltd.) to obtain a luminance value in a desired area in the image. Furthermore, a bright field image is recorded in the same field as the luminescent image, and the luminescent image and the bright field image are superimposed using the image analysis software described above to identify the luminescent cells.

According to the description described in the above embodiment, it can be understood that the present invention is an invention expressed in the following supplementary items.

Additional Item 1
A method for analyzing gene expression in specific cells,
Introducing into the cell an incomplete reporter gene operably linked to a promoter region of a gene expressed in a specific cell;
Introducing into the cell an incomplete reporter gene operably linked to the promoter region of the gene whose expression is induced by the stimulus of contacting a substance with the cell;
Maintaining the cells in a state where they can continue to survive;
A step of bringing the substance into contact with a cell for stimulation;
Optically imaging a detectable signal generated by binding of the incomplete reporter genes expressed in each stimulated cell;
Quantitatively determining the amount of signal generated by the reporter gene at a high S / N ratio;
A method for analyzing gene expression in a specific cell, comprising a step of identifying a cell in which a signal generated by the reporter gene is detected.
Additional Item 2
Item 2. The method according to Item 1, wherein the promoter region of a gene expressed in the specific cell is a promoter region of a gene expressed specifically in a nerve cell.
Additional Item 3
Item 3. The method according to Item 2, wherein the nerve cell-specific gene is an NSE (neuron-specific enolase) gene.
Additional Item 4
Item 2. The method according to Item 1, wherein the promoter region of a gene expressed in the specific cell is a promoter region of a gene expressed specifically in glial cells.
Additional Item 5
Item 5. The method according to item 4, wherein the glial cell-specific expression gene is a GFAP (glial fibrillary acidic protein) gene.
Additional Item 6
Item 2. The method according to Item 1, wherein the promoter region whose expression is induced by the stimulation is a promoter region of an early gene.
Additional Item 7
Item 7. The method according to Item 6, wherein the earliest gene is a human-derived c-fos gene.
Additional Item 8
Item 2. The method according to Item 1, wherein the incomplete reporter gene comprises a part of the gene sequence of the photoprotein.
Additional Item 9
Item 9. The method according to Item 8, wherein the photoprotein is firefly-derived luciferase.
Additional Item 10
Item 9. The method according to Item 8, wherein the photoprotein is Renilla luciferase.
Additional Item 11
Item 2. The method according to Item 1, wherein the incomplete reporter gene operably linked to the promoter region is introduced into a cell.
Additional Item 12

Item 2. The method according to Item 1, wherein the substance that contacts the cell is a ligand of a G protein-coupled receptor.
Additional Item 13
Item 2. The method according to Item 1, wherein the substance to be contacted with cells is serum.
Additional Item 14
Item 2. The method according to Item 1, wherein a signal generated by combining the incomplete reporter genes is optically imaged.
Additional Item 15
Item 15. The method according to Item 14, wherein imaging is performed with a light emission microscope in the optical imaging.
Additional Item 16
Item 2. The method according to Item 1, wherein the intensity of the signal generated by the reporter gene is converted into a luminance value.
Additional Item 17
Item 2. The method according to item 1, wherein a bright-field image is captured with a light emission microscope in the cell.
Additional Item 18
Item 2. The method according to Item 1, wherein a cell producing a signal is identified by superimposing an optical imaging image of a signal generated by the reporter gene and a bright field image of the cell.

The method and apparatus of the present invention are useful for application to other embodiments as described below.

TECHNICAL FIELD This aspect of the present invention relates to a method for analyzing a change in gene expression level in a single cell by optical imaging of a signal emitted by a reporter gene in the present invention.

Background Art As a conventional method for analyzing gene expression by optical imaging, a fluorescent protein is used as a reporter gene (special table 2004-5000576). A gene expression vector in which green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (RFP) genes are linked downstream of the promoter region is prepared and introduced into cells. The amount of fluorescence obtained by irradiating the excited light is measured. Since the amount of fluorescence is proportional to the expression level of the gene, the expression level of the target gene can be observed. On the other hand, as a gene expression analysis method using bioluminescence, a promoter of a gene whose expression is induced by stimulation via G protein, as disclosed in JP-A-2005-111050, JP-A-2000-354500, and JP-A-36443288. A gene expression vector in which a luciferase gene is linked downstream of the region is prepared and introduced into a cell, and light emitted from the cell after stimulation via G protein is detected with a luminometer. Since the amount of luminescence is proportional to the expression level of the gene, it is possible to observe the expression level of the gene whose expression is induced by stimulation.

