JP7045680B2 - Biological activity evaluation method for substances using nematodes - Google Patents

Description

The present invention relates to a nematode that can be used for evaluating the physiological activity of a substance, and a method for evaluating a substance or the like using the nematode.

C. elegans is used as a model for toxicology tests and drug efficacy tests. Among them, Caenorhabditis elegans (C. elegans) has biological characteristics similar to those of higher organisms, although they have a simple structure consisting of about 1,000 somatic cells. In addition, since the entire genome sequence has been decoded, it is used for large-scale forward or reverse genetics screening and chemical genetic screening. Since C. elegans responds to various compounds, they have been used to identify many bioactive substances and elucidate their mechanism of action. For example, the action of existing compounds such as acetylcholine receptor agonists (levamisol, nicotine, morantel), anesthetics (halotan), GABA-related compounds (GABA, musimol), serotonin-related drugs (serotonin, imipramine) using C. elegans. While the mechanism was clarified, a novel physiologically active compound acting on a disease gene was identified, and the drug efficacy was evaluated (Non-Patent Document 1 and Non-Patent Document 2).

By the way, the pharyngeal muscle of nematodes consists of three parts, corpus, isthmus and terminal bulb, and feeds bacteria and the like into the intestine by contraction movement called pumping and peristalsis generated by isthmus. In the medicinal efficacy test using nematodes, in addition to the lifespan and exercise amount of nematodes, the number of pumping of the pharyngeal muscle is often used as an index for evaluating the medicinal efficacy. Until now, the number of pumping counts was often counted by the experimenter while looking at the image (Non-Patent Document 3 and Non-Patent Document 4). However, this method is a labor-intensive task, and human counting errors are likely to occur, often resulting in inaccurate results.
In addition to the method of visually counting the number of pumping, there is a method of measuring using an electrophysiological method, but only one animal can be measured, and proficiency in the measuring operation is required. In addition to the initial investment in measuring equipment, consumables are also expensive and costly.

When the pharyngeal muscle of the nematode contracts, the intracellular calcium concentration increases due to the influx of calcium ions from the outside of the cell and the release of calcium ions from the sarcoplasmic reticulum inside the muscle cell. Therefore, the increase in intracellular calcium ion concentration corresponds well with the contraction of the pharyngeal muscle. So far, the physiological mechanism of pharyngeal muscle pumping has been analyzed by detecting changes in calcium ion concentration with a calcium probe.
Kerr et al. Reported that a fluorescent calcium probe, cameleon, was expressed in muscle cells of the pharyngeal muscle to image changes in calcium ion concentration (Non-Patent Document 5). Chameleon is a calcium probe composed of four domains (CFP, calmodulin, M13 (calmodulin binding domain) and YFP), which can detect changes in calcium ions in the pharyngeal muscle. However, the amount of change in the fluorescence ratio due to chameleon (YC2.1) is as small as 4.31 ± 0.16% in the wild type strain (Non-Patent Document 5), and since chameleon is a 1-wavelength excitation 2-wavelength photometric type, 2 wavelengths are measured. A measuring device that can be used is required, which is costly. Furthermore, since chameleon contains two GFP mutants, CFP and YFP, there is a problem that recombination is likely to occur between these fluorescent proteins and genes are likely to become unstable. Be careful when using it for the purpose.

As described above, the method of pumping the pharyngeal muscle of the nematode using a calcium probe is highly expected as a simple and inexpensive method, but has not yet been put into practical use. Therefore, even at present, in most cases, the number of pumping of the pharyngeal muscle is visually counted (Non-Patent Document 3, Non-Patent Document 4, etc.).

