WO2016117135A1 - Method for visualizing eukaryotic cell, and modified reporter gene and expression vector for visualizing eukaryotic cell - Google Patents

Abstract

Provided are a method for visualizing an eukaryotic cell, whereby the eukaryotic cell can be specifically identified from a sample in which the eukaryotic cell is mixed with a prokaryote, and a modified reporter gene. The method according to the present invention for visualizing an eukaryotic cell comprises: a step for preparing a sample in which one or more eukaryotic cells are mixed with one or more prokaryotes; a step for introducing one or more modified reporter genes, which contain a sequence encoding a light-related protein and have been modified so that the quantity of light derived from the light-related protein is reduced in the prokaryotes, into the eukaryotic cells and the prokaryotes; a step for placing the sample under such conditions as enabling the photodetection of the light-related protein; and a step for photodetecting the light-related protein in the sample. The modified reporter gene according to the present invention contains a sequence encoding a light-related protein and an intron spliced in an eukaryotic cell, wherein the intron is inserted into the sequence encoding the light-related protein.

Description

Eukaryotic cell visualization method, and modified reporter gene and expression vector for eukaryotic cell visualization

The present invention relates to a method for visualizing eukaryotic cells, and a modified reporter gene and expression vector for visualizing eukaryotic cells.

Conventionally, reporter assay techniques and cell imaging techniques have been widely used for the purpose of observing various life phenomena. In particular, in cell imaging technology, biological cells are modified using reporter genes that encode light-related proteins such as fluorescent proteins and photoproteins, and various biological phenomena in biological cells are analyzed using fluorescence and luminescence as indicators. Has been done. This technique is very useful in that various life phenomena including the expression and activation state of a target gene in living cells can be monitored and analyzed over time visually.

In order to perform such imaging with high accuracy, it is necessary to specifically detect light derived from a reporter (light-related protein) in a target biological cell. Therefore, efforts are being made to reduce backgrounds other than light derived from reporters in cells to be observed. In general, the sample or test system to be tested is designed to be configured with the minimum necessary elements, and can be pre-purified so as not to contain impurities such as cells other than the object to be observed, if necessary.

However, in the sample to be tested, it may be difficult to separate the cells to be observed from other cells. As an example, there may be mentioned a case where a plant cell or animal cell to be observed (that is, a eukaryotic cell) is infected with a bacterium (prokaryote). For example, in a reporter assay of a living plant cell, if the plant cell is infected with bacteria, the plant cell and the bacteria are mixed, and the reporter gene is introduced not only into the plant cell but also into the bacterium. As a result, light indicating the activation of the reporter is also generated from the mixed bacteria, the background is increased, and the test accuracy can be lowered.

For this reason, even in such a sample, a method is considered in which the reporter is activated only by the plant cell to be observed, the background due to bacteria is reduced, and the test accuracy of the reporter assay is improved. For example, in a reporter assay of a plant cell in which soil bacteria are mixed due to infection, the intron of the potato LS1 gene is inserted into the luc + gene (luminescence reporter gene), whereby the reporter gene incorporated into the soil bacteria It has been reported that the background from soil bacteria could be reduced by suppressing the activity (Non-patent Document 1).

However, the method of Non-Patent Document 1 is a method limited to plant cells performed using an old-type luminescent reporter gene. In a new type of luminescent reporter gene developed in recent years, true methods such as plant cells and animal cells are used. No method has been proposed that can be used in common with nuclear cells. In recent years, it has been suggested in biological mechanisms that the activation of target genes in animal cells to be observed may be affected by prokaryotes. For example, as described in Non-Patent Document 2, it has been reported that the on / off of the function of a clock gene in a biological clock of a mammalian intestine is maintained by a bacterial flora in the intestine. Using this as a trigger, eukaryotic cells and prokaryotes can coexist (for example, in which mammalian intestinal cells and intestinal flora are mixed), eukaryotic cells can be specifically identified. There is a need for possible reporter assays.

The present invention has been made in view of the above problems, and can visualize eukaryotic cells that can specifically identify eukaryotic cells in a sample in which eukaryotic cells and prokaryotes are mixed. It is an object to provide methods, and modified reporter genes and expression vectors for visualization of eukaryotic cells.

The present inventor uses a modified reporter gene modified so that the amount of light derived from light-related proteins in prokaryotes is reduced, so that eukaryotes are mixed in a sample in which eukaryotic cells and prokaryotes (prokaryotes) are mixed. It has been found that signals derived from cells can be detected with high accuracy. The present invention has been realized by conducting earnest research based on these findings.

That is, the eukaryotic cell visualization method of the present invention comprises:
Preparing a sample in which one or more eukaryotic cells and prokaryotes are mixed;
One or more modified reporter genes comprising a coding sequence of a light-related protein and modified so that the amount of light derived from the light-related protein in the prokaryote is reduced in the eukaryotic cell and the prokaryote A process to be introduced in,
Placing the sample under conditions capable of photodetecting the light-related protein;
Photodetecting the light-related protein in the sample;
It is characterized by including.
According to the eukaryotic cell visualization method of the present invention, eukaryotic cells can be specifically visualized in a sample in which eukaryotic cells and prokaryotes are mixed.

In the eukaryotic cell visualization method of the present invention, the modified reporter gene preferably includes an intron spliced in the eukaryotic cell in the coding sequence of the light-related protein. According to this configuration, light derived from a light-related protein can be specifically generated in a eukaryotic cell by a relatively simple principle.

In the eukaryotic cell visualization method of the present invention, the intron is preferably a base sequence that is commonly spliced between eukaryotic species, and more preferably the base sequence of SEQ ID NO: 1. According to these configurations, since it can be used in combination with a plurality of eukaryotic cells including animal cells and / or plant cells and prokaryotes, it is excellent in versatility and economy (for example, reasonable as a reagent). A method can be provided.

