JP2003254902A - Method and device for observing three dimensional localization of in vivo expressed gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To observe localization of an in vivo expressed gene in the whole of one individual of an organism such as an animal or a plant. <P>SOLUTION: A gene recombined organism sample having a marker detectable when a specific gene is expressed is sequentially cut, and a sectional image forming the image of a cut surface is picked up for every cut of the organism sample. The three dimensional localization of the expressed gene in the organism sample is observed by observing the three dimensional organism sample based on the image picked up for every cut. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and apparatus for observing the three-dimensional localization of a gene expressed in a living body, and more specifically, when observing the expression status of various genes in a living body such as an animal or a plant. The present invention relates to a method for observing the three-dimensional localization of an in vivo expressed gene and a device therefor.

[0002]

BACKGROUND OF THE INVENTION Conventionally, in observing the localization of genes expressed in vivo, that is, genes expressed in vivo,
A living body to be a sample (in the present specification, the “living body to be a sample” will be appropriately referred to as a “biological sample”) is sectioned, and the sectioned biological sample is observed, thereby A method of observing the two-dimensional arrangement of expressed genes has been adopted.

When performing the above-mentioned observation method, a gene (recombinant) biological sample having a marker (label) which can be detected at the time of expression is incorporated into the gene to be observed, and the gene having the marker is incorporated. It is becoming possible to separate the expression site of a specific gene in a biological sample by observing.

As the marker, a fluorescent substance, a luminescent substance, a dye or the like can be used, and specific examples thereof include GFP, EGFP, YFP, RFP, luciferin and melanin dye.

For these observations, a stereomicroscope is used for relatively large macro observations, but the observable range is limited because the entire biological sample is seen through, and it is also possible to observe minute parts. It was difficult and the resolution was poor.

Therefore, according to the conventional observation method,
There has been a problem that it is extremely difficult to observe the localization of a gene expressed in a living body as a whole.

As a method for observing the expressed gene by sectioning, "in-situ Hybridizati" is used.
However, with this method, it is necessary to permeate a marker such as a fluorescent substance or a dye into the inside of the biological sample, and it is difficult to uniformly stain the inside of the biological sample.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the problems of the above-mentioned conventional techniques, and its object is to provide a single living organism such as an animal or a plant. An object of the present invention is to provide a method and apparatus for observing the three-dimensional localization of an in vivo expressed gene, which makes it possible to observe the localization of an in vivo expressed gene.

[0009]

To achieve the above object, in the method for observing the three-dimensional localization of an in vivo expressed gene or its apparatus according to the present invention, a marker that can be detected when a specific gene is expressed. In the biological sample body by continuously cutting a genetically modified biological sample having the following, and observing a cross-sectional image that is an image of the cut surface for each cutting, to reconstruct a three-dimensional structure inside the biological sample. The three-dimensional localization of the expressed gene is observed.

That is, the invention according to claim 1 of the present invention continuously cuts a genetically modified biological sample having a marker that can be detected when a specific gene is expressed, and every time the biological sample is cut, By taking a cross-sectional image that is an image of the cut surface and observing the biological sample three-dimensionally based on the image taken for each cutting, the three-dimensional localization of the expressed gene in the biological sample body is confirmed. It was made to be observed.

Further, the invention according to claim 2 of the present invention comprises a genetically modified biological sample having a marker which can be detected when a specific gene is expressed, and a cutting means for continuously cutting the biological sample. An image pickup unit that picks up a cross-sectional image that is an image of the cut surface every time the biological sample is cut, and an image processing unit that generates a three-dimensional image of the biological sample based on the image picked up by the image pickup unit. By observing the three-dimensional image of the biological sample generated by the image processing means, the three-dimensional localization of the expressed gene in the biological sample body is observed.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION An embodiment of the method for observing the three-dimensional localization of an in vivo expressed gene and the apparatus therefor according to the present invention will be described below in detail with reference to the accompanying drawings.

FIG. 1 is a conceptual structural explanatory view showing an example of an embodiment of an apparatus for observing the three-dimensional localization of a gene expressed in vivo according to the present invention.

