New promoter

Abstract

Provided is a promoter comprising any one DNA of (1) to (4) below. (1) a DNA including the base sequence represented by any one of SEQ ID NOs: 1 to 3; (2) a DNA having promotor activity and including a base sequence in which one or more bases are substituted, added, or deleted in the base sequence represented by any one of SEQ ID NOs: 1 to 3; (3) a DNA having promotor activity and including a base sequence that has 90% or more of identity to the base sequence represented by any one of SEQ ID NOs: 1 to 3; and (4) a DNA having promotor activity and including a base sequence that hybridizes with the base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 1 to 3 under a stringent condition.

Description

[Technical Field]

[0001] This invention relates to a novel promoter. [Background technology]

[0002] Aquatic invertebrates, including mollusks, exhibit remarkable biological characteristics in regeneration, adaptation, reproduction, and aging, and elucidating the underlying gene regulation is of great academic significance. However, until now, elucidation of gene function in aquatic invertebrates has been extremely insufficient due to the lack of assay systems such as transfection using HeLa cells or transgenic mice.

[0003] On the other hand, bivalves are classified as the second largest class within the phylum Mollusca and are also important as a food source. In bivalves, studies have been conducted on CMV IE (Cytomegalovirus immediate early) promoters (Non-patent documents 1, 2, 3), but no practical promoter has been found to date.

[0004] To advance the elucidation of gene function in aquatic invertebrates such as bivalves, the establishment of cultured cell lines and the discovery of powerful gene promoters that highly express introduced genes are essential. Therefore, there has been a strong need to develop analytical methods that can quantitatively evaluate gene action with high reproducibility using these methods. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Front. Physiol. 9, 1-8 (2018) [Non-Patent Document 2] Mar. Biotechnol. 3, 322-335 (2001) [Non-Patent Document 3] Fish Shellfish Immunol. 103, 438-441 (2020) [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of this invention is to provide a novel promoter. [Means for solving the problem]

[0007] In this specification, the singular form (a, an, the, etc.) includes both singular and plural forms unless otherwise explicitly stated or the context clearly contradicts it.

[0008] To address the above challenges, the inventors conducted extensive research and discovered that the oshv117 promoter derived from Ostreid herpesvirus 1 (OsHV-1) exhibits potent promoter activity in cells of various animal species, including mollusk cells.

[0009] This invention was completed based on these findings and includes the following broad embodiments of the invention. [Section 1] A promoter consisting of any of the following DNAs (1) to (4): (1) DNA containing a base sequence represented by any of sequence numbers 1 to 3, (2) DNA having promoter activity, which includes a base sequence in which one or more bases are substituted, added, or deleted in any of the base sequences represented by Sequence ID No. 1 to 3. (3) DNA having promoter activity and containing a base sequence that has 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 3 (4) DNA having promoter activity, which contains a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by any of sequence numbers 1 to 3. [Section 2] An expression vector containing the promoter described in item 1. [Section 3] The expression vector according to item 2, in which the expression target gene is integrated under the control of the promoter according to item 1. [Item 4] A transformant comprising the expression vector according to item 2 or 3. [Item 5] The transformant according to item 4, which is an aquatic invertebrate. [Item 6] A method for producing a target substance encoded by an expression target gene, the method comprising the step of culturing the transformant according to item 4 or 5. [Advantages of the Invention]

[0010] According to the present invention, a novel promoter can be provided. Further, an expression vector containing the novel promoter and a transformant containing the expression vector can be provided. [Brief Description of the Drawings]