In the eukaryotic cell visualization method of the present invention, the one or more eukaryotic cells may include plant cells. According to this configuration, for example, in a plant cell in which soil bacteria are mixed due to infection, activation of a target gene in the plant cell can be specifically visualized.

In the eukaryotic cell visualization method of the present invention, the one or more eukaryotic cells may include animal cells. According to this configuration, for example, activation of a target gene in an animal cell can be specifically visualized in an animal cell in which pathogenic bacteria are mixed due to infection. The animal cell preferably includes an animal cell derived from a mammal. According to these configurations, life phenomena can be elucidated for the purpose of treating or preventing diseases and trauma in animal species, particularly mammalian species, and elucidating and improving body functions.

In the eukaryotic cell visualization method of the present invention, it is preferable that the light-related protein is a photoprotein, and the amount of luminescence is adjusted according to the type of the photoprotein. According to this configuration, the light emission amount can be adjusted and optimized by appropriately selecting the type of photoprotein (biological origin or modification), irrespective of the detection principle based on introns. The photoprotein preferably has the amino acid sequence of SEQ ID NO: 3 having high luminescence intensity.

The modified reporter gene for visualization of eukaryotic cells of the present invention is
A coding sequence of a light-related protein;
An intron spliced in a eukaryotic cell;
Including
A modified reporter gene for visualization of eukaryotic cells, characterized in that the intron is inserted into the coding sequence of the light-related protein.
It is characterized by. The modified reporter gene can be suitably used in the visualization method of the present invention.

In the modified reporter gene of the present invention, the intron preferably has a base sequence that is commonly spliced between eukaryotic species, and more preferably has the base sequence of SEQ ID NO: 1. According to these configurations, a modified reporter gene excellent in versatility and economy (for example, reasonable as a reagent) can be provided because it can be used in combination with a plurality of eukaryotic cells including mammals and prokaryotes. can do.

The modified reporter gene of the present invention can be used for visualization of plant cells. According to this configuration, the visualization method of the present invention can be suitably used when the visualization target is a plant cell.

The modified reporter gene of the present invention can be used for visualization of animal cells. According to this configuration, the visualization method of the present invention can be suitably used when the visualization target is an animal cell. The modified reporter gene of the present invention is preferably used for visualization of mammalian cells. According to this configuration, it can be suitably used for elucidating life phenomena for the purpose of treating or preventing diseases and trauma in animal species, particularly mammalian species, and elucidating and improving body functions.

In the modified reporter gene of the present invention, it is preferable that the light-related protein is a photoprotein, and the amount of luminescence is adjusted according to the type of the photoprotein. According to this configuration, it is possible to provide a modified reporter gene in which the amount of luminescence is regulated and optimized by appropriately selecting the type of photoprotein (derived from organism or modified). The photoprotein preferably has the amino acid sequence of SEQ ID NO: 3 having high luminescence intensity.

An expression vector for visualizing eukaryotic cells of the present invention is characterized by including the modified reporter gene of the present invention. The expression vector can be suitably used in the visualization method of the present invention.

According to the present invention, visualization of eukaryotic cells that can specifically identify eukaryotic cells in a sample in which one or more eukaryotic cells and one or more prokaryotes are mixed. Methods, modified reporter genes and expression vectors for visualization of eukaryotic cells can be provided.

The result which introduce | transduced the modified reporter gene which concerns on one Embodiment of this invention into soil bacteria, and was measured with the luminometer is shown. The result of having introduced the modified reporter gene which concerns on one Embodiment of this invention into soil bacteria, and performed the imaging is shown. The result of having introduced the modified reporter gene concerning one embodiment of the present invention into a plant, and having performed imaging is shown. The result which introduce | transduced the modified reporter gene which concerns on one Embodiment of this invention into colon_bacillus | E._coli, and measured with the luminometer is shown. The result of having imaged by introduce | transducing the modified reporter gene which concerns on one Embodiment of this invention in colon_bacillus | E._coli is shown. The result of having introduced the modified reporter gene concerning one embodiment of the present invention into a human cell, and performing imaging is shown.

Hereinafter, the present invention will be specifically described based on the embodiments.

<Visualization method for eukaryotic cells>
The eukaryotic cell visualization method of the present invention comprises:
Preparing a sample in which one or more eukaryotic cells and one or more prokaryotes are mixed;
One or more modified reporter genes comprising a coding sequence of a light-related protein and modified so that the amount of light derived from the light-related protein in the prokaryote is reduced in the eukaryotic cell and the prokaryote A process to be introduced in,
Placing the sample under conditions capable of photodetecting the light-related protein;
Photodetecting the light-related protein in the sample;
It is characterized by including.

The visualization method of the present invention uses one or more modified reporter genes that contain a coding sequence of a light-related protein and are modified so that the amount of light derived from the light-related protein is reduced in a prokaryote (prokaryotic cell) To do. When the modified reporter gene is introduced into a prokaryote, the amount of light derived from the light-related protein in the prokaryote is reduced, whereas when it is introduced into a eukaryotic cell, it is derived from the light-related protein in the eukaryotic cell. The amount of light is not reduced. Therefore, the detection intensity of the light quantity in the eukaryotic cell becomes relatively strong, and the eukaryotic cell can be detected and visualized with high accuracy.

The visualization method of the present invention can be used, for example, in a test environment (for example, a culture chamber) in which prokaryotes having eukaryotes as hosts are mixed. Moreover, the test environment may be in vivo or in vitro. According to the visualization method of the present invention, light derived from the modified reporter gene taken up by the prokaryotic organism is not detected, and substantially only light from the modified reporter gene taken up by the eukaryotic cell is detected. Therefore, it is possible to accurately detect and visualize the on / off of a biological function derived from a biological interaction with a eukaryotic cell alone or with a prokaryote.