Japanese Patent Application No. 2000-356047 Japanese Patent Application No. 2009-289789 Japanese Patent Application No. 2010-232788 Special Request 2012-137434 WO2015 / 190083

Rand et al., In Carnorhabditis elegans Modern Biological Analysis of Organism. 48 eds H.F. Estein & D.C. Shakes. Academic Press, Inc. Ch. 8, 187-204 1995 O’Reilly et al., Adv. Drug Deliv. Rev. 2014 Apr; 69-70: 247-53. Doi: 10.1016 / j.addr.2013.12.001. Epub 2013 Dec 12. Li et al., J. Appl. Toxicol. 36, 60-67 2016 Hao et al., J. Vis. Exp. 84, e50864 2014 Kerr et al., Neuron, 26, 583-594 2000

In view of the above circumstances, the present invention relates to a nematode that can easily count the number of pumping of the pharyngeal muscle, and the physiology of the test substance such as toxicity evaluation or drug efficacy evaluation of the test substance using the number of pumping of the nematode as an index. To develop a method for assessing activity.

So far, the inventors have developed proteinaceous fluorescent calcium probes G-CaMP and R-CaMP (G-CaMP and R-CaMP are collectively referred to as "CaMP"). CaMP is composed of (1) linker, (2) functional molecule 1 (3) linker, (4) modified fluorescent protein (fluorescent protein is referred to as FP), (5) linker, and (6) functionality from the N-terminal side. It is a protein based on a structure composed of molecule 2 (Patent Documents 1 to 5). Here, the modified FP is a protein in which a linker is linked to an FP such as modified GFP, modified RFP, or modified mApple, and specifically, from the N-terminal side, it consists of a partial sequence of FP, a linker, and a partial sequence of FP. .. Further, the functional molecule 1 is a myosin light chain kinase or a calmodulin kinase kinase having a binding ability to calmodulin (CaM) or a partial sequence thereof, and the functional molecule 2 is calmodulin and a variant thereof. The proteins corresponding to the functional molecule 1 and the functional molecule 2 can be exchanged. The amount of change in fluorescence intensity when calcium ions bind to CaMP changes more significantly than the fluorescent calcium probe made of previously known proteins. In addition, CaMP is a one-wavelength excitation one-wavelength metering type that contains only one molecule of GFP, RFP, or mApple as a fluorescent protein (measurement is possible with a measuring device that can measure one wavelength), so it is a complicated detection system for fluorescence detection. Is not necessary and is cost effective. Furthermore, due to the nature of CaMP, which contains only one molecule of GFP, RFP or mApple as a fluorescent protein, the rate of DNA recombination is relatively lower than that of chameleon, and stable expression of CaMP is expected. Therefore, it also has an advantage in the maintenance of gene-introduced nematodes into which CaMP has been introduced.
As described above, the inventors have come up with the idea that since good results have been obtained from the experimental results, they can be used for drug screening and the like.

Therefore, the present invention is the following (1) to (7).
(1) A nematode in which one or more of CaMP, which is a fluorescent calcium probe, is expressed or can be expressed in the muscle cells of the pharyngeal muscle.
(2) The line according to (1) above, wherein the CaMP is a fluorescent calcium probe selected from the group consisting of the fluorescent calcium probes shown in (a), (b) or (c) below. insect.
(A) A fluorescent calcium probe consisting of the amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.
(B) One or several amino acids are added, substituted, inserted or added to the amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. Fluorescent calcium probe consisting of the deleted amino acid sequence,
(C) Consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. Fluorescent calcium probe (3) The nematode according to (1) or (2) above, wherein the nematode is C. elegans, C. briggsae or C. remanei.
(4) A method for evaluating the physiological activity of a test substance, which comprises the following steps (a) and (b).
(A) The step of bringing the test substance into contact with the nematode according to any one of (1) to (3) above.
(B) The method according to (4) above, further comprising the steps (5) of the following steps (c) and (d) for counting the number of changes in fluorescence intensity from CaMP expressed in the nematode.
(C) The step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode according to any one of (1) to (3) above.
(D) A step of comparing the count number of the step (c) with the count number of the step (b) of the above (4) (6) The radiation to the living body including the steps (a) and (b) below. How to find out the impact.
(A) The step of irradiating the nematode according to any one of (1) to (3) above with radiation.
(B) The step of counting the number of changes in fluorescence intensity from CaMP expressed in the pharyngeal muscle of the nematode (7). The step according to (6) above, further comprising the steps (c) and (d) below. Method.
(C) The step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode according to any one of (1) to (3) above.
(D) A step of comparing the count number of the step (c) with the count number of the step (b) of the above (4).

