WO2006106752A1

WO2006106752A1 - Noninvasive analysis method

Info

Publication number: WO2006106752A1
Application number: PCT/JP2006/306506
Authority: WO
Inventors: Tomomi Asai; Shigeaki Nishii; Ritsuko Shimizu; Masayuki Yamamoto; Yoshihiro Ohmiya
Original assignee: Toyo Boseki Kabushiki Kaisha; National Institute Of Advanced Industrial Science And Technology
Priority date: 2005-03-30
Filing date: 2006-03-29
Publication date: 2006-10-12
Also published as: JPWO2006106752A1

Abstract

A method whereby a biological process in a mammalian subject is more effectively analyzed or measured with the use of noninvasive imaging. More specifically speaking, a method of conducting more effective optical noninvasive imaging by using a luminescent enzyme which exhibits a luminescent activity in the long wavelength side with a high tissue permeability and shows little change in the luminescent spectrum depending on pH.

Description

Specification

Non-invasive analysis method

Technical field

[0001] The present invention relates to a method for more effectively analyzing and measuring a biological process in a mammalian subject by noninvasive imaging. More specifically, the present invention relates to a method for performing more effective optical non-invasive imaging using a luminescent enzyme that exhibits luminescence activity on the long wavelength side with high tissue permeability and has substantially no fluctuation in emission spectrum due to pH. . Background art

Conventionally, cultured cell systems have been widely used as an in vivo assay system for examining various biological processes. However, it is not possible to construct a system that can be studied well with a simplified system that is different from that in the living body, or the results examined in such an environment do not necessarily reflect the phenomenon in the living body. Sometimes. Therefore, it is desirable to observe (non-invasive imaging) in a living mammal individual in recent years, that is, various fluorescent probes, that is, low-molecular fluorescent compounds such as fluorescein and green fluorescent protein ( Non-patent literature: Fluorescent proteins such as 1 and 2) have been developed and are being applied in various research fields. However, since it is necessary to irradiate the excitation light to detect the fluorescent protein, the background signal due to autofluorescence in the animal tissue is high, and the excitation light itself must pass through the inside of the individual. In other words, it is not suitable for detection in the deep part of an individual. Non-Patent Literature l: Chalfie, M., Tu, Y "Euskirchen, G" Ward, WW, and Prasher, DC; Green fluorescent protein as a marker for gene expression, Science. 1994, Feb 丄丄; 263 (5148): 802-805.

Patent Document 2: Morin, J.G. and Hastings, J.W .; Biochemistry of the Dioluminescence of f colonial hydroids and other coelenterates, J. Cell.Physiol. 1971 Jun; 77 (3): 305-12.

[0003] On the other hand, bioluminescent molecules do not require irradiation with excitation light to emit light, and there is almost no background signal compared to fluorescence detection. Such bioluminescent molecules include the luciferase family and have been identified in various prokaryotes and eukaryotes. Prokaryotic luciferases (lux) include Vibrio and Photobacterium Enzymes derived from marine bacteria, terrestrial bacteria belonging to the genus Xenorhabdus are known. Further, as eukaryotic type noluciferases (luc), luciferases derived from beetles such as Photinus pyralis and Pyrophorus plagioph thalamus or marine organisms such as Renilla reniformis (Sea pansy) are known.

[0004] Prokaryotic luciferase (lux) is a heterodimeric luciferase, which is cumbersome to handle, whereas eukaryotic luciferase is more widely used because it also has the power of one protein. . Among eukaryotic luciferases, luciferases derived from luminescent beetles such as Photinus pyralis and Pyrop horus plagiophthalamus convert beetle luciferin (D-luciferin) into ATP-dependent oxyluciferin, while Renilla luciferase ( Rluc) uses coelenterazine as a substrate and converts to coelenteramide independent of ATP. The latter is unstable and has high self-luminous properties, so it is not a knock ground signal. Recently, coelenterazine force TP—binding cassette transporter family, especially multidrug resistance MDRl P—glycoprotein (ABCA1; Pgp) is recognized as a substrate and is excreted from the cell, so Pgp-expressing cells However, there is a report that the luminescence of Rluc cannot be detected in tissues and the range of use is limited (Non-Patent Document 3). On the other hand, since D-luciferin is not recognized by Pgp, it is not excreted from cells.