The visualization method of the present invention includes a step of preparing a sample in which one or two or more eukaryotic cells and one or two or more prokaryotes are mixed. “One or more” means that it may be only a single type or a mixture of different types.

The one or more eukaryotic cells include eukaryotic cells visualized by the visualization method of the present invention. The one or more eukaryotic cells are not particularly limited, and may include plant cells and animal cells alone or in combination. The animal cells preferably include mammalian cells. Elucidation of life phenomena is often performed for the purpose of treating or preventing diseases and trauma in mammalian species and elucidating and improving body functions, and animal cells derived from mammals are widely used for such life clarification. is there.

The prokaryote is not particularly limited, and may be any of bacteria and algae. Specific examples include those that use eukaryotic cells to be visualized as hosts, those that can affect the life phenomena of the eukaryotic cells, and those that are difficult to separate from the eukaryotic cells. . Examples include soil bacteria that infect plant cells and pathogenic bacteria that infect animal cells.

When the test environment is in vitro, the sample is preferably prepared under conditions where viable eukaryotic cells can survive. Examples of such conditions include medium composition and pH, atmospheric composition (eg, oxygen concentration), temperature, luminous intensity, container, and the like. These can be appropriately selected depending on the eukaryotic cell to be visualized. Although the example of a container is not specifically limited, A culture chamber, a petri dish, a well plate, etc. are mentioned. The prepared sample may be subjected to the next step without taking time, or may be subjected to the next step after a certain period of time has elapsed. Alternatively, if necessary, the next step may be performed after culturing eukaryotic cells or prokaryotes in the sample. The sample is preferably placed under conditions that allow the eukaryotic cells to be visualized to survive in the subsequent steps.

The visualization method of the present invention includes the step of introducing one or more modified reporter genes into one or more eukaryotic cells and one or more prokaryotes. The modified reporter gene includes a coding sequence for a light-related protein. The light-related protein means a protein whose presence can be detected directly or indirectly by light, and examples thereof include a photoprotein and a fluorescent protein.

Photoprotein means an enzyme that catalyzes a chemical reaction in which luminescence occurs. Examples include luciferase, aequorin, and mutants thereof. Luciferase catalyzes the oxidation reaction of luciferin as a substrate when ATP is present. During the reaction, luciferin emits light. Aequorin is a protein complex that contains coelenterazine, a substrate, in the molecule, and aequorin itself emits light when triggered by calcium ions.

Fluorescent protein means a protein that emits fluorescence by itself or absorbs excitation energy when irradiated with excitation light. Examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and variants thereof.

As the light-related protein, any of known luminescent proteins and fluorescent proteins can be used, but luminescent proteins are more preferable, and luciferase is more preferable among them. Luciferase can control the on / off of luminescence using enzyme-substrate reaction such as luciferase-luciferin reaction, the species-specific combination of enzyme and substrate is strong, the half-life is compared with the half-life of fluorescent protein This is because it has many advantages such as short and short, auto-fluorescence emitted by cells does not become a background, and a wide dynamic range. Luciferase may be derived from various organisms such as fireflies and bacteria. As the luciferase, for example, commercially available products such as luc2 luciferase, Eluc luciferase, CRB luciferase, and Renilla luciferase can be used.

The modified reporter gene is modified so that the amount of light derived from the light-related protein is reduced in prokaryotes. For example, it has been modified so that expression of a modified reporter gene in prokaryotes can be inhibited or reduced by taking advantage of differences in gene expression mechanisms between prokaryotes (prokaryotes) and eukaryotic cells. Such alteration can be performed by genetic techniques such as intron introduction, mutation introduction, and modification. Such modifications may be introduced into commercially available vectors that already contain the coding sequence for the light-related protein. Examples of commercially available vectors containing a luciferase gene in advance include pGL4 vector (Promega), Eluc vector (Toyobo), CRB vector (Promega) and Renilla vector (Promega).

In the present invention, the modified reporter gene preferably includes an intron that is spliced in a eukaryotic cell to be visualized in the coding sequence of the light-related protein. Including an intron in the coding sequence means that the intron has been inserted in a manner that disrupts the coding sequence. When the modified reporter gene is transcribed in a eukaryotic cell, an mRNA precursor containing the complementary sequence of the intron is formed. Only the intron region is spliced from the mRNA precursor. Subsequently, the complementary sequences of the separated exon sequences are ligated to form a mature mRNA that contains the complete sequence of the complementary sequence of the light-related protein coding sequence. This mature mRNA is translated to form a polypeptide chain containing the full-length amino acid sequence of the light-related protein, and the light-related protein is formed in a complete form. That is, the light-related protein is normally expressed. In this way, in eukaryotic cells, normal expression of light-related proteins can be detected as light, that is, light can be detected.

On the other hand, in a prokaryote (prokaryotic cell), the modified reporter gene is transcribed into mRNA containing the intron region. Since splicing is a phenomenon unique to eukaryotic cells, the mRNA is translated without the intron region being spliced. When the intron region contains a stop codon (for example, UAA, UAC, UGA, etc.), transcription of the modified reporter gene is terminated at the position of the stop codon, so that only a part of the coding sequence of the light-related protein is translated. Not. The polypeptide chain thus translated contains only a partial fragment on the N-terminal side of the full-length amino acid sequence of the light-related protein. Therefore, the light-related protein cannot be formed in a complete form.

On the other hand, if the intron region does not contain a stop codon, or if the stop codon does not function due to mutation or the like, in prokaryotes, transcription is completed without terminating in the middle of the modified reporter gene, and the intron region is also translated. Is done. The polypeptide chain thus translated contains the full-length amino acid sequence of the light-related protein, but also contains an intron translation sequence so as to disrupt the amino acid sequence. Therefore, normal folding of the polypeptide chain becomes difficult, and the light-related protein cannot be formed substantially. Thus, in prokaryotes, light-related proteins are not normally expressed, and light derived from light-related proteins is not substantially detected.

