WO2016117135A1 - Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d'expression pour la visualisation de cellule eucaryote - Google Patents

Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d'expression pour la visualisation de cellule eucaryote Download PDF

Info

Publication number
WO2016117135A1
WO2016117135A1 PCT/JP2015/052781 JP2015052781W WO2016117135A1 WO 2016117135 A1 WO2016117135 A1 WO 2016117135A1 JP 2015052781 W JP2015052781 W JP 2015052781W WO 2016117135 A1 WO2016117135 A1 WO 2016117135A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
reporter gene
related protein
intron
cells
Prior art date
Application number
PCT/JP2015/052781
Other languages
English (en)
Japanese (ja)
Inventor
和史 合田
Original Assignee
オリンパス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by オリンパス株式会社 filed Critical オリンパス株式会社
Priority to PCT/JP2015/052781 priority Critical patent/WO2016117135A1/fr
Priority to JP2016570469A priority patent/JPWO2016117135A1/ja
Publication of WO2016117135A1 publication Critical patent/WO2016117135A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a method for visualizing eukaryotic cells, and a modified reporter gene and expression vector for visualizing eukaryotic cells.
  • reporter assay techniques and cell imaging techniques have been widely used for the purpose of observing various life phenomena.
  • 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.
  • 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.
  • 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.
  • a plant cell or animal cell to be observed that is, a eukaryotic cell
  • a bacterium prokaryote
  • 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.
  • Non-Patent Document 1 is a method limited to plant cells performed using an old-type luminescent reporter gene.
  • 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.
  • it has been suggested in biological mechanisms that the activation of target genes in animal cells to be observed may be affected by prokaryotes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the one or more eukaryotic cells may include animal cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • E._coli, and measured with the luminometer is shown.
  • 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.
  • 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.
  • a prokaryote prokaryotic cell
  • 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.
  • the sample 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.
  • fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and variants thereof.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intron examples include, for example, a second intron (hereinafter referred to as ST-LS1 intron) possessed by the LS1 gene of potato (Solanum tuberosum).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the base sequence of the light-related protein adjacent to the 5 ′ end of the intron is AG, and the 3 ′ end of the intron (splice acceptor 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • conditions vary depending on the light-related protein used. Those skilled in the art can appropriately select appropriate conditions.
  • 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.
  • 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.
  • light can be detected by imaging using an imaging device such as a CCD or CMOS.
  • the light detected can be visualized by amplifying it with a photomultiplier such as a photomultiplier.
  • 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.
  • 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.
  • 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.
  • a light-related protein such as a commercially available photoprotein or fluorescent protein
  • any genetic technique can be produced. Examples of the genetic techniques include restriction enzyme digestion, ligase ligation, and commercially available recombinant techniques.
  • the intron may be inserted into a commercially available vector previously containing a photoprotein or fluorescent protein coding sequence.
  • commercially available vectors containing a luciferase gene in advance include pGL4 vector (Promega), Eluc vector (Toyobo), CRB vector (Promega) and Renilla vector (Promega).
  • 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.
  • 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.
  • Expression vectors can be constructed.
  • 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. .
  • Example 1 Construction of mammalian cell expression vector having modified reporter gene
  • ST-LS1 intron 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.
  • 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
  • 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.
  • 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 ⁇ L.
  • the nucleotide sequence of the luc2 amplification primer is shown below. PCR reaction conditions were as follows.
  • 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.
  • 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.
  • Example 2 Construction of expression vectors for plant cells and soil bacteria
  • 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 ⁇ L.
  • 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.
  • 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.
  • 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.
  • 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.
  • the light emission amount on the vertical axis of the graph is an integrated value for 15 seconds.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DMEM containing 10% fetal bovine serum (FBS) was used as the medium. It was prepared to contain 2.8 ⁇ g of plasmid DNA and 8.3 ⁇ L of FuGENE® HD reagent in 128 ⁇ L 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.
  • FBS fetal bovine serum
  • FIG. 6 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.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé pour la visualisation d'une cellule eucaryote, grâce auquel la cellule eucaryote peut être spécifiquement identifiée à partir d'un échantillon dans lequel la cellule eucaryote est mélangée avec un procaryote, et un gène rapporteur modifié. Le procédé selon la présente invention pour la visualisation d'une cellule eucaryote comprend : une étape consistant à préparer un échantillon dans laquelle une ou plusieurs cellules eucaryotes sont mélangées avec un ou plusieurs procaryotes ; une étape consistant à introduire un ou plusieurs gènes rapporteurs modifiés, qui contiennent une séquence codant pour une protéine associée à la lumière et ont été modifiés de sorte que la quantité de lumière dérivée de la protéine associée à la lumière est réduite dans les procaryotes, dans les cellules eucaryotes et les procaryotes ; une étape consistant à placer l'échantillon dans des conditions permettant la photodétection de la protéine associée à la lumière ; et une étape consistant à réaliser une photodétection de la protéine associée à la lumière dans l'échantillon. Le gène rapporteur modifié selon la présente invention contient une séquence codant pour une protéine associée à la lumière et un intron épissé dans une cellule eucaryote, l'intron étant inséré dans la séquence codant pour la protéine associée à la lumière.
PCT/JP2015/052781 2015-01-23 2015-01-23 Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d'expression pour la visualisation de cellule eucaryote WO2016117135A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2015/052781 WO2016117135A1 (fr) 2015-01-23 2015-01-23 Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d'expression pour la visualisation de cellule eucaryote
JP2016570469A JPWO2016117135A1 (ja) 2015-01-23 2015-01-23 真核生物細胞の可視化方法、並びに真核生物細胞の可視化用の改変レポーター遺伝子及び発現ベクター