Non-Patent Literature 3: Andrea P., June LP, and David PW ,; Imaging reversal of multidrug r esistance in living mice with bioluminescence: MDRl P—glycoprotein transports coel enterazine, Proc. Natl. Acad. Sci. USA, 2004 Feb; 101 (6): 1702— 1707

[0005] On the other hand, the most widely used luciferase derived from Photinus pyralis as a luciferase using D-luciferin as a substrate is characterized in that the emission spectrum fluctuates due to a force pH that is said to have an emission wavelength peak at 562 nm. There is. As detection sensitivity, about 10 ³ luciferase-expressing cells are required, and detection of a smaller number of cells is difficult.

Disclosure of the invention

Problems to be solved by the invention

[0006] An object of the present invention is to provide a method for more effectively analyzing and measuring a biological process in a mammal by non-invasive imaging. Furthermore, it exhibits luminescence activity on the long wavelength side with high tissue permeability, and the influence of its luminescence spectrum by pH. The present invention relates to a method for performing optical non-invasive imaging with high accuracy and more effectively using a luminescent enzyme that is substantially free from luminescence.

Means for solving the problem

[0007] The present inventors have succeeded in cloning a red luminescence luciferase derived from a railroad worm as a luciferase having a maximum emission wavelength on the longest wavelength side among the luciferases found so far. (Patent Document 1). After that, there was an example where it was possible to express it in mammalian cells by modifying the structure of the railworm photoprotein gene (Patent Document 2), but noninvasive imaging was performed using living mammalian subjects. I have not done it. The present inventors have previously disclosed a method for measuring the three promoter activities simultaneously using the expression of the red luciferase of the railway worm, the green and orange luciferases derived from Irimotobotaru in mammalian cells. (Patent Literature 3). As a result of further intensive research, the present inventors have detected a probe in a living mammal more efficiently using a luciferase having a maximum emission wavelength of 600 or more, such as a railroad worm red-emitting luciferase. In other words, we found a more efficient non-invasive imaging method for living mammals and completed the present invention.

Patent Document 1: US2002 / 0119542-A1

Patent Document 2: WO2003-016839

Patent Document 3: WO2004 / 099421

[0008] That is, the present invention has the following constitutional power.

1. A noninvasive imaging method comprising administering a luciferase having a maximum emission wavelength of 600 nm or more and luciferin to a mammal and detecting the luminescence.

2. A noninvasive imaging method characterized by detecting luminescence of the luciferase in a mammal expressing a luciferase gene having a maximum emission wavelength of 600 or more under the control of a specific promoter.

3. It is characterized by transplanting a mammalian cell expressing a luciferase gene having a maximum emission wavelength of 600 or more under the control of a specific promoter to a mammal individual and detecting the luminescence of the luciferase in the mammal individual. Non-invasive image Method.

4. The method according to any one of Items 13 to 13, wherein the maximum emission wavelength of luciferase is 620 nm or more.

5. The method according to Item 4, wherein the luciferase gene is a luciferase gene derived from a luminescent beetle having a maximum emission wavelength of 620 or more.

6. The method according to Item 5, wherein the luminescence spectrum of the luciferase gene encoded by the luciferase gene is luciferase in which the emission spectrum is not substantially affected by pH.

7. The method according to item 6, wherein the luciferase is a luciferase derived from the firefly family, the mosquito family, the rice family, or the Iriomote family.

8. The method according to item 7, wherein the luciferase is a red light-emitting luciferase derived from a railroad worm.

9. The method according to item 1 or 2, which comprises detecting luciferase luminescence to examine changes in ATP concentration in a specific tissue.

10. The method according to item 3, wherein the specific promoter is a constitutive expression promoter, and the number of transplanted cells is monitored by detecting luminescence.

11. The method according to item 10, wherein the cells are selected from the group consisting of tumor cells, normal cells, ES cells, and stem cells.