The intron is not particularly limited, and even if it is a base sequence commonly spliced among eukaryotic species, it is a base sequence specifically spliced according to the organism origin of the eukaryotic cell to be visualized. May be. The intron is preferably a base sequence that is commonly spliced among eukaryotic species. Since it can be used with a sample containing a combination of plant cells and / or animal cells and prokaryotes derived from a plurality of eukaryotes regardless of species, the method is excellent in versatility and economy (for example, reasonable as a reagent). Can be provided. Examples of such introns include those in which the base sequence on the 5 'end side (splice donor site) is GT and the base sequence on the 3' end side (splice acceptor site) is AG.

Examples of the intron include, for example, a second intron (hereinafter referred to as ST-LS1 intron) possessed by the LS1 gene of potato (Solanum tuberosum). The ST-LS1 intron contains a typical splice junction (ie, GT at the intron 5 ′ end (splice donor site) and AG at the intron 3 ′ end (splice acceptor site)) and multiple stop codons (in all translation reading frames). And at least one stop codon), and has a base sequence having a length of 189 bp represented by SEQ ID NO: 1.

The present inventor conducted a test method using a reporter gene having an ST-LS1 intron, which has been used only in a combination of plant cells and soil bacteria in the prior art (for example, Prior Art Document 1), on mammalian cells as well. I found out that it could be applied. That is, it was found that a reporter gene having an intron designed for plant observation can be applied to animal cells (for example, derived from mammals) as it is. Furthermore, the present inventors have replaced the combination of the conventional luminescent reporter gene with low luminescent brightness and the ST-LS1 intron, which has been studied in the prior art for the purpose of visualizing plant cells, to emit luminescence with stronger luminescence intensity. It was found that a combination of the gene luc2 luciferase and the ST-LS1 intron can be used. In splicing in eukaryotic cells, the RNA that undergoes the splicing reaction is a single-stranded nucleic acid, and is therefore expected to form a higher-order three-dimensional structure in the cell. Under these circumstances, considering differences in the coding sequences of light-related proteins and differences between plant cells and animal cells, whether the target splicing reaction proceeds as expected is similar to the consensus sequence before and after the intron. Prediction based only on the degree is difficult even for those skilled in the art. In fact, when comparing the sequences in the vicinity of introns between primate-derived animal cells and plant cells, the inventor found that the frequencies of occurrence of bases of −2, −1, and +3 on the 5 ′ side and −6 on the 3 ′ side, The knowledge that the appearance frequency of bases of −5, −4, and −3 is different has been obtained. This fact suggests that different gene expression mechanisms are working between plant cells and animal cells even in sequences near introns. Based on these facts, the novel modified reporter gene and visualization method of the present invention that can be commonly used in eukaryotic cells regardless of plant cells and animal cells provide a significant advance over the prior art. is there.

The site for inserting an intron in the coding sequence of light-related protein is not particularly limited, but it is inserted so that a base sequence that appears frequently in the vicinity of the intron in the eukaryotic cell species to be visualized is located in the vicinity of the intron. It is preferable. For the purpose of visualizing plant cells, for example, the base sequence of the light-related protein adjacent to the 5 ′ end (splice donor site) of the intron is AAG or CT, and the light related protein is adjacent to the 3 ′ end (splice acceptor site) of the intron. Examples include a site where the base sequence of the protein is G. In mammalian cells (eg, from primates), for example, the base sequence of the light-related protein adjacent to the 5 ′ end of the intron (splice donor site) is AG, and the 3 ′ end of the intron (splice acceptor site). The site | part from which the base sequence of the light related protein adjacent to is G is mentioned. The site for inserting the intron is, for example, AG, the base sequence of the light-related protein adjacent to the 5 ′ end (splice donor site) of the intron, and the base of the light-related protein adjacent to the 3 ′ end (splice accepting site) of the intron. A site where the sequence is G is preferred. In this example, an intron is inserted between AG and G at the site having the base sequence of AGG in the coding sequence of the light-related protein. In the coding sequence of the light-related protein, an intron may be inserted into a site of a desired base sequence that originally exists, or an intron may be inserted into a site of a desired base sequence generated by introducing a mutation.

As an example of a preferred combination of a light-related protein and an intron, a luciferase luc2 derived from North American firefly (Phototinus pyralis) encoded by the base sequence of SEQ ID NO: 2 and having the amino acid sequence of base sequence 3, and an ST-LS1 intron And the combination. A preferred example of the modified reporter gene is the base sequence of SEQ ID NO: 4 in which an ST-LS1 intron is inserted into the luciferase luc2. In the modified reporter gene having the base sequence of SEQ ID NO: 4, the base sequence of the ST-LS1 intron (SEQ ID NO: 1) is inserted between the 478th base and the 489th base of luc2.

Modulating light in eukaryotic cells and prokaryotes by changing the type of light-related protein, the type of intron, the combination of light-related protein and intron, the arrangement of introns and light-related protein coding sequences (for example, Generation, augmentation, suppression, or stopping). A person skilled in the art can determine the optimal combination depending on the origin or sequence type of the intron and / or light-related protein.

The modified reporter gene may be a DNA strand or an RNA strand. The modified reporter gene of the present invention may be composed only of the coding sequence of the light-related protein and the intron, or as long as the function and expression of the modified reporter gene and the light-related protein are not impaired. It may have a mutation such as substitution, deletion or addition of a base sequence, or an additional base sequence.

Also, the visualization method of the present invention includes a step of placing a sample in which eukaryotic cells and prokaryotes are mixed under conditions that allow light detection of the light-related protein. Such conditions vary depending on the light-related protein used. Those skilled in the art can appropriately select appropriate conditions. For example, when the light-related protein is luciferase, luciferin that reacts with the luciferase is added to the sample. Calcium ions are added to the sample when the light-related protein is iculione. When the light-related protein is a fluorescent protein, the sample is irradiated with excitation light having a specific wavelength that can excite the fluorescent protein.