The nematode of the present invention can detect the contraction of muscle cells of the pharyngeal muscle as a change in fluorescence intensity from CaMP. Therefore, the effects of compounds on living organisms (for example, toxicity tests, drug efficacy tests, etc.), the effects of introduced or functionally modified (functional deficient) genes, etc., temperature and humidity, gravity, lighting, presence or absence of food, mechanical stimulation , When investigating the influence of environmental factors such as the presence or absence of other organisms (for example, natural enemies) or social connection with other individuals, the number of pumping of nematodes is evaluated easily, quickly and accurately using the change in fluorescence as an index. Is possible.

The fluorescence micrograph (left figure) of the C. elegans wild-type N2 strain expressing G-CaMP2 in the pharyngeal muscle and the result of measuring the number of pumping of the pharyngeal muscle using this nematode (right figure) are shown.

The first embodiment of the present invention is a nematode in which CaMP, which is a fluorescent calcium probe, is expressed or can be expressed in the muscle cells of the pharyngeal muscle.
Any type of nematode used in the embodiments of the present invention can be used as long as it is easily genetically engineered, for example, C. elegans, C. briggsae or C. remanei. Can be mentioned, preferably C. elegans.
Further, in the present specification, the fluorescent calcium probe is a protein whose fluorescence intensity changes when calcium is bound. In the present invention, CaMP is used as the fluorescent calcium probe. As described above, CaMP is a fluorescent calcium probe developed by the inventors and has a characteristic that its fluorescence characteristics (for example, fluorescence intensity) change when it binds to calcium ions. For example, in the cell. It can be used to detect changes in calcium ion concentration. Examples of CaMP are (a) SEQ ID NO: 2 (G-CaMP2), SEQ ID NO: 4 (G-CaMP4), SEQ ID NO: 6 (G-CaMP6), SEQ ID NO: 8 (G-CaMP7), SEQ ID NO: 10 (G-). CaMP8), a fluorescent calcium probe consisting of the amino acid sequences represented by SEQ ID NO: 12 (R-CaMP1.07) and SEQ ID NO: 14 (R-CaMP2), (b) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6. Fluorescent calcium probe consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequences represented by No. 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, (c) SEQ ID NO: 2. A fluorescent calcium probe consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. Can be mentioned. The CaMP is particularly preferably a fluorescent calcium probe comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.
In addition, in this specification, when it simply describes as "CaMP", it means a CaMP protein.

In the present specification, when "an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted" is described, the number of amino acids added, substituted, inserted or deleted is not particularly limited, but for example. , 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 pieces are preferable. Further, in the present specification, the "amino acid sequence having 80% or more sequence identity" may be any percentage as long as it is an amino acid sequence having 80% or more sequence identity, but for example, 90% or more. , 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more.

In order to express or make CaMP expressive in the muscle cells of the pharyngeal muscle of the nematode, it is necessary to introduce an expression plasmid carrying the nucleic acid encoding CaMP into the nematode. be. Examples of the gene encoding CaMP include SEQ ID NO: 1 (G-CaMP2), SEQ ID NO: 3 (G-CaMP4), SEQ ID NO: 5 (G-CaMP6), SEQ ID NO: 7 (G-CaMP7), and SEQ ID NO: 9 (SEQ ID NO: 9). G-CaMP8), a gene consisting of the base sequences represented by SEQ ID NO: 11 (R-CaMP1.07) and SEQ ID NO: 13 (R-CaMP2) can be mentioned. Further, it is stringent with a nucleic acid having a sequence complementary to the nucleic acid consisting of the base sequences represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13. Examples thereof include genes consisting of nucleic acids that hybridize under conditions, in which the protein encoded by this gene has activity as a fluorescent calcium probe.