The invention's effect

[0009] The method of the present invention reduces the rate of tissue absorption of luciferase, which is a reporter of a specific biological process within a living animal individual, thus affecting the type or depth of surrounding tissue. It has become possible to detect more directly and more effectively without being affected. Furthermore, the present method can maintain the luminescence tissue permeation efficiency constant by using luciferase that is not affected by the emission spectrum strength H, so that detection with higher accuracy is possible.

Brief Description of Drawings

[0010] [Fig.l] Lucinase from Photinus pyralis (HLuc, maximum emission wavelength of about 560 nm) Luciferase from Ragopht halmus ohbai (Rol, maximum emission wavelength of about 550 nm) Mutant luciferase from Ragophthalmus oh bai (Rol T226N, maximum emission wavelength of about 560 nm) 580 nm), Phrixothrix hi Among the luminescence of various luciferases using a lysate of HeLa S3 cells that expressed rtus luciferase (PhRE, maximum emission wavelength of about 630 nm), Phrixothrix hirtus luciferase (PhRE) is particularly suitable for tissue permeability. These are data and graphs showing that the emission ratio in the long wavelength region (600 nm or more) is high V.

[Fig.2] Good luminescence detection with a high-sensitivity CCD camera in living cells expressing Ragophthalmus ohbai-derived noreluciferase (Rol), Ragophthalmus ohbai-derived variant luciferase (Rol T226N), and Phrixothrix hirtus-derived luciferase (PhRE) It is an image, a measured value, and a graph showing that is recognized.

[Figure 3] Ragophthalmus ohbai-derived noreluciferase (Rol), Ragophthalmus ohbai-derived variant luciferase (Rol T226N), and Phrixothrix hirtus-derived luciferase (PhRE) were injected subcutaneously with D-luciferin, and Phrixothrix hirtus It is an image, a measured value, and a graph which show that the light emission of origin luciferase (PhRE) is detected very strongly.

[Fig. 4] A graph suggesting that the luminescence of Phrixothrix hirtus-derived luciferase (PhRE) is detected at a very high efficiency compared to other luciferases.

FIG. 5 shows the structure of the G1HRD-PhRE transgene constructed in Example 4.

[Fig. 6] After anemia was induced with PHZ (phenylhydrazine), the luminescence signal derived from PhRE was significant in the spleen, the hematopoietic tissue of G1HRD-PhRE TG mice, compared to the negative control (G1HRD-PhRE non-introduced mice, left) It is an image showing that it was observed. BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The present invention is described in detail below.

[0012] The "noninvasive imaging method" in the present invention refers to the detection and measurement of luciferase emission, which is a reporter of a specific biological process, in a living mammal using a living mammal. In this method, the position and relative intensity of luminescence is examined, and the biological process in the animal is analyzed and measured. The method is used from basic scientific analysis such as transcription control, signal transduction pathway, protein-protein interaction, cell traffic, to applied science fields such as disease detection and treatment monitoring. Typical detection (imaging) methods include high-sensitivity CCD (charge-coupled device) such as Xenogen's IVIS Imaging System and BERTHOL TEC HNOLOGIES 'NightOWL LB981 system. ) Examples include, but are not limited to, methods of imaging using a system equipped with a camera. The CCD camera converts the photons that hit the silicon wafer into electrons, encrypts the projected photon density spatially into a charge pattern, and generates an image. For bioluminescence imaging, the CCD camera can be cooled to reduce noise caused by heat, and by installing it in a dark box, the sensitivity of the camera that detects bioluminescence, which is faint light, can be increased. . Non-invasive methods include bioimaging optical imaging, PET (positoron emission tomography) and MRI (magnetic resonance imaging) systems, which are less three-dimensional than other systems. However, this point is also being improved now, and it is not necessary to use radioactive compounds, so it is attracting attention as a simpler system, especially for PET systems. In addition to the safety of these radioactive compounds, the half-life of this compound is as short as a few minutes, and in the facility equipped with an automated synthesizer for synthesizing the compound On the other hand, the luciferase luminescence reaction requires intravenous injection, intraperitoneal injection, or target site injection of luciferase as a luciferase substrate in the individual animal. Injection, sustained release of D-luciferin It can be detected by administering it with a sustained-release preparation, etc. The beetle luciferin (D-luciferin) is a substrate for biomolecules such as Cytochrome P450 or Caspase, as well as natural luciferin derived from fireflies. And a D-luciferin derivative that is modified to D-luciferin by the active molecule.