Problems arising from the above background Conventional techniques using fluorescent proteins do not take into account phototoxicity to cells caused by irradiation with excitation light, and cannot cope with maintaining cells in a state where they can continue to survive. Therefore, paying attention to this point, it is an object to provide a method for analyzing gene expression in a single cell using bioluminescence, in which there is no need to irradiate excitation light, and thus there is little damage to cells. In addition, the conventional technology using fluorescent proteins does not consider the background of excitation light itself or autofluorescence of intracellular substances, and quantitatively determines the amount of signal generated by the reporter gene with a high S / N ratio. I cannot cope with the point of doing. Therefore, it is also an object to provide a method for analyzing gene expression in a single cell using bioluminescence, which uses a photoprotein that does not require excitation light and has a relatively low background. Furthermore, in the prior art using a photoprotein, it is not considered to observe a signal generated by a reporter gene in a single cell, and it is possible to cope with optical imaging of a detectable signal generated by a reporter gene. I can't. Therefore, focusing on this point, it is also an object to provide a method for analyzing gene expression in a single cell using bioluminescence, in which imaging is performed with a luminescence microscope so that cells emitting a signal generated by a reporter gene can be identified. Become.

In order to achieve the purpose of solving the above-mentioned problems with respect to means and action effects to solve the above-mentioned problems, as described later, a luciferase gene such as firefly or Renilla is used as a reporter gene to excite the generation of a signal. The present inventors have found that optical imaging can be performed in a state where there is no need to irradiate light, damage to cells is reduced, and background due to autofluorescence of intracellular substances is reduced. In addition, in a method of analyzing gene expression level in cells using a luciferase gene as a reporter gene, when observation is performed using a luminescence microscope, expression of the reporter gene can be optically imaged in a single cell by adding a ligand. I found out. In addition, by using the promoter region of the human c-fos gene as a promoter to control the expression of the reporter gene, it was found that when ATP or serum was added as a ligand, increased expression of the reporter gene product could be optically imaged in a single cell. . Furthermore, by capturing bright-field images of cells in the same field of view as the luminescent image, overlaying the images, and identifying the cell from which the luminescent image was obtained, the change in gene expression over time in a single cell is obtained. We have found that this is possible and have completed the invention.

A. Definition of phrase <Promoter region of a gene whose expression is induced by stimulation>
Examples of the promoter region in the present invention include an early gene promoter. Examples of the promoter of the early gene used in the present invention include the c-fos promoter region. In addition, the promoter used in the present invention includes a counterpart of any animal species of the promoter. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Reporter gene>
The reporter gene in the present invention means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example.

<Substance>
The “substance” used in the invention described below means a natural substance existing in nature or any substance prepared artificially. Specifically, for example, any compound synthesized chemically can be mentioned. The type and molecular weight of the compound are not particularly limited. When the substance is a protein or peptide, those isolated from living tissues and cells and those prepared by genetic recombination or chemical synthesis are also included. Furthermore, those chemical modifications are also included.

<Ligand>
The ligand of the G protein-coupled receptor used in the present invention means any agonist that interacts with the G protein-coupled receptor or any antagonist of the receptor.

<Optical imaging>
Optical imaging in the present invention means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted by a reporter gene in a cell into which the reporter gene has been introduced. In order to achieve optical imaging, the intensity of the signal emitted by the reporter gene must be high enough so that the signal can be analyzed from outside the cell. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously.

B. Optical imaging method of gene expression <Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The gene-introduced cells used in the present invention include both transiently expressing cells and stable expressing cells.

<Optical imaging of gene expression by stimulation>
The present invention can be implemented, for example, as follows.
A reporter gene (preferably a firefly or a reporter gene) operably linked to a cell constant (for example, a promoter region of a gene whose expression is induced by stimulation with which the substance is brought into contact with the cell (preferably, the promoter region of the c-fos gene)) A luciferase derived from Renilla, etc. is introduced into a cell using the above-described gene introduction method, and constants (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ^{3 to} 1 × 10 ⁶ cells) of the obtained cells into which the gene has been introduced are obtained. ) Is cultured in a desired nutrient medium (eg, D-MEM medium) using an apparatus capable of culturing the desired cell (eg, petri dish, multi-plate having a large number of wells, etc.) In advance, keep the sample at a temperature optimal for the cells (for example, 25 to 37 ° C, preferably 35 to 37 ° C), and inject water to prevent the sample from drying. A luminescence image is recorded by a digital camera through an objective lens in a sample observation section of the luminescence microscope, and a substance for stimulating the sample by contacting the cell (for example, , Compound) at a desired concentration (eg, 1 pM to 1 M, preferably 100 nM to 1 mM), and recording a luminescent image at a desired time interval (eg, 5 minutes to 5 hours, preferably 10 minutes to 1 hour). The brightness value in the desired area in the image is acquired using commercially available image analysis software (for example, MetaMorph; manufactured by Universal Imaging Co., Ltd.), and the bright field image is recorded in the same field of view as the emission image. Then, the light emitting image and the bright field image are superimposed using the image analysis software to identify the light emitting cells.