The apparatus for observing the three-dimensional localization of an in-vivo expressed gene according to the present invention (hereinafter appropriately referred to as "the present apparatus") is detected as a biological sample as an observation target when a specific gene is expressed. A gene recombinant having a gene incorporating a marker that enables

Here, when producing a gene recombinant having a marker that can be detected when the above-mentioned specific gene is expressed, for example, recombinant DN is added to egg cells.
A method of injecting A may be used, or a method of producing a recombinant gene body by an assembly chimera may be used.

That is, the gene recombinant used in the present invention as a biological sample may be produced by any method, and the present invention is not limited to the production method.

As the marker, a conventionally known fluorescent substance, luminescent substance, dye or the like can be used. Specifically, GFP, EGFP, YFP, RFP,
Luciferin, melanin pigment, etc. may be used. However, the markers are not limited to those described above, and substances other than those described above can be used as appropriate as long as they can be detected when a specific gene is expressed.

Incidentally, in detecting the marker, for example, electromagnetic waves may be used. Specifically, when white light is used as the electromagnetic wave, it is to detect a specific wavelength reflected by applying light of a wide wavelength, so when using a dye as a marker, it is possible to observe with white light. It will be possible. That is, when a dye is used as a marker, an electromagnetic wave having a non-specific wavelength is irradiated and an electromagnetic wave having a specific wavelength is received and detected.

When a substance that emits light is used as the marker, it is not necessary to irradiate the electromagnetic wave, and the electromagnetic wave having a specific wavelength is received for detection.

Further, when a fluorescent dye is used as the marker, an electromagnetic wave having a specific wavelength is irradiated and an electromagnetic wave having a specific wavelength is received and detected.

The present apparatus shown in FIG. 1 is a fluorescent substance which, when expressed in a living body as a marker, emits light or develops color when excited by irradiation with light having a predetermined wavelength, and behaves differently from other genes. A biological sample composed of a genetically modified substance having a gene incorporating a substance (hereinafter appropriately referred to as "marker-incorporated genetically modified biological sample") is cut with a knife to prepare a cross section of the marker-incorporated genetically modified biological sample. Cross-section creation device (slicer) 10 as cutting means, and cross-section creation device 1
The light source 12 for irradiating the cross section of the marker-incorporated genetically modified biological sample created by 0 with light of a predetermined wavelength for exciting the marker of the marker-incorporated genetically modified biological sample, and the cross-section preparing device 10 are prepared. In addition, the cross-sectional image observing section serving as an imaging unit configured by a CCD camera 14 or the like for taking a cross-sectional image of the marker-incorporated genetically modified biological sample irradiated with light of a predetermined wavelength by the light source 12 It is composed of an image recording device 16 that stores an image of an imaged cross-section of a genetically modified biological sample incorporating a marker, a cross-section creation device 10, and a personal computer that outputs a control signal for controlling the operation of the image recording device 16. Markers recorded in the control device 18 and the image recording device 16 An image processing device (graphic work station) 20 as an image processing means for performing image processing on an image of a cross section of an integrated genetically modified biological sample to generate a processed image such as an arbitrary cross section or a stereoscopic image (three-dimensional image); An image of a cross-section of the marker-incorporated genetically modified biological sample recorded in the recording device 16 or an arbitrary cross-section or three-dimensional image processed by the image processing device 20 (3
The observer 24 observes the monitor 22 to observe the expression state of the marker-incorporated gene in the marker-integrated gene recombinant biological sample.

When a luminescent substance or the like is used as the marker, it is not necessary to provide the light source 12 in this device.

Here, the cross-section creation device 10, the cross-section image observation unit, the image recording device 16, the control device 18, and the image processing device 2 are provided.
0 and the monitor 22 are, for example, Japanese Patent Publication No. 7-109384 “Automatic inspection device equipped with a sample surface cutting device” proposed by the inventors of the present application, Japanese Patent Laid-Open No. 10-2.
An apparatus such as that disclosed in Japanese Patent No. 6586, "Sample Observation Method and Apparatus Therefor" or Japanese Patent Application Laid-Open No. 10-265586, "Sample Observation Method and Apparatus therefor" can be used.