[0013] D-luciferin is commercially available from a number of manufacturers and is readily available. Also, there are no reports of dangers such as acute toxicity.

[0014] Luciferases to be detected include those administered to mammals as standard products by injection, those incorporated and expressed in the animal DNA as transgenic mammals, and introduced into mammalian cells and expressed. In which the cells are transplanted into a mammal individual. ATP is required for the reaction of luciferin by luciferase. The injection of a luciferase standard product gives the luminescence proportional to the amount of ATP present at the site when a sufficient amount of luciferase standard product and luciferin are administered. It can be used as a measurement method. The amount of ATP is transformed with a luciferase-expressing mammalian cell or a luciferase linked to an inducible promoter such as an organ or tissue-specific promoter, time-specific or cold-inducible promoter. It can also be detected in transgenic mammals.

[0015] By monitoring the ATP concentration, the activity state of the site can be monitored. For example, it is possible to screen improvers by monitoring ATP levels in brains whose activity has been reduced by Alzheimer. In addition, the myocardial cerebral nerve cell has a function of opening and self-protecting the ATP-sensitive K channel depending on the intracellular ATP concentration during ischemia. -Ng can be expected to be applied to ischemic heart disease Also, intracellular A

As for TP concentration, spleen j8 cells work to regulate insulin secretion, and are an effective index when screening diabetes drugs.

[0016] On the other hand, when the luciferase gene is incorporated into the animal DNA, it is possible to analyze what transcription control is given by the transcription control sequence in the upstream region within a certain yarn and tissue. It is. An evaluator of pharmacological effects in a living body using such an animal. The column f shows luciferase genes linked to the promoters of genes that change expression in association with obesity, such as Peroxisome proliferators—activated receptor y (PPAR lucose Transporter 4 (GLUT4), lipoprotein lipase ゝ adiponectin, TNF- Combining with normal animals, as well as obesity models and diabetes model animals, it is possible to evaluate drugs that improve diabetes and obesity using the luminescence of the expressed luciferase as an index, or inflammatory site strength (eg, IL- 1, IL-6, IL-11, TNF-α, etc.) and the luciferase gene can be linked to evaluate anti-inflammatory agents, as well as PCNA, cdc2 kinase, c-myc, c Cell cycle regulation gene promoters such as -myb are linked to the luciferase gene to evaluate the inhibitory effect on cell growth inhibition, thereby inhibiting myocardial treatment The ability to be used as a vascular restenosis inhibitor and anti-cancer agent evaluation system is not limited to these, but transplantation of mammalian cells into which a luciferase gene has been introduced into a mammal individual Compared with the assembly by the cultured cell system, analysis that reflects the phenomenon in the living body is possible. Become. Furthermore, when mammalian cells into which a luciferase gene under the control of a constitutive expression promoter is introduced are transplanted into the same or different types of mammalian animals, the luminescence mainly reflects the growth state of the cells. For example, using tumor cells into which a luciferase gene has been introduced is effective as a system for monitoring the growth, metastasis, etc. of tumor cells. In addition, if the growth of normal cells, ES cells, and stem cells (mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, etc.) into which the luciferase gene has been introduced is monitored, it can be used as an evaluation system for transplantation.

[0017] When luciferase (protein) is administered to a mammal, it may be administered in an amount of about 100 to 1000 (^ _§ / body weight 1 ^ g. In addition, when a mammalian cell expressing luciferase is transplanted into an animal, the 10 ² to 10 ^omega pieces about the cell can be transplanted. the present invention, because it uses luciferase maximum emission wavelength of above 600 nm, are possible imaging even amount luciferase is very small For example, in the case of tumor cells expressing luciferase, even about 1 X 10 ³ cells can be detected by imaging sufficiently, so it is not necessary to sacrifice animals and make tissue sections as in the past. It is also possible to image a small number of tumor cells (eg 1000 or less, preferably 100 or less) that cannot be recognized by the naked eye. It is very suitable for grayed.