Example 1
<Construction of c-fos promoter-luciferase reporter gene>
A c-fos promoter-luciferase reporter gene was constructed as follows. Using human genomic DNA as a template, PCR was performed using primer fos pro Fw (5'-AGCTCGAGAGCAGTTCCCGTCAATCCCT-3 ') and primer fos pro Rv (5'-CAAAGCTTTGCAGAAGTCCTAGAACAA-3'), and the promoter region of the c-fos gene (transcription) -363 to +1,067) was amplified relative to the start site. The amplified DNA fragment was digested with restriction enzymes XhoI and HindIII.
The expression vector pGL3-Basic vector (Promega) was digested with restriction enzymes XhoI and HindIII, and the above DNA fragment was cloned into the pGL3-Basic vector. (Hereinafter, it may be described as a reporter gene expression vector (pGL3-fos).)

Example 2
<Change in expression level of reporter gene when cells are stimulated with ATP as a ligand>
An example of measuring the change in the expression level of a reporter gene when ATP is used as a ligand for cell stimulation is shown below.
HeLa cells were spread on a glass bottom dish (diameter 35 mm; manufactured by IWAKI) at 1 × 10 ⁵ cells / dish. D-MEM containing 10% fetal bovine serum was used as the growth medium. After culturing the cells at 37 ° C. for 1 day, a reporter gene expression vector was introduced into the cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After about 2 hours, the gene transfer solution was discarded, and a growth medium was added, followed by culturing at 37 ° C. for 16 hours or more. The growth medium was replaced with a serum-free medium (D-MEM not containing fetal calf serum) and cultured at 37 ° C. for 16 hours or more. The incubator in which the cells were cultured was placed in a culture apparatus part of a luminescence microscope that had been set to 35 to 37 ° C. in advance, and the sample observation part was operated to focus the cells. Luciferin was added to a final concentration of 1 mM and allowed to stand for 1 hour or longer, and then ATP was added to a final concentration of 100 μM. Using a digital camera (DP30; Olympus) operation software (DP controller; Olympus), bright-field images of the cells were captured and recorded. The luminescence image was obtained by adding 5 times of exposure to a digital camera for 1 minute. Luminescent images obtained every 10 minutes were captured by a computer and recorded. The luminance value of the luminescent image was obtained using image analysis software (Metamorph; manufactured by Universal Imaging), and the luminescent image and the bright field image were superimposed to identify the luminescent cells.

FIG. 4 shows a superimposed image when identification is performed in the second embodiment. Here, the bright field image of the cell is shown in FIG. 4-1, the emission image acquired in the same field is shown in FIG. 4-2, and the bright field image and the emission image superimposed on the image analysis software are shown in FIG. 4-3. It was. From this superimposed image, the position and form of the luminescent cells could be confirmed.

As shown in FIG. 5, it was observed that the activity of luciferase, which is a reporter gene product, transiently increased in response to a stimulus that contacted ATP in a single cell. However, it was clarified that the time required for the luciferase activity to reach its peak after stimulation and the amount of activity at the peak varied between cells.

Example 3
<Change in expression level of reporter gene when cells are stimulated with serum>
An example of measurement of change in the expression level of a reporter gene when fetal bovine serum is used as a ligand for cell stimulation is shown below. Other conditions were the same as in Example 2 except that the ligand used was different. Fetal calf serum stimulation was performed at a final concentration of 10%.

FIG. 6 shows a superimposed image when identification is performed in the third embodiment. Here, a bright field image of a cell is shown in FIG. 6-1, a luminescence image acquired in the same field is shown in FIG. 6-2, and an image obtained by superimposing the bright field image and the luminescence image on image analysis software is shown in FIG. It was. The position and form of the luminescent cells were confirmed.

As shown in FIG. 7, it was observed in single cells that the activity of luciferase, which is a reporter gene product, increased transiently in response to stimulation with fetal calf serum. However, it was clarified that the time required for the luciferase activity to reach the peak after stimulation and the amount of activity at the peak varied among the cells as in the ATP stimulation of Example 2.

According to the description described in the first to third embodiments, it can be understood that the present invention is an invention expressed in the following supplementary items.