The visualization method of the present invention includes a step of photodetecting a light-related protein in a sample. Photodetection can be performed using any device capable of detecting light from the light-related protein used. For example, light can be detected by imaging using an imaging device such as a CCD or CMOS. In addition, the light detected can be visualized by amplifying it with a photomultiplier such as a photomultiplier. By using a photoprotein, particularly luciferase, as the light-related protein, light detection and visualization can be performed with high sensitivity, high accuracy, and short time. An apparatus capable of performing photodetection and visualization of a photoprotein is also called a luminescence imaging system, and examples thereof include a luminescence imaging system LV200 (manufactured by Olympus Corporation).

Imaging with a light image sensor can be performed over time, that is, continuously at an arbitrary interval. For example, imaging is performed at intervals of 5 minutes to 1 hour. Moreover, the time of one imaging is arbitrarily set according to the light quantity of light related protein. One imaging time can be adjusted so that a sufficient amount of light can be detected. Visualization over time can be performed by imaging over time. Therefore, it is very significant in that life phenomena in eukaryotic cells can be monitored over time.

The method for visualizing eukaryotic cells of the present invention can be carried out in vitro or in vivo. Further, in a sample containing a plurality of different types of eukaryotic cells, a plurality of modified reporter genes having different light-related proteins for each type can be used to visualize each type of eukaryotic cell. Furthermore, by using a luminescent protein such as luc2 such as luc2 as the light-related protein, high sensitivity and / or shortening of detection time can be achieved. In addition, the visualization method of the present invention can be modified in various ways such as a modified reporter gene, light detection conditions, a modified reporter gene introduction method and the like based on common general technical knowledge.

<Modified reporter gene for genetic visualization of eukaryotic cells>
The modified reporter gene for eukaryotic cells of the present invention is
A coding sequence of a light-related protein;
An intron spliced in a eukaryotic cell;
Including
The intron is inserted in the coding sequence of the light-related protein. This modified reporter gene can be suitably used in the eukaryotic cell visualization method of the present invention.

The constitution and preferred embodiment of the modified reporter gene of the present invention are as described above. This modified reporter gene inserts an intron that is spliced in a eukaryotic cell to be visualized into the coding sequence of a light-related protein, such as a commercially available photoprotein or fluorescent protein, using any genetic technique Can be produced. Examples of the genetic techniques include restriction enzyme digestion, ligase ligation, and commercially available recombinant techniques.

For example, the intron may be inserted into a commercially available vector previously containing a photoprotein or fluorescent protein coding sequence. Examples of commercially available vectors containing a luciferase gene in advance include pGL4 vector (Promega), Eluc vector (Toyobo), CRB vector (Promega) and Renilla vector (Promega).

<Expression vector for visualization of eukaryotic cells>
The expression vector for visualization of eukaryotic cells of the present invention is characterized by including a modified reporter gene for visualization of eukaryotic cells of the present invention. The expression vector of the present invention only needs to contain the modified reporter gene so that it can be expressed. For example, in the expression vector, the modified reporter gene is linked to the downstream of the promoter gene region so that it can be expressed. The type of vector is not particularly limited as long as it can be expressed in eukaryotic cells, and commercially available products can be used. Examples include plasmid vectors, phage vectors, cosmids and the like.

A person skilled in the art will select an appropriate vector based on various conditions such as cloning sites, promoter genes and other regulatory factors, and the origin of the eukaryotic cell to be expressed, in accordance with common general technical knowledge. Expression vectors can be constructed. Alternatively, as described above, introns may be inserted into commercially available vectors that already contain coding sequences for photoproteins and fluorescent proteins.

The expression vector of the present invention can also be provided in the form of a kit containing a reagent for introduction into eukaryotic cells, a reagent necessary for light detection of the formed light-related protein, and / or an assay protocol. .

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

[Experimental Example 1: Construction of mammalian cell expression vector having modified reporter gene]
As a modified reporter gene according to one embodiment of the present invention, an intron (ST-LS1 intron) of a potato-derived ST-LS1 gene in the base sequence (SEQ ID NO: 2) of a luciferase gene luc2 derived from North American firefly (Photinus pyralis) IntGL4 (SEQ ID NO: 4) in which the base sequence (SEQ ID NO: 1) was inserted was constructed. Specifically, the luc2 gene is inserted downstream of the CMV promoter of the plasmid pcDNA3.1 for transformation of mammalian cells to produce pcDNA3.1 (+)-GL4, and in pcDNA3.1 (+)-GL4 A mammalian cell expression vector pcDNA3.1 (+)-intGL4 having a modified reporter gene intGL4 was constructed by inserting the ST-LS1 intron into the luc2 gene. The experimental procedure is as follows.

[Preparation of pcDNA3.1 (+)-GL4]
First, using the plasmid pGL4.14 (Promega) having the nucleotide sequence of the luc2 gene as a template, the DNA of the luc2 gene was amplified by PCR. The PCR reaction solution was prepared as follows according to the standard protocol attached to PrimeSTAR (registered trademark) polymerase kit (TAKARA). Primeng HS DNA polymerase attached buffer (100 mM Tris-HCl buffer, 10 mM KCl, 6 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgCl ₂ , 0.1% Triton X-100, 0.001% BSA), 98 ng of pGL4 .14 plasmid DNA, each dNTP (final concentration: 0.2 mM), 2.5 U PrimeSTAR HS DNA polymerase, luc2 amplification primer (each final concentration: 300 nM) were added to a final volume of 40 μL. The nucleotide sequence of the luc2 amplification primer is shown below.