[0018] Luciferin can be imaged by administering to a mammal an amount of about 10 to 1000 mg / kg body weight.

[0019] Important characteristics as the detectability of the luciferase used include luminescence intensity, depth of the luminescent site from the body surface, wavelength, and stability of the emission spectrum. The luminescence intensity is preferably high in mammalian cells when detecting the expression of a transgene in which the abundance ratio of luciferase is high. Examples of this include luciferase genes modified to have high V and codon usage in mammalian cells, luciferase genes with sequences such as Kozak sequences added to increase translation efficiency, and constitutive expression profiles. Motors include, but are not limited to, luciferase genes linked to promoters with high expression and expression activity such as CAG promoter and CMV promoter. [0020] The wavelength is related to the tissue permeability of light. Most animal tissues contain a lot of water, and water is known to transmit long wavelength light (red region light) better than short wavelength light. Therefore, a luciferase gene having a high emission ratio at a wavelength of 6 00 or longer is preferable. That is, a luciferase gene having a maximum emission wavelength of 600 nm or more, more preferably 620 nm or more is preferred. The upper limit of the maximum emission wavelength of luciferase is not particularly limited, but is usually about 650 nm, particularly about 640 nm.

[0021] Also, the present inventors. Certain luciferases have been confirmed to shift to shorter wavelengths with increasing pH. For example, Photinus pyralis reports that the maximum emission wavelength at pH 6 is 617 nm, whereas that at pH 8 is 565 nm. In other words, if the pH increases, the emission spectrum shifts to the short wavelength side, and the detection efficiency is expected to decrease. This type of luminescent beetle luciferase is from the family Firefly, and luciferases that are not affected by pH are preferred, and more specifically luciferases from the family Firefly, Family, and Iriomotepoota. ! /

[0022] In the present specification, the luciferase having a maximum emission wavelength of 600 nm or more means a luciferase having a maximum emission wavelength of 600 nm or more in the whole physiological pH range (pH 59), The luciferase derived from Photinus pyralis is not included in the “luciferase whose maximum emission wavelength is 600 nm or more”. In addition, “luciferase whose emission spectrum is substantially unaffected by pH near pH 59” means that the change in the maximum emission wavelength in this pH range is 10 nm or less, preferably 5 nm or less, more preferably 3 nm or less.

[0023] From the characteristics of the stability of the wavelength and emission spectrum, the most preferred V-luciferase includes a red-emitting luciferase derived from the firefly, particularly Phrixothrix hirtus, and mutants thereof. The luciferase has a maximum emission wavelength around 630 nm (which may vary slightly depending on the measurement equipment), and the emission spectrum is stable in pH in the pH 59 region (non- (Patent Document 4)

[0024] Variants of red-emitting luciferase include base substitutions that do not involve alteration of amino acids such as codon usage, amino acids that retain pH-insensitive enzyme characteristics with a maximum emission wavelength of 620 nm or more and an emission spectrum. Power including substitutions, and additions of proteinaceous tag sequences such as myc tag, histidine tag, GST tag, etc. Not limited to this! Non-Patent Document 4: Viviani V "Uchida, A" Suenaga, N "Ryuluku, M., and Ohmiya, Y .; Th r226 Is a Key Residue for Bioluminescence Spectra Determination in Beetle Lucifera se .; Biochemical and Biophysical Research Communications. 2001 ; 280, 1286-1291 Examples of mammals include humans, mice, rats, mice, horses, hidges, monkeys, pigs, mice, mussels, guinea pigs, rabbits, and nu, preferably mice and rats. It is preferable to shave the hair that corresponds to the desired detection site in the case of colored hair, in which nude and white hair are preferred in mice and rats, and express luciferase that is introduced into mammals. Examples of mammalian cells include human and monkey cells (particularly tumor cells) in addition to mice and rats.