[0011] [Figure 1] Shows the results of examining the optimization of the culture conditions of scallop cardiomyocytes. [Figure 2] Shows an overview of the electroporation of scallop cardiomyocytes. [Figure 3] Shows the results of examining the optimization of electroporation conditions in Example 1. [Figure 4] Shows the results of the electroporation of luciferase mRNA in Example 1. Appearance of cardiomyocytes 3 days after electroporation. [Figure 5] Shows the results of the electroporation of luciferase mRNA in Example 1. Luciferase expression level 3 days after electroporation. [Figure 6] Shows the results of the electroporation of luciferase mRNA in Example 1. Cell viability after electroporation. [Figure 7] Shows the luciferase vector in Example 2. [Figure 8]The correlation between luciferase expression levels and the number of adherent cells two days after electroporation in Example 3 is shown. [Figure 9] The results of the promoter activity evaluation in Example 4 are shown. [Figure 10] The results of the optimization study of electroporation conditions in Example 5 are shown. A: Expression level when the poring pulse voltage is changed. B: Cell viability when the poring pulse voltage is changed. C: Expression level when the impedance is changed. D: Cell viability when the impedance is changed. E: Expression level when the plasmid vector concentration is changed. F: Cell viability when the plasmid vector concentration is changed. G: Expression level when the culture period after electroporation is changed. H: Cell viability when the culture period after electroporation is changed. [Figure 11] The EGFP vector used in Example 6 is shown. [Figure 12] The GFP assay results for scallop cardiomyocytes in Example 6 are shown. [Figure 13] The results of the GFP assay of scallop hemocytes in Example 6 are shown. [Figure 14] The GFP assay results for HEK293 in Example 6 are shown. [Figure 15] The results of the GFP assay of zebrafish embryos in Example 6 are shown. [Figure 16] The arrangement of oshv117 and its adjacent genes in Example 7 is shown. [Figure 17] The sequence analysis results of Poshv117 using NNPP and NSite in Example 7 are shown. [Figure 18] The promoter deletion analysis results for Poshv117 in Example 8 are shown. [Figure 19] This shows the sequence of arrays 1 through 3. [Figure 20] The sequences of promoters No. 1 to No. 5 in Example 8 are shown. [Modes for carrying out the invention]

[0012] In one embodiment, the present invention provides a promoter comprising any of the following DNAs (1) to (4). (1) DNA containing a base sequence represented by one of sequence numbers 1 to 3. (2) DNA having promoter activity, which contains a base sequence in which one or more bases are substituted, added, or deleted in any of the base sequences represented by Sequence ID No. 1 to 3. (3) DNA containing a base sequence that has 90% or more identity with the base sequence represented by any of sequence numbers 1 to 3, and which has promoter activity. (4) DNA having promoter activity, which contains a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by any of sequence numbers 1 to 3.

[0013] In this specification, "promoter activity" means the activity that promotes the transcription of DNA (genes) into mRNA. Therefore, in the present invention, "having promoter activity" means exhibiting activity that promotes the transcription of DNA (genes) into mRNA in any one or more experimental systems. In the present invention, if a DNA exhibits the above activity in at least one of the experimental systems using cells belonging to mollusks, mammals, fish, etc. (preferably in a system using at least one type of cell from aquatic invertebrates), then the DNA is interpreted as having promoter activity.

[0014] Promoter activity can be confirmed by whether or not protein expression is detected using a suitable reporter gene. For example, promoter activity can be confirmed by introducing DNA containing DNA encoding a detectable protein under promoter control (a reporter gene) into cells and measuring the production amount of the gene product of that reporter gene. Examples of reporter genes include luciferase genes, fluorescent protein genes such as GFP (Green Fluorescent Protein) genes, β-galactosidase (LacZ) genes, β-glucuronidase (GUS) genes, and β-lactamase genes. More specifically, promoter activity can be measured by the method described in the examples of this application, which will be discussed later.

[0015] In one embodiment, the promoter of the present invention has at least twice (more preferably three times, even more preferably five times, and particularly preferably seven times) promoter activity compared to the conventional promoter, the CMV IE (Cytomegalovirus immediate early) promoter.

[0016] Furthermore, in one embodiment, the promoter of the present invention has sufficient promoter activity to enable fluorescence observation using a fluorescence microscope when the GFP gene is used as the reporter gene.

[0017] The promoter of the present invention may consist of a nucleotide sequence substantially identical to the nucleotide sequence shown in any of sequence numbers 1 to 3 of the sequence listing, as long as it has promoter activity. The "promoter activity" of the present invention causes a gene under the control of the promoter (typically, for example, located downstream) to be transcribed into mRNA.