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2015/052781 WO2016117135A1 (fr) 2015-01-23 2015-01-23 Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d'expression pour la visualisation de cellule eucaryote

Publications (1)

Publication Number Publication Date
WO2016117135A1 true WO2016117135A1 (fr) 2016-07-28

Family

ID=56416700

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/052781 WO2016117135A1 (fr) 2015-01-23 2015-01-23 Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d'expression pour la visualisation de cellule eucaryote

Country Status (2)

Country Link
JP (1) JPWO2016117135A1 (fr)
WO (1) WO2016117135A1 (fr)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ERIKO OGURA ET AL.: "Idenshi Hatsugen Kaiseki ni Okeru Intron Sonyugata Hakko Reporter Idenshi no Oyo", JAPANESE SOCIETY FOR PLANT CELL AND MOLECULAR BIOLOGY TAIKAI SYMPOSIUM KOEN YOSHISHU, vol. 31, 2013, pages 144 *
ERIKO OGURA ET AL.: "Idenshi Hatsugen Kaiseki ni Okeru Intron Sonyugata Hakko Reporter Idenshi no Riyo Oyobi Kohatsugen Vector Cis Hairetsu no Saitekika ni Tsuite", JAPANESE SOCIETY FOR PLANT CELL AND MOLECULAR BIOLOGY TAIKAI SYMPOSIUM KOEN YOSHISHU, vol. 32, 2014, pages 77 *
HIDETO HAYAKAWA ET AL.: "Novel intron-containing luciferase genes for quantitative analysis of mRNA levels in transient gene expression assays", PLANT BIOTECHNOLOGY, vol. 29, no. 5, 2012, pages 505 - 509 *
JOSE S. GIL ET AL.: "A method to rapidly and accurately compare relative efficacies of non- invasive imaging reporter genes in a mouse model, and its application to luciferase reporters", MOL IMAGING BIOL., 2013, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813453/pdf/nihms-521075.pdf> *
S. LUKE MANKIN ET AL.: "Introduction of a plant intron into the luciferase gene of Photinus pyralis", PLANT MOLECULAR BIOLOGY REPORTER, vol. 15, no. 2, 1997, pages 186 - 196 *

Also Published As

Publication number Publication date
JPWO2016117135A1 (ja) 2017-10-26

Similar Documents

Publication Publication Date Title
Minko et al. Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas
WO2013044792A1 (fr) Sonde fluorescente codant pour un gène nicotinamide adénine dinucléotide, son procédé de préparation et son application
JP4669180B2 (ja) ルシフェラーゼ発現カセットおよび使用法
US8206961B2 (en) Modified Luciola cruciata luciferase protein
JP2008054689A (ja) 単離されたルシフェラーゼおよびその使用
Mitani et al. Novel gene encoding a unique luciferase from the fireworm Odontsyllis undecimdonta
JP6025566B2 (ja) ホタル由来ルシフェラーゼ
KR102018248B1 (ko) 생물발광 강도가 증폭된 변이 가우시아 루시퍼라아제
JP2012235756A (ja) ホタル由来ルシフェラーゼ
Shimizu et al. Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei
JP6032861B2 (ja) ホタル由来ルシフェラーゼ
JP6006070B2 (ja) ホタル由来ルシフェラーゼ
EP3037534B1 (fr) Polypeptide présentant des propriétés fluorescentes et son utilisation
WO2016117135A1 (fr) Procédé de visualisation de cellule eucaryote, et gène rapporteur modifié et vecteur d&#39;expression pour la visualisation de cellule eucaryote
US9328334B2 (en) Luciferase derived from lucidina accensa
JP5969757B2 (ja) ホタル由来ルシフェラーゼの変異体
CN108659110B (zh) 一种改进的红色荧光蛋白及其应用
EP3505625B1 (fr) Luciférase modifiée
JP5980608B2 (ja) ホタル由来ルシフェラーゼ
JP5860651B2 (ja) ホタル由来ルシフェラーゼ
EP2686424B1 (fr) Luciférase du ver étoilé
Yu Development of a bioassay for engineered nanoparticles (ENPs) in the environment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15878826

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2016570469

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15878826

Country of ref document: EP

Kind code of ref document: A1