Example

[0025] Hereinafter, the effects of the present invention will be made clearer by illustrating examples of the present invention.

[0026] Example 1 Measurement of luciferase luminescence frequently in a nursing street shattering system

HeLa S3 cells 1 X 10 ⁵ cells were seeded on 12 well plates and cultured in Dulbecco's modified Eagle medium (Nissui Pharmaceutical) containing 10% FCS. The next day, pGL3-control (Promega, Photinus pyralis luciferase (ffLuc), maximum emission wavelength about 560 nm), pSLG-SV40 control (Toyobo, Ragophthalmus ohbai luciferase (Rol), maximum emission wavelength about 55 Onm), pSLO- SV40 control (Toyobo, Ragophthalmus ohbai-derived mutant luciferase (Rol T226N), maximum emission wavelength of about 580 nm), pSLR-SV40 control (Toyobo, Phrixo thrix hirtus-derived luciferase (PhRE), maximum emission wavelength of about 1 μg of each plasmid (630 nm) is diluted with 100 μ 1 Opti-MEM (GIBCO), mixed with 2.5 μ 1 Lipofectamine 2000 (Invitrogen) diluted with 100 μ 1 Opti-MEM (GIBCO), and HeLa S3 cells I made a transfer. After 24 hours, the cells are rinsed with PBS (-), lysed with Pitscagene cell lysing agent Lc | 8 (Toyo Ink), luminescent substrate (Toyo Ink) is added, and green, orange and red luminescent luciferases are added. Using a luminometer with light separation function and AB-2250 luminescence sensor MCA (Atoichi), the total light intensity (without filter), 600 nm LP filter transmitted light, and 540 nm LP filter transmitted light 20 It was measured as an integrated value per second. Figure 1 shows the measured value under each condition and the ratio of the 600 nm LP filter transmitted light measurement value to the total light measurement value. Show.

[0027] Example 2 Detection of luciferase luminescence in a non-destructive system of a nursing street

COS cells 4.0 X 10 ⁴ cells were seeded on 24 well plates and cultured. On the next day, 0.5 μg of each plasmid of pSLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control was transfected. At that time, 1 mM and 0.5 mM D-luciferin were added, and the next day, exposure was performed for 10 seconds with IVIS Imaging System (Xenogen). Figure 2 shows the image and relative luminescence intensity values and graphs quantified for each well.

Example 3 Detection of luminescence by subcutaneous injection of luciferase-expressing cells

Each luciferase-expressing cell of Example 2 was recovered by scraping with a 2-well scraper and concentrated to 100 l (in a medium containing D-ludferin) by centrifugation. This cell solution was injected subcutaneously in the vicinity of the mouse limbs and exposed for 10 seconds with the IVIS Imaging System. Figure 3 shows the image and relative luminescence intensity values and graphs quantified for each well. FIG. 4 shows the ratio of the measurement value at the time of subcutaneous injection to the measurement value at the time of cell detection in Example 2 for each luciferase and a graph thereof.

[0029] Example 4 Preparation of a vector for producing a transgenic mouse

The transcription factor GATA-1 expressed in erythrocytes is upstream of the blood cell-specific first exon IE 3.9 kb to the second exon (GIHRD; GATA-lHematopoietic Regulatory Domain) (Non-Patent Documents 5 and 6) downstream The SLR gene (PhRE) of pSLR-test (Toyobo) was inserted. As described in Non-Patent Document 7, the IE3.9int plasmid vector was digested with the restriction enzyme Notl (Toyobo) to open the IE3.9int plasmid vector. In addition, the SLR gene (PhRE) was amplified using oligonucleotide 1 (SEQ ID NO: 1) and oligonucleotide 2 (SEQ ID NO: 2) in the form of a 5'-site addition of pSLR-tesU restriction enzyme Notl. The PhRE gene fragment was obtained by digestion with I and inserted into the IE3.9int plasmid vector (GIHRD-PhRE). Figure 5 shows the structure of the constructed GIHRD-PhRE transgene.