The apparatus disclosed in each of the above publications is an apparatus for reconstructing the three-dimensional structure inside the sample by repeatedly cutting the sample to be observed and observing the cross-sectional image of the cut surface.

An example of the operation of the above-described device having the above-mentioned structure will be described. As the marker-incorporated gene recombinant biological sample, the marker-incorporated gene recombinant biological sample produced by various methods as described above is used. You can

As the light source 12 for exciting the marker, for example, a halogen light source or a xenon mercury light source,
A filter or the like can be appropriately combined and used. That is, as the light emitted from the light source 12, white light, fluorescence, or the like may be set appropriately.

That is, when a halogen light source and a xenon mercury light source using an optical filter are used as the light source 12, white light observation by the halogen light source and V, B, and G excitation fluorescence observation by the xenon mercury light source and the optical filter are performed. It is possible to observe simultaneously.

Here, the cross-section creating apparatus 10 is, for example,
"15 mm x 12 mm x 120 mm to 180 mm x 15
A marker-incorporated genetically modified biological sample having a size of “0 mm × 200 mm” can be cut to a minimum thickness of 10 μm.

The cross-section image observing unit observes the cross-section creation device 10 by irradiating the cut surface with light emitted from the light source 12 every time the marker-incorporated genetically modified biological sample is cut. This cross-sectional image observation unit, for example,
The cross section of the genetically modified biological sample incorporating the marker can be configured so that white light observation with a halogen light source and fluorescence observation of V, B, and G excitation with a xenon mercury light source and an optical filter can be performed simultaneously.

In the image recording device 16, when recording a cross-sectional image of an observed cut surface, a cross-sectional image showing observation with white light is first recorded for the same cut surface, and then a cross-section showing observation with fluorescent light is recorded. The image processing apparatus 20 controls to record an image.

The image processing device 20 performs image processing on the cross-sectional image recorded in the image recording device 16 to obtain an image of an arbitrary cross-section when observing white light, a stereoscopic image, or an image of an arbitrary cross-section when observing fluorescence. And data indicating a stereoscopic image are output to the monitor 22, and the monitor 22 displays an image based on the output data.

Next, the results of the experiments carried out by the above-mentioned device will be explained. As a marker-incorporated gene-recombinant biological sample, EGFP was added to the actin promoter.
Green mouse (G
reenMice (C57BL / 6TgN (act-E
GFP) OsbC14-Y01-FM131) (Reference 3 Ikawa et al. FEBS Lette).
rs 430 (1998) 83-87 "'Green
mice 'and ther potential
usage in biological rese
arch ")) was used.

Here, the marker-incorporated genetically modified biological sample was frozen-embedded after PFA fixation and PBS washing. The embedded marker-incorporated genetically modified biological sample is subjected to the cross-section preparation device 10 under the environment of “−45 ° C.”.
Was cut by. In addition, the cross-section creating apparatus 10 has a rotation speed of a knife used for cutting of 90 rpm and a cutting thickness of 30 rpm.
μm.

The observation by the light source 12 was carried out by using white light and fluorescent light as the observation light source, the number of cut sections was 1736, and the shooting time of the cut image was about 30 minutes.

2 to 4 show three images extracted from continuous cross-sectional images observed with white light. 2 is an image of the abdomen, FIG. 3 is an image of the chest, and FIG. 4 is an image of the head.

5 to 7 show the results of observing the same section with fluorescence as the images shown in FIGS. 2 to 4 by fluorescence. 5 is an image of the abdomen corresponding to FIG. 2, FIG. 6 is an image of the chest corresponding to FIG. 3, and FIG. 7 is an image of the head corresponding to FIG.

In FIGS. 5 to 7, the light-colored portion is G.
This is a portion in which FP is expressed (note that in FIGS. 5 to 7, the portion in which GFP is expressed actually develops green).

In the image processing device 20, a three-dimensional image is constructed by ray casting based on the sectional image recorded in the image recording device 16. FIG. 8 shows a stereoscopic image cut along the spine based on an image taken with white light. Further, FIG. 9 shows a stereoscopic image corresponding to FIG. 8 constructed on the basis of an image obtained by fluorescence observation under the same conditions.