[0018] In (1) above, DNA containing the nucleotide sequences represented by SEQ ID NOs: 1 to 3, and which possesses promoter activity, is included. DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 1 to 3 is a promoter derived from viruses belonging to the Malacoherpesviridae family. DNA consisting of the nucleotide sequence represented by SEQ ID NOs: 1 is the promoter of oshv117 of Ostreid herpesvirus 1. DNA consisting of the nucleotide sequence represented by SEQ ID NOs: 2 is the promoter of oshv117 of Ostreid herpesvirus 1 variants (Ostreid herpesvirus 1 strain microvariant A and Ostreid herpesvirus 1 strain microvariant B). DNA consisting of the nucleotide sequence represented by SEQ ID NOs: 3 is the promoter of oshv117 of Chlamys acute necrobiotic virus (AVNV). Note that SEQ ID NOs: 2 is a sequence in which two nucleotides have been substituted relative to SEQ ID NOs: 1, and SEQ ID NOs: 3 is a sequence in which five nucleotides have been substituted and one nucleotide has been inserted relative to SEQ ID NOs: 1.

[0019] In (2) above, the number of one or more bases to be substituted, deleted, added, or inserted is not particularly limited as long as it is an integer of one or more. For example, it can be about 1 to several tens of, preferably about 1 to 30, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. This includes nucleic acid molecules containing a base sequence in which one or more bases are substituted, added, or deleted in the base sequence represented by any of SEQ ID NOs: 1 to 3, and in which the DNA has promoter activity.

[0020] In one embodiment, the number of one or more bases to be substituted, added, or deleted can be about 1 to 107 such that the base sequence identity with the base sequence represented by any of SEQ ID NOs. 1 to 3 is 90% or more, preferably about 1 to 54 such that the identity is 95% or more, more preferably about 1 to 21 such that the identity is 98% or more, and particularly preferably about 1 to 11 such that the identity is 99% or more.

[0021] In (3) above, the identity of the base sequence can be 90% or more, preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more. The homology or identity of the base sequence can be less than 100%. The homology and identity between base sequences can be determined using known algorithms such as BLAST. This includes nucleic acid molecules containing a base sequence that has 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 3, and in which the DNA has promoter activity.

[0022] In one embodiment, it is preferable that the base sequence has a TATA box sequence (TATATAA) and is identical to the base sequence represented by any of sequence numbers 1 to 3 by 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more). Furthermore, in the above, the TATA box sequence (TATATAA) may have one (or two or three) base sequences substituted, deleted, added, or inserted.

[0023] In one embodiment, it is preferable that the nucleotide sequence has the TACGTGGG sequence and is identical to the nucleotide sequence represented by any of SEQ ID NOs: 1 to 3 by 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more). Furthermore, in the above, the TACGTGGG sequence may have one (or two or three) nucleotide sequences substituted, deleted, added, or inserted.

[0024] In one embodiment, it is preferable that the nucleotide sequence has the GGATTGGC sequence and is identical to the nucleotide sequence represented by any of SEQ ID NOs: 1 to 3 by 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more). Furthermore, in the above, the GGATTGGC sequence may have one (or two or three) nucleotide sequences substituted, deleted, added, or inserted.

[0025] In one embodiment, it is preferable that the nucleotide sequence has the GAGGGAAGGT (SEQ ID NO: 36) sequence and is identical to any of the nucleotide sequences represented by SEQ ID NOs: 1 to 3 by 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more). Furthermore, in the above, the GAGGGAAGGT sequence may have one (or two or three) nucleotide sequences substituted, deleted, added, or inserted.

[0026] In one embodiment, it is preferable that the nucleotide sequence has the ACACCATTACATT (SEQ ID NO: 37) sequence and is identical to any of the nucleotide sequences represented by SEQ ID NOs: 1 to 3 by 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more). Furthermore, in the above, the ACACCATTACATT sequence may have one (or two or three) nucleotide sequences substituted, deleted, added, or inserted.