Non-Patent Document 5: Onodera, K., Takahashi, S., Nishimura, S., Ohta, J., Motohashi, H., Yomogida, K., Hayashi, N., Engel, JD, Yamamoto, M. GATA- 1 transcription is co ntrolled by distinct regulatory mechanisms during primitive and definitive erythropie sis .: Proceedings of the Natitonal Academy of Sciences of the United States of Ameri ca. 1997; 94, 4487-4492

Non-Patent Document 6: Nishimura, S., Takahashi, S., Kuroha, T., Suwabe, N., Nagasawa, T., Trainor, C, Yamamoto, M. A GATA Box in the Network of GATA Factors and Sites That Regulate This Gene .: Molecular and Cellular Biology. 2000; 20, 713-723 Non-Patent Document 7: Shimizu, R. Takahashi, S "Ohneda, K., Engel, JD, Yamamoto, M. In vivo requirements functional domains during primitive and definitive erythropoiesis .:

The EMBO Journal. 2001; 20, 5250-5260

[0030] Example 5 Creation of Transgenic Mouse

For the DNA for producing a transgenic mouse, G1HRD-PhRE was digested with restriction enzyme Sal I (Toyobo), separated by agarose gel electrophoresis to remove the vector DNA, and the G1 HRD-PhRE transgene was purified. The purified DNA fragment was injected into a mouse fertilized egg according to a standard method (Non-patent Document 8). The prepared G1HRD-PhRE TG mice were administered D-luciferin, and then luminescence was observed with the IVIS system, and individuals with high luminescence intensity were selected. Figure 6 shows the luminescence signal derived from PhRE in the spleen, a hematopoietic tissue of G1HRD-PhRE TG mice, compared to the negative control (GlHRD-PhRE non-introduced mice, left) after induction of anemia with PHZ (phenylhydrazine). Non-special literature 8: Hogan, B., R. Beddington, F. Constantini, E. Lacy. Manipulating the mouse embryo. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY 199 4.

Industrial applicability

[0031] The noninvasive detection method in a mammalian animal using a luciferase having a wavelength of 600 nm or more in the present invention is a drug screening system as a system for more effectively monitoring a biological process in a mammalian animal. It can be used as an analysis system for tumors and infectious diseases, and as a transplant evaluation system, and contributes greatly to industries such as drug discovery and medical care.

Claims

The scope of the claims

[I] A noninvasive imaging method comprising administering luciferase and luciferin having a maximum emission wavelength of luminescence of 600 or more to a mammal and detecting the luminescence.

[2] A noninvasive imaging method characterized by detecting the luminescence of the luciferase in a mammal expressing the luciferase gene having a maximum emission wavelength of 600 or more under the control of a specific promoter.

[3] A luciferase gene with a maximum emission wavelength of 600 or more is identified as a specific promoter. Mammalian cells expressing under the control of a specific promoter are transplanted into a mammal individual, and the luciferase luminescence in the individual mammal is detected. A non-invasive imaging method characterized.

[4] The method according to any one of claims 1 to 3, wherein the maximum emission wavelength of luciferase is 620 nm or more.

[5] The method according to claim 4, wherein the luciferase gene force is a luciferase gene derived from a luminescent beetle having a maximum emission wavelength of 620 or more.

[6] The method according to claim 5, wherein the luminescence spectrum is a luciferase that is substantially unaffected by pH near the luciferase pH 5-9 encoded by the luciferase gene.

[7] The method according to claim 6, wherein the luciferase is a luciferase derived from the firefly family, the mosquito family, the rice family, or the Iriomote family.

[8] The method according to claim 7, wherein the luciferase is a red light-emitting luciferase derived from a railroad worm.

[9] The method according to claim 1 or 2, comprising detecting luciferase luminescence in order to examine a change in ATP concentration in a specific tissue.

[10] The method according to claim 3, wherein the specific promoter is a constitutive expression promoter, and the number of transplanted cells is monitored by detection of luminescence.

[II] The method according to claim 10, wherein the cells are selected from the group consisting of tumor cells, normal cells, ES cells and stem cells.