In the white light image shown in FIG. 8, the internal organs and the entire body can be seen. In addition, it can be seen from the fluorescence image shown in FIG. 9 that GFP is strongly expressed in sites such as cartilage and skin surface.

The marker-incorporated genetically modified biological sample used in this experiment was treated with GF by the expression of actin.
Since P fluoresces, it can be inferred that actin expression is active at these sites.

That is, according to the present apparatus, it becomes possible to observe the three-dimensional localization of the expressed gene in one individual biological sample at high speed with micron accuracy.

[0042]

EFFECTS OF THE INVENTION Since the present invention is configured as described above, the excellent effect that it becomes possible to observe the localization of an in vivo expressed gene in an entire living organism such as an animal or a plant. Play.

[Brief description of drawings]

FIG. 1 is a conceptual configuration explanatory view showing an example of an embodiment of an observation apparatus for three-dimensional localization of a gene expressed in vivo according to the present invention.

FIG. 2 is an image of an abdomen extracted from continuous cross-sectional images observed with white light.

FIG. 3 is an image of a chest extracted from continuous cross-sectional images observed with white light.

FIG. 4 is an image of a head extracted from a continuous cross-sectional image observed with white light.

FIG. 5 is an image of the abdomen of the same cross section as the image shown in FIG. 2 extracted from the continuous cross section images observed by fluorescence.

6 is an image of a chest having the same cross section as the image shown in FIG. 3 extracted from the continuous cross section images observed by fluorescence.

7 is an image of the head having the same cross section as the image shown in FIG. 4 extracted from the continuous cross section images observed by fluorescence.

FIG. 8 is a stereoscopic image reconstructed from a state taken along the spine based on an image taken with white light.

9 is a three-dimensional image obtained by reconstructing a state cut along the spine based on an image observed by fluorescence under the same conditions as shown in FIG.

[Explanation of symbols]

10 Cross Section Creating Device (Slicer) 12 Light Source 14 CCD Camera 16 Image Recording Device 18 Control Device 20 Image Processing Device (Graphic Workstation) 22 Monitor 24 Observer

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Jun Yoshiki             3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture             Institute for Chemical Research Tsukuba Research Center (72) Inventor Akitake Makinouchi             2-1, Hirosawa, Wako-shi, Saitama RIKEN             Within (72) Inventor Toshiro Higuchi             2-1, Hirosawa, Wako-shi, Saitama RIKEN             Within (72) Inventor Ryutaro Himeno             2-1, Hirosawa, Wako-shi, Saitama RIKEN             Within F term (reference) 2F065 AA04 BB27 BB30 CC16 FF04                       JJ03 JJ26 TT07                 2G043 AA03 BA16 DA02 EA01 JA02                       KA02 LA03 NA05                 2G059 AA05 AA06 BB12 CC16 DD12                       EE06 EE07 FF01 FF02 GG10                       JJ02 KK04 MM10 PP04                 4B024 AA11 AA20 CA03 DA02 FA10                       HA11                 4B029 AA23 BB20

Claims (2)

[Claims] 1. A genetically modified biological sample having a marker that can be detected when a specific gene is expressed is continuously cut, and a cross-sectional image that is an image of the cut surface is taken every time the biological sample is cut, A three-dimensional station of an in-vivo expressed gene for observing the three-dimensional localization of an expressed gene in the inside of the biological sample by observing the three-dimensional biological sample based on an image captured for each cutting. Current observation method. 2. A genetically modified biological sample having a marker that can be detected when a specific gene is expressed, a cutting means for continuously cutting the biological sample, and an image of the cut surface for each cutting of the biological sample. A three-dimensional image generated by the image processing unit, which has an image capturing unit that captures a barrel cross-sectional image and an image processing unit that generates a three-dimensional image of the biological sample based on the image captured by the image capturing unit. An apparatus for observing the three-dimensional localization of an in-vivo expressed gene for observing the three-dimensional localization of an expressed gene in the biological sample by observing an image of the biological sample.