[0027] In one embodiment, it is preferable that the base sequence has the CAATT box consensus sequence (T / C)GATTGG(T / C)(T / C)(G / A) (Sequence ID 38) and has 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any of Sequence IDs 1 to 3. Furthermore, in the above, the (T / C)GATTGG(T / C)(T / C)(G / A) sequence may have one (or two or three) base sequences substituted, deleted, added, or inserted.

[0028] In one embodiment, it is preferable that the base sequence has the Kozak consensus sequence (A / G)NNATG(A / G) and is identical to any of the base sequences represented by Sequence ID No. 1 to 3 by 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more). Furthermore, in the above, the (A / G)NNATG(A / G) sequence may have one (or two or three) base sequences substituted, deleted, added, or inserted.

[0029] In (4) above, "stringent conditions" refer to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. One example of such stringent conditions is "hybridization in 1×SSC (0.9M NaCl, 0.09M trisodium citrate) or 6×SSPE (3M NaCl, 0.2M NaH2PO4, 20mM EDTA·2Na, pH 7.4) at 42°C, followed by washing with 0.5×SSC at 42°C," but this is not the only example. Such conditions are described, for example, in J. Sambrook et al., Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

[0030] The method for obtaining the promoter of the present invention is not particularly limited and can be obtained by conventional genetic engineering methods or chemical synthesis methods. For example, for DNA consisting of the base sequence represented by Sequence ID No. 1, the genomic DNA of Ostreid herpesvirus 1 can be extracted from shellfish such as Pacific oysters (Crassostrea gigas) infected with Ostreid herpesvirus 1, and DNA consisting of the base sequence represented by Sequence ID No. 1 can be cloned. For DNA consisting of the base sequence represented by Sequence ID No. 2, the genomic DNA of Ostreid herpesvirus 1 strain microvariant A can be extracted from shellfish such as Pacific oysters (Crassostrea gigas) infected with Ostreid herpesvirus 1 strain microvariant A, or the genomic DNA of Ostreid herpesvirus 1 strain microvariant B can be extracted from shellfish such as Pacific oysters (Crassostrea gigas) infected with Ostreid herpesvirus 1 strain microvariant B, and DNA consisting of the base sequence represented by Sequence ID No. 2 can be cloned. Regarding the DNA consisting of the base sequence represented by Sequence ID No. 3, the genomic DNA of Chlamys acute necrobiotic virus (AVNV) can be extracted from mollusks such as Chlamys farreri infected with the AVNV, and the DNA consisting of the base sequence represented by Sequence ID No. 3 can be cloned. Furthermore, the promoter DNA of the present invention can be artificially synthesized based on the base sequences shown in Sequence IDs No. 1 to 3.

[0031] The promoter of the present invention exhibits significantly higher promoter activity in bivalve cells compared to conventional promoters. For example, the promoter consisting of the nucleotide sequence represented by Sequence ID No. 1 shows approximately 25 times higher promoter activity compared to the conventional CMV IE (Cytomegalovirus immediate early) promoter when the luciferase gene is used as the reporter gene (Figure 9). Furthermore, when the GFP gene is used as the reporter gene for the promoter consisting of the nucleotide sequence represented by Sequence ID No. 1, GFP fluorescence can be observed using a fluorescence microscope. This promoter is particularly useful in bivalves.

[0032] In another embodiment, the present invention provides an expression vector containing the promoter described above.

[0033] The expression vector of the present invention can be constructed by incorporating the promoter of the present invention into an expression vector that enables the expression of a target gene. In the expression vector of the present invention, the target gene is incorporated under the control of the DNA of the present invention. Typically, in the expression vector of the present invention, the target gene is operably ligated downstream of the DNA of the present invention.

[0034] The expression vector into which the promoter of the present invention is incorporated is not particularly limited as long as it can be stably maintained and proliferated within host cells. For example, plasmid vectors, viral vectors, cosmid vectors, fosmid vectors, and artificial chromosome vectors can be appropriately selected depending on the host cell. Among these, plasmid vectors are preferred.