Here, the nucleic acid that hybridizes with any nucleic acid under stringent (low stringent and high stringent) conditions is, for example, the base sequence of the complementary strand of the arbitrary nucleic acid and preferably 80% or more of the nucleic acid sequence. Examples thereof include nucleic acids composed of sequences having the same identity. The nucleic acid that hybridizes with any nucleic acid under high stringent conditions is the base sequence of the complementary strand of the arbitrary nucleic acid, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, Examples thereof include nucleic acids having nucleic acid sequence identity of 95% or more, 96% or more, 97 or more, 98% or more, or 99% or more.
In addition, stringent conditions are hybridization conditions easily determined by those skilled in the art, and are generally empirical experimental conditions that depend on probe length, washing temperature, and salt concentration. Generally, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. The formation of nucleic acid-to-nucleic acid hybrids depends on the ability of the complementary strand to reanneal in an environment slightly below its melting point. More specifically, low stringent conditions are, for example, 5 × SSC (1x SSC solution (1 times concentration)) under temperature conditions of 37 ° C to 42 ° C in the washing step of the filter after hybridization. The composition of × SSC) can be, for example, 150 mmol / L sodium chloride and 15 mmol / L sodium citrate), conditions for washing in a 0.1% SDS solution, and the like. Further, the high stringent condition includes, for example, a condition of cleaning in 0.1 × SSC and 0.1% SDS under a temperature condition of 60 ° C. to 70 ° C., preferably 65 ° C. in the cleaning stage. Furthermore, by raising the stringent conditions such as raising the temperature, nucleic acids with high homology can be obtained.

Introduction of foreign DNA, such as a plasmid for expression into gonads, can usually be performed by microinjecting the DNA into the gonad of an adult (hermaphrodite). Since the adult gonads are polynuclear and have two anterior and posterior gonads, microinjection into either or both of the anterior and posterior gonads can be performed once to obtain larvae (F1) transformed with foreign DNA. In addition to the foreign DNA of interest, DNA that serves as a marker for transformation may be injected at the same time. The resulting transformant carries extrachromosomal DNA extrachromosomally as a linear extrachromosomal array formed by recombination of a large number of foreign DNAs, and each extrachromosomal DNA array undergoes division. It is distributed to daughter cells. In order to stably insert the introduced foreign DNA onto the chromosome of the nematode, not as an extrachromosomal DNA array, for example, by irradiating with ultraviolet (UV) or γ-ray, the foreign DNA is inserted into the chromosome. Insects can be isolated.

When inherited and introduced into nematodes, for example, pFX_EGFPT vector (K. Gengyo-Ando, S. Yoshina, H. Inoue, and S. Mitani, “An efficient transgenic system by TA cloning vectors and RNAi for C. elegans,” Biochem. Biophys. Res. Commun., Vol. 349, pp. 1345-1350, 2006.) and The Fire Lab C. elegans Vector can be used. In order to express CaMP in a pharyngeal muscle cell-specific manner, a CaMP expression vector was prepared by inserting a DNA encoding a desired CaMP into a vector carrying a pharyngeal muscle-specific promoter in a state in which it can be expressed. Introduce this to nematodes. As the pharyngeal muscle-specific promoter of C. elegans, for example, the promoter of the myo-2 gene or the ceh-22 promoter can be used.
In the present specification, the "expressible state" is not only a state in which CaMP is constantly expressed, but also CaMP is expressed at a desired time by an inducible promoter such as a heat shock promoter. It also includes the state in which the DNA encoding CaMP is introduced into the cell in a possible state (Reference Bacaj T and Shaham S: Temporal Control of Cell-Specific Transgene Expression in Caenorhabditis elegans. Genetics 176: 2651-2655, 2007 .).

A second embodiment of the present invention is a method for evaluating the physiological activity of a test substance, which comprises the following steps (a) and (b).
(A) A step of bringing the test substance into contact with the nematode according to the first embodiment,
(B) Step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode.

The bioactivity evaluation method according to the second embodiment of the present invention (in the present specification, "physiological activity" refers to an action having some effect on a living body) is a line according to the first embodiment. This is a method of using an insect as a model organism, investigating the effect of the test substance (substance subject to bioactivity evaluation) on the number of pumping of the pharyngeal muscle, and evaluating the effect of the test substance on the living body based on the result. The presence or absence of the influence of the test substance can be evaluated, for example, by comparing with the number of times of pumping of the nematode according to the first embodiment which is not in contact with the test substance.
That is, a method for evaluating the physiological activity of the test substance, which comprises the following steps (a) to (d), is also included in the second embodiment of the present invention.
(A) A step of bringing the test substance into contact with the nematode according to the first embodiment,
(B) A step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode.
(C) A step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode according to the first embodiment.
And (d) a step of comparing the count number of the step (c) with the count number of the step (b).