Additional Item 19
A method for analyzing changes in gene expression level in response to a stimulus in a single cell,
Introducing into the cell a reporter gene operably linked to the promoter region of the gene whose expression is induced by the stimulus of contacting a substance with the cell;
Maintaining the cells in a state where they can continue to survive;
A step of stimulating cells by contacting the substance with the substance,
Optical imaging of a detectable signal produced by the reporter gene expressed in each stimulated cell;
Quantitatively determining the amount of signal generated by the reporter gene at a high S / N ratio;
A method for analyzing a change in gene expression level in a single cell, comprising a step of identifying a cell in which a signal generated by the reporter gene is detected.
Additional Item 20
Item 20. The method according to Item 19, wherein the promoter region is a promoter region of an early gene.
Additional Item 21
Item 20. The method according to Item 20, wherein the initial gene is a human-derived c-fos gene.
Additional Item 22
Item 201. The method according to Item 19, wherein the reporter gene is a photoprotein.
Additional Item 23
Item 22. The method according to Item 22, wherein the photoprotein is firefly-derived luciferase.
Additional Item 24
Item 22. The method according to Item 22, wherein the photoprotein is Renilla luciferase.
Additional Item 25
Item 20. The method according to Item 19, wherein the reporter gene operably linked to the promoter region is introduced into a cell.
Additional Item 26
Item 20. The method according to Item 19, wherein the substance brought into contact with the cell is a ligand of a G protein-coupled receptor.
Additional Item 27
Item 27. The method according to Item 26, wherein the ligand of the G protein-coupled receptor is a nucleic acid.
Additional Item 28
Item 27. The method according to Item 26, wherein the nucleic acid is adenosine triphosphate.
Addendum 29
Item 20. The method according to Item 19, wherein the substance that is brought into contact with the cell is serum.
Additional Item 30
Item 20. The method according to Item 19, wherein the signal generated by the reporter gene is optically imaged.
Additional Item 31
Item 31. The method according to Item 30, wherein imaging is performed with a light emission microscope in the optical imaging.
Additional Item 32
Item 201. The method according to Item 19 wherein the intensity of the signal generated by the reporter gene is converted into a luminance value.
Additional Item 33
Item 20. The method according to item 19, wherein a bright-field image is captured with a light emission microscope in the cell.
Additional Item 34
Item 20. The method according to Item 19, wherein a cell producing a signal is identified by superimposing an optical imaging image of a signal generated by the reporter gene and a bright field image of the cell.

It is a figure which shows the outline | summary of the apparatus for enforcing the analysis method of this invention. In the embodiment of the present invention, it is a diagram showing the interaction between the Id gene site in the NLuc-Id gene expressed in cells and the MyoD gene site in the MyoD-CLuc gene. It is a figure which shows the induction | guidance | derivation using the stimulating substance in the cell B in which NLuc-Id gene (or MyoD-CLuc gene) is expressing. It is a figure which shows the bright-field image at the time of identifying in Example 2 in another aspect. It is a figure which shows the light emission image at the time of identifying in Example 2. FIG. It is a figure which shows the image which overlap | superposed each image of FIGS. 2-1 and 2-2. It is a figure which shows the result of having analyzed the luciferase activity for every elapsed time after the stimulation by ATP. 6 is a diagram showing a bright field image when identification is performed in Example 3. FIG. It is a figure which shows the light emission image at the time of identifying in Example 3. FIG. It is a figure which shows the image which overlap | superposed each image of FIGS. 4-1 and FIGS. 4-2. It is a figure which shows the result of having analyzed luciferase activity for every elapsed time after stimulation by serum.

Explanation of symbols

1: Sample 2: Objective lens 3: Condensing lens 4: CCD camera 5: Monitor 6: Zoom lens

Claims (10)

Can be expressed in the first incomplete reporter gene operably linked to the promoter region of a gene expressed in a specific cell, and in the promoter region of a gene whose expression is induced by a stimulus that contacts a substance with the cell Placing a sample containing cells into which a second incomplete reporter gene linked to is within the imaging field of the imaging means;
Performing the stimulation by bringing the substance into contact with the sample;
The imaging means optically images a detectable signal based on the interaction between the first reporter gene and the second reporter gene expressed in the stimulated cell, and the amount of the signal generated by the reporter gene is determined. An optical processing step for quantitative determination,
The weak light analysis method, wherein the optical processing step further includes a step of generating an image capable of image analysis of weak light. The weak light analysis method according to claim 1, wherein the promoter region of the first reporter gene is a promoter region of a gene that is specifically expressed in a nerve cell. The weak light analysis method according to claim 2, wherein the gene specifically expressed in the nerve cell is an NSE (neuton-specifec enolase) gene. The weak light analysis method according to any one of claims 1 to 3, wherein the reporter gene is a photoprotein. The weak light analysis method according to claim 4, wherein the intensity of a signal generated by the reporter gene is converted into a luminance value. The weak light analysis method according to claim 4, wherein an optical imaging image of a signal generated by the reporter gene and a bright field image of a cell are superimposed. 2. The objective lens according to claim 1, wherein the imaging means uses an objective lens having a square value of (NA ÷ β) expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 or more. The weak light analysis method in any one of -6. The weak light analysis method according to claim 7, wherein the square of (NA ÷ β) is 0.039 or more. The weak light analysis method according to claim 8, wherein the square of (NA ÷ β) is 0.071 or more. The weak light analysis method according to claim 7, wherein the optical imaging is performed at a short time interval using an objective lens having a high numerical aperture (NA).