[0035] Commercially available cloning and expression vectors can be used as expression vectors, for example, pNL1.1 / 1.2 / 1.3, pNL2.1 / 2.2 / 2.3, pNL3.1 / 3.2 / 3.3, pGL4, pGEM T / T easy, pGEMEX-1 (all from Promega Corporation), T-Vector pMD20 / pMD19, pUC18 / 19, pUC118 / 119, pBR322, pMW218 / 219 (all from Takara Bio Inc.), pCR4-TOPO, pCR-XL-2-TOPO, pCR-BluntII-TOPO, pcDNA3.1 / V5-His-TOPO, pcDNA3.4-TOPO, pcDNA3.3-TOPO, pENTR / D-TOPO, pCR8 / GW / TOPO, pENTR / SD / D-TOPO, pcDNA3.1 Examples include V5-His A / B / C, pPICZ A / B / C (both from Thermo Fisher Scientific, Inc.), pDRIVE, pQE (both from QIAGE NV), pBluescript II SK(+ / -) (from Stratagene), pSC-A-amp / kan, pSC-A-amp / kan (both from Agilent Technologies, Inc.), pET, pGEX (from Merck KGaA), pACYC184 (from Nippon Gene Co., Ltd.), adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, and Sendai virus vectors.

[0036] The target gene to be expressed downstream of the promoter of the present invention in the expression vector described above is not particularly limited. For example, the target gene to be expressed is a gene that codes for a target substance or an enzyme involved in its synthesis. The target gene to be expressed can also be rephrased as a nucleic acid molecule (e.g., DNA, RNA) that codes for these target substances. The target gene to be expressed may be a heterologous gene that codes for a heterologous expression product, a gene that codes for an expression product that is naturally present in the host cell, or any other gene that codes for any protein, peptide, nucleic acid, etc. Examples of target substances coded by the target gene to be expressed include reporter fluorescent proteins such as GFP, growth factors, maturation factors, sterilization factors, disease resistance factors, Cas9 protein, enzymes, hormones, cytokines, toxins such as diphtheria, other physiologically active peptides, transporters, non-coding RNA, and shRNA (small hairpin RNA or short hairpin RNA) in RNA interference.

[0037] In another embodiment, the present invention provides a transformant comprising the above-mentioned expression vector.

[0038] The host cells into which the expression vector of the present invention is introduced are not particularly limited as long as the promoter of the present invention functions as a promoter. Examples include aquatic invertebrates (shellfish, squid, octopuses, etc.), mammals (humans, mice, rats, rabbits, dogs, monkeys, chimpanzees, horses, donkeys, hamsters, guinea pigs, etc.), fish (zebrafish, medaka, rainbow trout, salmon, sea bream, yellowtail, tuna, etc.), annelids (earthworms, polychaetes, leeches, etc.), flatworms (planarians, etc.), cnidarians (jellyfish, corals, sea anemones, etc.), echinoderms (sea urchins, starfish, sea cucumbers, etc.), arthropods (shrimp, crabs, insects, etc.), and tunicates (sea squirts, etc.). Among these, aquatic invertebrates are preferred, and aquatic mollusks are more preferred. Examples of aquatic mollusks include shellfish such as gastropods and bivalves, and cephalopods such as octopuses and squid. Among these, shellfish are preferred, and bivalves are particularly preferred. Examples of gastropods among shellfish include abalone and turban shells. Examples of bivalves include scallops, Pacific oysters, mussels, Manila clams, freshwater clams, cockles, ark clams, cockles, surf clams, razor clams, pen shells, and pearl oysters.

[0039] Transformed organisms can be obtained by introducing an expression vector containing the promoter of the present invention into host cells using a general transformation method, such as electroporation, lipofection, microinjection, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium. In this specification, "introduction into host cells" includes not only introduction into cells collected from living organisms or established cell lines, but also introduction into cell aggregates or tissues collected from living organisms; introduction into cell aggregates or tissues constructed using cells collected from living organisms; and introduction into living organisms (for example, non-human organisms).