PCR reaction conditions were as follows. 1st step: 95 ° C for 5 minutes, 2nd step: 94 ° C for 15 seconds (denaturation), 55 ° C for 15 seconds (annealing), 72 ° C for 3 minutes (extension), 40 cycles, 3rd step: 7 minutes at 72 ° C. This PCR product was subjected to agarose electrophoresis to confirm the presence of the target amplification product of about 1660 bp. After confirmation, a gel of the same size was cut out and purified using Wizard (registered trademark) SV Gel and PCR Clean up system (Promega) to recover the DNA fragment of the luc2 gene. This was further digested with restriction enzymes EcoRI and SalI, and a DNA fragment of the luc2 gene for pcDNA3.1 insertion of about 1650 bp was recovered using agarose electrophoresis and Wizard SV Gel and PCR Clean up system.
A plasmid pcDNA3.1 (Lifetechnologies) for mammalian cell transformation was digested with restriction enzymes EcoRI and Xhol, and then a DNA fragment of about 5500 bp was recovered using agarose electrophoresis and Wizard SV Gel and PCR Clean up system. . The pcDNA3.1 DNA fragment and the pcDNA3.1 insertion DNA fragment were ligated to obtain a plasmid pcDNA3.1 (+)-GL4 in which the luc2 gene was inserted downstream of the CMV promoter. The insertion of the luc2 gene was confirmed by decoding the nucleotide sequence with a sequencer.

[Amplification and purification of ST-LS1 intron]
The DNA of ST-LS1 intron was totally synthesized by a gene synthesis service provided by Operan. In order to insert this ST-LS1 intron DNA into the luciferase gene luc2, it was amplified by PCR reaction using PrimeSTAR HS DNA polymerase. A PCR reaction solution was prepared as follows according to the standard protocol attached to PrimeSTAR HS DNA polymerase. PrimeSTAR HS DNA polymerase attached buffer, 0.5 ng ST-LS1 intron DNA, each dNTP (final concentration: 0.2 mM), 2.5 U PrimeSTAR HS DNA polymerase, ST-LS1 intron amplification primer (each final concentration) : 300 nM) to a final volume of 40 μL. The base sequence of the primer for ST-LS1 intron amplification is shown below.

These primers were designed by adding sequences homologous to the insertion site in the luc2 base sequence. Further, the 5 ′ forward primer (SEQ ID NO: 7) contains a silent mutation in which the 476 to 479th nucleotide sequence AGGG of luc2 is changed to AAGG in the modified reporter gene intGL4 in order to enhance recognition as an intron site. Designed. PCR reaction conditions were as follows. 1st step: 95 ° C for 5 minutes, 2nd step: 94 ° C for 20 seconds (denaturation), 56 ° C for 20 seconds (annealing), 72 ° C for 1 minute (extension), 50 cycles, 3rd step: 7 minutes at 72 ° C. This PCR product was subjected to agarose electrophoresis to confirm the presence of the target amplification product. After confirmation, a gel of the size of the target amplification product is excised, purified using the Wizard SV Gel and PCR Clean up system, and the DNA fragment of the ST-LS1 intron for insertion into pcDNA3.1 (+)-GL4 Got.

[Construction of pcDNA3.1 (+)-intGL4]
The restriction enzyme site of Bsp14071 present at position 492 of the nucleotide sequence of the luc2 gene in the pcDNA3.1 (+)-GL4 plasmid was cleaved, and pcDNA3.1 was used using agarose electrophoresis and Wizard SV Gel and PCR Clean up system. A DNA fragment of (+)-GL4 was recovered. Using GeneART® Seamless cloning kit (Lifetechnologies), the DNA fragment of ST-LS1 intron was cloned into the DNA fragment of pcDNA3.1 (+)-GL4 and pcDNA3.1 having the modified reporter gene intGL4 (+)-IntGL4 was obtained. It was confirmed that the target modified reporter gene intGL4 (SEQ ID NO: 3) was obtained by decoding the base sequence with a sequencer.
Gene transfer into human cells and E. coli and confirmation of the luminescence signal will be described later.

[Experimental Example 2: Construction of expression vectors for plant cells and soil bacteria]
For expression of the modified reporter gene intGL4 in plants, a plant transformation plasmid pRI201AN (TAKARA) was used, and between the restriction enzyme sites Ndel and SalI downstream of the 35S promoter derived from cauliflower mosaic virus (CaMV). The intGL4 gene was inserted to construct a plant cell expression vector pRI201AN-intGL4. The specific procedure is as follows.
DNA of intGL4 gene was amplified by PCR using plasmid pcDNA3.1 (+)-intGL4 having the base sequence of intGL4 gene constructed in Experimental Example 1 as a template. The PCR reaction solution was prepared as follows according to the standard protocol attached to PrimeSTAR HS DNA polymerase. PrimeSTAR HS DNA polymerase attached buffer with 98 ng pGL4.14-intGL4 DNA, each dNTP (final concentration: 0.2 mM), 2.5 U PrimeSTAR HS DNA polymerase, intGL4 amplification primer (each final concentration: 300 nM) Added to a final volume of 40 μL. The base sequence of the intGL4 amplification primer is shown below.

The conditions for the PCR reaction were as follows. First step: 95 ° C. for 5 minutes, second step: 95 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds (annealing), 72 ° C. for 2 minutes 30 seconds (extension), 40 cycles, Step: 7 minutes at 2 ° C.
The PCR product was subjected to agarose electrophoresis to confirm the presence of the target amplification product of about 1850 bp. After confirmation, a gel of the same size was cut out and purified using the Wizard SV Gel and PCR Clean up system, and the DNA fragment of the intGL4 gene was recovered. The recovered intGL4 gene DNA fragment was further digested with restriction enzymes Ndel and SalI, and a DNA fragment of about 1840 bp was recovered and used as a pRI201AN insertion intGL4 gene DNA fragment. Plasmid pRI201AN was digested with restriction enzymes Ndel and SalI, and a DNA fragment of approximately 10500 bp was recovered using agarose electrophoresis and Wizard SV Gel and PCR Clean up system. The pRI201AN-intGL4 DNA fragment was ligated to the pRI201AN-inserted intGL4 gene DNA fragment to obtain pRI201AN-intGL4. The insertion of the intGL4 gene was confirmed by decoding the base sequence with a sequencer.
As controls, plasmid pRI201AN and plasmid pRI201AN-GL4 in which only the luc2 gene was inserted downstream of the 35S promoter were also prepared. The introduction of genes into plants and soil bacteria and confirmation of luminescence signals will be described later.