Pumping of the pharyngeal muscles of C. elegans is a vital exercise for feeding. By detecting the effect of the test substance on the pumping motion, it is possible to predict the presence or absence of the effect of the test substance on the living body. As an example of a toxicity test using the number of pumping of the pharyngeal muscle of the nematode as an index, for example, as a result of contacting the nematode with acrylamide known as a neurotoxin in a concentration-dependent manner, the number of pumping of the pharyngeal muscle decreased. (Non-Patent Document 3). In addition, an example has been reported in which the number of times of pumping of nematodes is used as an index for evaluating the efficacy of an antipsychotic drug (Non-Patent Document 4).
As described above, the pumping of the pharyngeal muscle of the nematode is very effective in evaluating the effect of the test substance on the living body.

The test substance to be evaluated in the second embodiment is not particularly limited and may be anything. In the step of bringing the nematode into contact with the test substance, an appropriate amount of the test substance is added to the medium for culturing the nematode, or the bred nematode is directly immersed in a solution containing the test substance. Can be carried out. Alternatively, in the case of a chemical substance that easily vaporizes, a method of dyeing a filter paper or the like with the chemical substance and breeding nematodes on a medium is also used. When the chemical substance is a gas, for example, the medium is placed in a container filled with air containing the chemical substance and bred. C. elegans can be reared by methods known to those of skill in the art, see, for example, "The Nematode Caenorhabditis elegans" (WBWood, ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988. Usually, nematodes are NGM agarose plates (Nematode growth media, eg 0.25% tryptone (2.5 g / L), 0.3% NaCl (3.0 g / L), 1.5% agar (15.0 g / L), 1 mM CaCl 2, 1 mM CaCl ₂ , A culture medium of Escherichia coli OP50 to be fed may be applied on 1 mM DDL ₄ , 25 mM KPO ₄ pH 6.0, 5 μg / mL cholesterol) and bred on a plate cultured overnight at room temperature.

The fluorescence from CaMP expressed in the pharyngeal muscle of the nematode of the present invention can be imaged by a CCD camera attached to a fluorescence microscope, a cMOS camera, or the like, and the change in fluorescence intensity can be recorded. One or a plurality of nematodes to be observed may be distributed to, for example, a multi-well plate and observed one by one, or a plurality of nematodes may be observed together.
Since the fluorescence intensity of the nematode of the present invention changes each time pumping is performed, the number of changes in the fluorescence intensity can be detected as the number of pumping (the number of one peak of the fluorescence intensity is defined as the number of pumping). Can be done. See Examples).
Therefore, for example, when the number of changes in fluorescence intensity per unit time differs depending on the presence or absence of the test substance, it can be determined that the test substance has some effect on the movement of the pharyngeal muscle of the nematode.

A third embodiment of the present invention is a method for examining the effect of radiation on a living body, which comprises the following steps (a) and (b).
(A) A step of irradiating the nematode according to the first embodiment with radiation.
(B) Step of counting the number of changes in fluorescence intensity from CaMP expressed in the pharyngeal muscle of the nematode The method according to the third embodiment of the present invention uses the nematode according to the first embodiment as a model organism. This is a method of investigating the effect of radiation on the number of pumping of the pharyngeal muscle and evaluating the effect of radiation on the living body based on the result. The presence or absence of the influence of radiation can be evaluated by comparing with the number of pumping of nematodes according to the first embodiment not irradiated with radiation.
That is, a method for investigating the effect of radiation on a living body including the following steps (a) to (d) is also included in the third embodiment of the present invention.
(A) A step of irradiating the nematode according to the first embodiment with radiation.
(B) A step of counting the number of changes in fluorescence intensity from CaMP expressed in the pharyngeal muscle of the nematode.
(C) A step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode according to the first embodiment.
And (d) a step of comparing the count number of the step (c) with the count number of the step (b).