[0040] The conditions for electroporation vary depending on the host cells and expression vector, and are not particularly limited, but for example, the poreing pulse voltage is 100 to 250 V, preferably 175 to 200 V. The impedance is also not particularly limited, but is 30 to 100 Ω, preferably 50 to 70 Ω. The osmotic pressure of the expression vector solution is also not particularly limited, but is 300 to 1500 mOsm / kg, preferably 800 to 1200 mOsm / kg. The amount of expression vector added is 1 × 10⁶ cells per host cell. 6 The recommended dosage per cell is 5 to 35 μg, preferably 10 to 30 μg.

[0041] In another embodiment, the present invention provides a method for producing a target substance encoded by an expression target gene, comprising the step of culturing the above-mentioned transformants.

[0042] The transformant (host cell) transformed with an expression vector in which the target gene is linked downstream of the promoter of the present invention is cultured in a suitable medium to express the target gene under the control of the promoter of the present invention and produce the target substance. The culture conditions for the host cell are not particularly limited as long as they allow for the proliferation of the host cell and the production of the target substance. After the culture is complete, the target substance can be obtained by recovering it from the culture. Examples of the target substance include the substance encoded by the target gene described above, and the products synthesized by the action of the substance.

[0043] By using the promoter of the present invention, genetic modification and genome editing can be efficiently performed in aquatic invertebrates such as bivalves, thereby accelerating the elucidation of gene functions in aquatic invertebrates such as bivalves. This is expected to lead to applications in ecosystem conservation, such as genetic sterilization technology. Furthermore, for bivalves and other organisms used for food, this technology is expected to be applied to the creation of superior varieties by modifying their taste and nutritional composition. In addition, it is expected to be applied to imparting water purification functions and producing functional proteins. [Examples]

[0044] The present invention will be described in more detail using manufacturing examples and embodiments, but the present invention is not limited to these embodiments.

[0045] <Culture of scallop cells> Scallops (Mizuhopecten yessoensis) aged 1-2 years were obtained from aquaculture farms in Onagawa Bay (Miyagi Prefecture), Mutsu Bay (Aomori Prefecture), or Funka Bay (Hokkaido). (1) Culture of cardiomyocytes The ventricles were extracted from scallops and washed with artificial seawater (Instant Ocean, Aquarium Systems) filtered through a sterile filter with a pore size of 0.22 μm. After washing, the obtained cardiac tissue was finely cut with ethanol-disinfected forceps and scissors, and then rinsed with DBSS (Table 1) using a shaker at room temperature for 30 minutes at 50 rpm. After removing the DBSS, the cardiac tissue fragments were enzymatically digested in trypsin-EDTA solution (Table 1) at 4°C for 18-21 hours. Enzymatic digestion was then stopped by adding the same volume of 10% fetal bovine serum (FBS) supplemented growth medium (Table 1), and fine cells and debris were removed. The cardiac tissue fragments after enzymatic digestion were explanted into multi-well plates (Sumitomo Bakelite Co., Ltd.) or T-flasks (Nunc, Thermo Fisher Scientific, Inc.) and cultured at 20°C. At the start of cell culture, cardiac tissue fragments were first attached to the substrate with a small amount of growth medium (Table 1), and the following day, an appropriate amount of medium was added (75 cm³). 2 In the case of a T flask, 6 ml was added at seeding and 9 ml the following day. During this time, adherent primary cultured cells were obtained by allowing cells released from the explanted cardiac tissue fragments to adhere to and spread on the bottom surface of the T flask. The growth medium was replaced with fresh medium every 10 days or when the pH of the medium decreased. When the adherent primary cultured cells in the T flask reached confluence, the cells were detached from the T flask using a cell scraper, and growth medium was added to a new T flask for subculturing. Primary cardiomyocytes derived from these ventricular cells survived for 3-4 weeks at 20°C and then tended to gradually degenerate, although some cells survived for more than 3 months. Additional supply of CO2 and humidity was not necessary.

[0046] [Table 1]

[0047] All of the solutions listed in Table 1 were adjusted to pH 7.4 using hydrochloric acid (HCl) or sodium hydroxide aqueous solution (NaOH) before being used in the experiment.