[Experimental Example 3: Introduction of modified reporter gene into soil bacteria and detection of luminescent signal]
The plant cell expression vector pRI201AN-intGL4 was introduced into Agrobacterium tumefaciens LBA4404 Electro-Cells (TAKARA) using an electroporation method. MicroPulser (BioRad) was used for electroporation. For comparison, similar to pRI201AN-intGL4, plasmids pRI201AN and pRI201AN-GL4 were also introduced alone. The gene transfer was performed according to an experimental protocol (http://catalog.takara-bio.co.jp/PDFS/9115_j.pdf) shown by TAKARA.

The detection of the luminescence signal in the soil bacteria was performed by two methods: luminometer measurement of the cultured cells and imaging of the fungal colonies.

In the luminometer measurement of the microbial cell, first, soil bacteria picked up from the colony were cultured with shaking at 28 ° C. for about one night. On the next day, 500 μL of a culture solution showing almost the same growth was taken and centrifuged at 4 ° C. and 10000 rpm for 10 minutes to collect bacteria. The supernatant was discarded, and 300 μL of the pellet and the substrate solution after redissolving in Bright-Glo ™ Luciferase Assay system (Promega) were mixed. After thoroughly mixing, 150 μL was taken from the mixed solution and measured with a luminometer (Luminescence JNRII AB-2300 manufactured by ATTO). The results are shown in FIG. The light emission amount on the vertical axis of the graph is an integrated value for 15 seconds.
In soil bacteria into which the plasmid pRI201AN-GL4 into which only the luciferase gene luc2 was inserted was introduced, a high amount of luminescence was detected. On the other hand, in the soil bacteria into which pRI201AN-intGL4 into which the modified reporter gene was inserted was introduced, when only the reagent was introduced, a very low light emission amount was detected as in the case of introducing plasmid pRI201AN.

In the imaging of fungal colonies, a 2.5 mM luciferin aqueous solution was sprayed on the colonies and allowed to stand in the dark for about 5 minutes, and then a luminescence signal was obtained with Image Quant LAS4000mini from GE. The measurement conditions were “Ultra” in chemili mode and the exposure time was 15 seconds. The results are shown in FIG.
In soil bacteria into which the plasmid pRI201AN-GL4 into which only the luciferase gene luc2 had been inserted was introduced, a luminescence signal could be detected. On the other hand, in the soil bacteria into which pRI201AN-intGL4 into which the modified reporter gene was inserted was introduced (inside the black thick frame in FIG. 2), the luminescence signal could not be detected as in the case of introducing only the reagent and the plasmid pRI201AN. . From these results, it was suggested that the luc2 gene fragment contained in the modified reporter gene of the present invention did not show a luminescence signal in soil bacteria, and the intron in the luc2 gene was not spliced.

[Experimental Example 4: Introduction of modified reporter gene into plant and detection of luminescent signal]
Gene introduction into a plant (Arabidopsis thaliana) was performed using a FAST (Fast Agro-Mediated Seeding Transformation) method. FAST method of protocol, Jian-Feng Li, et al., "The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seeding of Arabidopsis and other plant species", Plant Methods, 2009 years, Vol. 5 No. 6 As described in the issue. The soil bacteria used for gene introduction are cells having pRI201AN (negative control) and cells having pRI201AN-intGL4. Plants after introduction of each gene were subjected to luminescence imaging to confirm the luminescence signal. The observation conditions for luminescence imaging were the same as the method used in Experimental Example 3 except that the exposure time was changed to 10 seconds.

Fig. 3 shows the detection result of the luminescence signal in the plant. The left figure of FIG. 3 is an image under white illumination after luciferin addition, the left half is a plant into which pRI201AN as a negative control is introduced, and the right half is a plant into which pRI201AN-intGL4 is introduced. The right figure in Fig. 3 is a photograph of the luminescence signal taken by adding luciferin to cause luciferase to emit light. The left half is a plant into which pRI201AN as a negative control was introduced, and the right half is a plant into which pRI201AN-intGL4 was introduced. In the photograph of the right figure of FIG. 3, it turns out that light emission is imaged in the position corresponding to the plant body which introduce | transduced pRI201AN-intGL4 shown by the photograph of the left figure of FIG. From this result, it was shown that in plants into which the modified reporter gene of the present invention was introduced, significant luminescence was detected and visualized in plant cells by the visualization method of the present invention.

From the above experiment, it can be seen that by using the modified reporter gene intGL4, plant cells can be visualized significantly in a sample in which plant cells and soil bacteria are mixed. Next, the following experiment was performed to confirm that human cells, which are animal cells, can be visualized significantly even in a sample in which human cells and E. coli are mixed using the modified reporter gene intGL4.

[Experimental Example 5: Introduction of modified reporter gene into E. coli and detection of luminescent signal]
In order to confirm the luminescence signal, a plasmid was introduced into E. coli DH5α strain (TAKARA) by the heat shock method. Details of the protocol are as shown on the TAKARA website (http://catalog.takara-bio.co.jp/PDFS/9057_j.pdf). The detection of the luminescence signal in E. coli was carried out by two methods: luminometer measurement of cultured cells and imaging of bacterial colonies.