Since pumping of the pharyngeal muscle of C. elegans is an essential exercise for feeding that is important for life support, it is possible to predict the presence or absence of the effect of radiation on the living body by detecting the effect of radiation on the pumping movement. can. In recent years, a lot of knowledge about radiation has been accumulated, and its use is advancing in various fields. For example, in the field of medicine, it is used for the treatment of cancer, and while its therapeutic effect is expected, it also causes serious side effect problems. In addition, in fields such as space engineering, astronauts are already increasing the opportunities to work outside the spacecraft, and research on the effects of various cosmic rays on the human body is underway. Under such circumstances, nematodes are considered to be useful model organisms for investigating the effects of various radiations on living organisms.
Here, "radiation" is particle radiation such as material particles (ion, electron, neutron, proton and intermediate) that flow with high kinetic energy, such as α-ray, β-ray, electron beam, proton beam, neutron beam and heavy particle beam. ) And high-energy electromagnetic waves (electromagnetic radiation such as γ-rays and X-rays), and are not particularly limited.

The disclosures of all references cited herein are incorporated herein by reference in their entirety. Also, throughout the specification, when the singular words "a", "an", and "the" are included, not only the singular but also multiple, unless the context clearly indicates otherwise. It shall include things.
Hereinafter, the present invention will be described with reference to examples, but the examples are merely examples of embodiments of the present invention and do not limit the scope of the present invention.

1. 1. Creation of transgenic nematodes
Transgenic nematodes expressing G-CaMP2 (SEQ ID NO: 2) were prepared according to the microinjection method (Mello et al., EMBO J. 10, 3959-3970, 1991). In this method, transformation is performed by microinjecting a DNA solution into the reproductive foci of adult nematodes hermaphroditic. The following experimental equipment was used.
Experimental device
(1) Stereomicroscope: Used for mounting nematodes. The nematodes were mounted on an agar pad (a thinly stretched agar on a slide glass) while observing at a magnification of about 0.7-15 times.
(2) Inverted microscope: Used for microinjection of nematodes. A grind type stage was attached to facilitate the movement of nematodes. The objective lens used was a lens for differential interference contrast, 40x and 4x low magnification (to bring the needle and nematode close to each other).
(3) Needle puller: A glass tube was heated and pulled thinly to create a needle for microinjection.
(4) Manipulator: A device that is attached to the stage of an inverted microscope to operate a needle for microinjection.
(5) Microinjection manipulator: A device that controls the pressure during microinjection.

As the nematode host, C. elegans wild-type N2 strain was used. DNA was injected into a glass needle for microinjection and set in a microinjection manipulator. Hermaphrodite adults were mounted on the agar pad, a glass needle was promptly inserted into the gonad, and DNA was injected into the gonad. After the injection, the insects were collected and cultured in a solid culture medium for nematodes (NGM medium). Three to four days after microinjection, transformants (F1) were identified and isolated using CaMP fluorescence and markers (mainly fluorescence) as indicators and cultured in fresh NGM medium. After 3-4 days, transformants (F2) were sorted to obtain transgenic lines that transmit CaMP fluorescence and marker expression. The transgenic nematode obtained by this method is a chimera having two types of cells in the body, a cell having an extrachromosomal DNA array and a cell having no extrachromosomal DNA array (Ex line). Therefore, a transgenic line (Is line) that stably expresses CaMP was prepared according to the UV method (Mitani Development Growth & Differentiation 37 (5) 551-557, 1995). In this method, the Ex line is irradiated with short-wavelength ultraviolet light (254 nm) to induce DNA recombination, and the DNA array is inserted into the chromosome. The Is line also backcrossed several times with the wild-type strain (N2). The obtained transgenic nematodes were used for analysis, and frozen stock was prepared and stored.

DNA
The expression vector of G-CaMP4 used for microinjection was constructed as follows.
After adding restriction enzymes Not I and Bgl II to the pFX_EGFPT vector and reacting at 37 ° C. for 2 hours, the reaction product was electrophoresed with 1% agarose. After electrophoresis, the position of the DNA fragment on the gel was confirmed with a gel imaging device, and the gel fragment was cut out. Use the Mag Extractor PCR & Gel Cleanup kit (Toyobo Life Science Division) or FastGene Gel / PCR Extraction kit (Nippon Genetics) to recover DNA fragments from the gel, and operate according to the kit manual, and do not include the EGFP part. A vector fragment was obtained. The DNA fragment of G-CaMP4 was prepared by the following method. Using the pN1-G-CaMP4 vector as a template, a Not I site was added to the 5'end side and a Bgl II site was added to the 3'end side by the PCR method. The following primer pairs were used for the PCR reaction.
GCaMP4 # F1: 5'-TGCGGCCGCATGCGGGGTTCTCATCATCA-3'(SEQ ID NO: 15)
GCaMP4 # R2: 5'-TAGATCTTCACTTCGCTGTCATCATTTG-3' (SEQ ID NO: 16)
After the PCR reaction, the reaction solution was electrophoresed on a 1% agarose gel, and the amplified PCR product was recovered using the MagExtractor PCR & Gel Cleanup kit or the FastGene Gel / PCR Extraction kit. The recovered PCR product was operated using the Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific) according to the manual of the kit, and was subcloned into the pCR Blunt II vector (Thermo Fisher Scientific). The subcloned DNA was extracted from Escherichia coli using a Plasmamid mini prep kit (Qiagen) or a FastGene® plasmid mini kit (Nippon Genetics), and the procedure was performed according to the manual of the kit. The DNA sequence of PCR-amplified G-CaMP4 was confirmed using DNA sequencing (GenomeLab DTCS Quick Start Kit Beckman Coulter). After adding restriction enzymes Not I and Bgl II to the PCR-amplified pCR Blunt II vector with G-CaMP4 and reacting at 37 ° C for 2 hours, use MagExtractor PCR & Gel Cleanup kit or FastGene Gel / PCR Extraction kit. DNA (G-CaMP4 cDNA) was recovered from the gel. Using DNA Ligation kit ver 2.1 (Takara Bio), operate according to the manual of the kit, pFX_EGFPT vector containing no EGFP part cleaved with Not I / Bgl II, and G- CaMP4 cleaved with Not I / Bgl II. The PCR product was bound.

The Ligation reactant is transformed into One shot Top10 chemically competent E. coli, cultured overnight at 37 ° C, and then plasmid using the Plasmamid mini prep kit or FastGene® plasmid mini-kit. Extraction was performed. The plasmid was confirmed by enzymatic treatment by double digestion using Xho I and Bgl II, and double digestion using Not I and Bgl II, and by electrophoresis on a 1% agarose gel. Subsequently, restriction enzymes BamH I and Not I were added to the plasmid and reacted at 37 ° C. for 2 hours, and then the reaction product was electrophoresed with 1% agarose. After electrophoresis, the position of the DNA fragment on the gel was confirmed with a gel imaging device, and the gel fragment was cut out. DNA fragments were recovered from the gel using the Mag Extractor PCR & Gel Cleanup kit or the FastGene Gel / PCR Extraction kit. The integration of the myo-2 promoter (pharyngeal muscle-specific promoter) sequence was performed as follows. The pFX-myo2 :: venus vector was treated with the restriction enzymes Bam HI and Not I in the same manner as above to obtain a DNA fragment containing the myo-2 promoter sequence. A DNA fragment containing this myo-2 promoter sequence was mixed with a pFX_EGFPT vector containing G-CaMP4 cleaved with Bam HI and Not I, and a Ligation reaction was performed.
The Ligation reaction was transfected into One shot Top10 chemically competent E. coli or DH5α chemically competent E. coli (Takara Bio), and the plasmid was recovered by the same method as above, and the plasmid for G-CaMP expression Pmyo-2 :: Obtained G-CaMP.