The luminometer measurement of the bacterial cells was the same as the method used in Experimental Example 4 except that the accumulated amount of light emission was changed to 5 seconds. The results are shown in FIG.
A high amount of luminescence was detected in the colon bacterium into which the plasmid pcDNA3.1 (+)-GL4 into which only the luciferase gene luc2 was inserted was introduced. On the other hand, in the colon bacterium introduced with pcDNA3.1 (+)-intGL4 into which the modified reporter gene is inserted, when only the reagent is introduced, when plasmid pcDNA3.1 (pcDNA3.1 (+)) as a negative control is introduced As with, very low light emission was detected.

The observation conditions for fungal colony imaging were the same as those used in Experimental Example 4 except that the exposure time was changed to 10 seconds. The detection result of the luminescence signal is shown in FIG.
In Escherichia coli introduced with the plasmid pcDNA3.1 (+)-GL4 into which only the luciferase gene luc2 was inserted, luminescence could be visualized. On the other hand, in E. coli into which pcDNA3.1 (+)-intGL4 into which the modified reporter gene has been introduced (FIG. 5 black frame), when only the reagent is introduced, the same as when pcDNA3.1 (+) is introduced. The luminescence was not visualized. From these results, in E. coli, no luminescence signal was shown by the luc2 gene fragment contained in the modified reporter gene of the present invention, suggesting that the intron in the luc2 gene was not spliced.

[Experimental Example 6: Introduction of modified reporter gene into cultured human cells and detection of luminescent signal]
The expression plasmid vector pcDNA3.1 (+)-intGL4 was transiently introduced into human cultured cell line Hela cells (obtained from ATCC (American Type Culture Collection)). FuGene HD (Promega) was used for gene transfer. The experimental protocol for gene transfer follows the protocol shown on the Promega's website (http://www.promega.com/techserv/tools/FugeneHdTool/) Hela cells, 35 mm dish. It was. Specifically, it is as follows. The day before gene introduction, Hela cells were seeded in a 35 mm dish at a cell number of 3 × 10 ⁵ . DMEM containing 10% fetal bovine serum (FBS) was used as the medium. It was prepared to contain 2.8 μg of plasmid DNA and 8.3 μL of FuGENE® HD reagent in 128 μL of OPTIMEM and incubated at room temperature for 5 minutes. Then, the gene was introduced into Hela cells by adding to the 35 mm dish.

Observation was performed with a luminescence imaging system LV200 (manufactured by Olympus Corporation) for 24 to 48 hours after introduction of the plasmid. An objective lens having a magnification of 40 times was used. The imaging camera was an ImageM camera (manufactured by Hamamatsu Photonics Co., Ltd.), and the image was taken in 1 minute exposure in the EMCCD mode.
The detection result of the luminescence signal in human Hela cells is shown in FIG. The left figure of FIG. 6 is a bright field image under a microscope after luciferin addition. The right side of FIG. 6 (in the thick black frame) is a photograph of the luminescence signal obtained by adding luciferin to cause luciferase to emit light. In the photograph on the right side of FIG. 6, it can be seen that the luciferase luminescence signal is captured at the position corresponding to the human cell shown in the photograph on the left side of FIG. From this result, it was shown that significant luminescence was detected and visualized in human Hela cells by the modified reporter gene of the present invention and the visualization method of the present invention.

From the results of the above experimental examples, it can be said that the visualization method of the present invention and the modified reporter gene and expression vector of the present invention can identify animal cells with high accuracy in a sample in which animal cells and prokaryotes are mixed.

Claims (18)

1. Preparing a sample in which one or more eukaryotic cells and one or more prokaryotes are mixed;
   One or more modified reporter genes comprising a coding sequence of a light-related protein and modified so that the amount of light derived from the light-related protein in the prokaryote is reduced in the eukaryotic cell and the prokaryote A process to be introduced in,
   Placing the sample under conditions capable of photodetecting the light-related protein;
   Photodetecting the light-related protein in the sample;
   A method for visualizing eukaryotic cells, comprising:
2. The visualization method according to claim 1, wherein the modified reporter gene contains an intron spliced in the eukaryotic cell in the coding sequence of the light-related protein.
3. The visualization method according to claim 2, wherein the intron has a base sequence commonly spliced between eukaryotic species.
4. The visualization method according to any one of claims 1 to 3, wherein the intron has the base sequence of SEQ ID NO: 1.
5. The visualization method according to any one of claims 1 to 4, wherein the one or more eukaryotic cells include plant cells.
6. The visualization method according to any one of claims 1 to 5, wherein the one or more eukaryotic cells include animal cells.
7. The visualization method according to claim 6, wherein the animal cells include mammalian cells.
8. The visualization method according to any one of claims 1 to 7, wherein the light-related protein is a photoprotein, and the amount of luminescence is adjusted according to the type of the photoprotein.
9. The visualization method according to claim 8, wherein the photoprotein has the amino acid sequence of SEQ ID NO: 3.
10. A coding sequence of a light-related protein;
    An intron spliced in a eukaryotic cell;
    Including
    A modified reporter gene for visualization of eukaryotic cells, characterized in that the intron is inserted into the coding sequence of the light-related protein.
11. The modified reporter gene according to claim 10, wherein the intron has a base sequence commonly spliced between eukaryotic species.
12. The modified reporter gene according to claim 11, wherein the intron has the base sequence of SEQ ID NO: 1.
13. The modified reporter gene according to any one of claims 10 to 12, which is used for visualization of plant cells.
14. The modified reporter gene according to any one of claims 10 to 13, which is used for visualization of animal cells.
15. The modified reporter gene according to claim 14, which is used for visualization of mammalian cells.
16. The modified reporter gene according to any one of claims 10 to 15, wherein the light-related protein is a photoprotein.
17. The modified reporter gene according to claim 16, wherein the photoprotein has the amino acid sequence of SEQ ID NO: 3.
18. An expression vector for visualizing eukaryotic cells, comprising the modified reporter gene according to any one of claims 10 to 17.

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