The G-CaMP expression plasmid (Pmyo-2 :: G-CaMP) prepared as described above was adjusted to 200 ng / μl, and after removing fine impurities with a 0.45 micron filter, it was used for microinjection. .. At this time, as a marker, a gene marker that causes an expression vector such as red fluorescent protein (RFP) or a genetic feature (used to distinguish between non-expressed nematodes and expressed nematodes based on morphology and behavior) is co-injected. be able to. For example, in the case of a red fluorescent protein (RFP) expression vector, it is adjusted to 200 ng / μl, and both expression vectors are mixed in a quantity ratio of CaMP: RFP = 1: 1 to 10: 1, respectively, and a 0.45 micron filter is used. It was used for microinjection after removing fine impurities.

Measuring method of pumping frequency An agar pad was prepared by spreading 3% agar thinly on a slide glass. CaMP-expressing nematodes were placed on the pharyngeal muscles on this agar pad, and observation was performed with or without a cover glass. Time-lapse photography was performed at 15 to 30 images / sec (512 x 512 pixel size) in the confocal mode of a laser microscope (A1RMP Nikon) using a 4x or 10x objective lens. Fluorescent images and transmitted light images were taken using a 488 nm laser as the excitation light. After shooting, offline analysis was performed using image analysis software (NIS Elements Nikon Instec). A region of interest (ROI) was taken in the pharyngeal muscle of the nematode, the fluorescence brightness was measured, and the change over time was graphed.

2. 2. Measurement results As a result of transducing the above Pmyo-2 :: G-CaMP vector into wild-type nematode N2, CaMP expression was confirmed in the pharyngeal muscle (Fig. 1, left). When the fluorescence of CaMP obtained from the pharynx of C. elegans was measured with a fluorescence microscope (laser confocal microscope), a change in fluorescence intensity was observed in response to the contraction movement of the pharyngeal muscle (Fig. 1, right). In addition, fluorescence measurement could also be performed with a high-sensitivity cMOS camera for research (ORCA-Flash 4.0 Hamamatsu Photonics) mounted on a fluorescence microscope (FN1 Nikon Instec).

In the nematode according to the present invention, CaMP is expressed in the muscle cells of the pharyngeal muscle. Using the nematodes, it is possible to investigate the effects of toxicity tests, drug efficacy tests, environmental changes such as radiation and temperature, gene mutations, epigenetic changes, and social behaviors between allogeneic and heterologous animals. In addition to the fields of pharmacy and medicine, it is expected to be used in fields such as space engineering and the environment.

Claims (6)

One or more of CaMP, which is a fluorescent calcium probe, is expressed specifically in the pharyngeal muscle in the muscle cells of the pharyngeal muscle, or can be expressed by an inducible promoter, and the CaMP is described in the following (a). ), A nematode characterized by being a fluorescent calcium probe selected from the group consisting of the fluorescent calcium probe group shown in (b) or (c).
(A) A fluorescent calcium probe group consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.
(B) One or several amino acids are added, substituted, inserted or added to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. Fluorescent calcium probe group consisting of deleted amino acid sequences,
(C) From an amino acid sequence having 90 % or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. Fluorescent calcium probe group The nematode according to claim 1, wherein the nematode is C. elegans, C. briggsae or C. remanei. A method for evaluating the physiological activity of a test substance, which comprises the following steps (a) and (b).
(A) The step of bringing the test substance into contact with the nematode according to any one of claims 1 or 2.
(B) Step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode. The method of claim 3, further comprising the steps (c) and (d) below.
(C) The step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode according to claim 1 or 2.
(D) A step of comparing the count number of the step (c) with the count number of the step (b) of claim 3. A method for investigating the effect of radiation on a living body, which comprises the following steps (a) and (b).
(A) The step of irradiating the nematode according to any one of claims 1 or 2 with radiation.
(B) Step of counting the number of changes in fluorescence intensity from CaMP expressed in the pharyngeal muscle of the nematode. The method of claim 5, further comprising the steps (c) and (d) below.
(C) The step of counting the number of changes in fluorescence intensity from CaMP expressed in the nematode according to claim 1 or 2.
(D) A step of comparing the count number of the step (c) with the count number of the step (b) of claim 5.

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