[0048] (2) Culture of blood cells Scallop hemolymph was directly extracted from the ventricle using a 22-gauge needle and immediately mixed 1:1 with prepared Alcever's solution (MAS (Table 1)). Without centrifugation, blood cells in the hemolymph-MAS mixture were separated into 2 × 10⁶ cells. 5 The cells were seeded in growth medium at a concentration of cells / ml, and the final medium composition was prepared to be approximately 2% FBS and 10% scallop hemolymph-MAS mixture. To prevent cell aggregation, the collected hematopoietic cells were placed on ice before seeding. Primary hematopoietic cells survived for two weeks at 20°C, and degeneration accelerated with medium changes. Additional CO2 and humidity supply were not required.

[0049] (3) Optimization of culture conditions for scallop cardiomyocytes We investigated the culture conditions for scallop cardiomyocytes. Using cardiac tissue samples enzymatically digested in trypsin-EDTA solution (Table 1) at 4°C for 18 hours, we cultured them under the following conditions (i) to (iv) regarding seeding density, culture temperature, osmotic pressure, and presence or absence of FBS. The number of adherent cells on day 2 was counted from five randomly taken microscope images at 100x magnification (N=20). The results are shown in Figure 1. Further investigation of FBS concentration revealed that 2% FBS was sufficient. Based on these results, the culture conditions were determined to be suitable for a seeding density of 4 × 10⁶. 5 The following experiments were conducted under optimal conditions: cells / ml, culture temperature 10°C, osmotic pressure 1100 mOsmol / kg, and FBS 2%.

[0050] (i) Seeding density 2×10 5 cells / ml or 4 × 10 5 cells / ml (ii) Culture temperature 10°C or 20°C (iii) Osmotic pressure of 900 mOsmol / kg or 1100 mOsmol / kg (iv) FBS 0% or 10%

[0051] <Culture of human HEK293 cells> Human HEK293 cells (JCRB9068) were provided by the JCRB Cell Bank. They were cultured in Dulbecco's modified Eagle medium (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% FBS in a humidified incubator at 37°C with 5% CO2 supply.

[0052] <Obtaining Zebrafish Embryos> The zebrafish were purchased as adults from either Meito Suien Co., Ltd. (Aichi Prefecture) or MASUKO Co., Ltd. (Saitama Prefecture). The purchased adults were placed in 60cm tanks and fed with commercial feed. Fertilized eggs were obtained by mating mature male and female zebrafish.

[0053] <Example 1: Investigation of electroporation conditions> Primary cardiomyocytes from scallops cultured for 1-2 weeks were used for electroporation. The growth medium was removed, and the cardiomyocytes cultured in FBS-free growth medium were washed and collected using a cell scraper (Sumitomo Bakelite Co., Ltd.). The collected cardiomyocyte cell suspension was filtered through a sterile 125 μm pore diameter steel mesh (Yikai Metal Products Co., Ltd.) to remove any cardiac tissue fragments used during explantation. The resulting cultured cell suspension was centrifuged at 400 × g for 5 minutes at 20°C, and the precipitated cultured cells were collected. Cell count and viability were measured using a hemocytometer and trypan blue exclusion method.

[0054] Electroporation was performed using a NEPA21 electroporator and a 2mm gap electroporation cuvette (manufactured by Neppageen Co., Ltd.). 100 μl of Opti-MEM (Gibco, Thermo Fisher Scientific, Inc.) containing 10 μg of plasmid DNA or 10 μg of mRNA was used to sample 1 × 10⁶ cardiomyocytes cultured for 1-2 weeks. 6 The cells were resuspended. Since the osmotic pressure of Opti-MEM is 300 mOsmol / kg unadjusted, it was adjusted to 1050 mOsmol / kg by adding 256.7 mg / ml of sucrose. The impedance of the cell suspension was 50-60 Ω. The electroporation parameters were set as follows. For poring pulses, the pulse voltage is variable, the pulse length is 2.5 ms, the pulse interval is 50 ms, the number of pulses is 2, the attenuation rate is 10%, and the polarity is switched to +. For transfer pulses, the pulse voltage is 20V, pulse length is 50ms, pulse interval is 50ms, number of pulses is 5, attenuation rate is 40%, and polarity is switched + / -.

[0055] (1) Examination of electroporation conditions in the absence of mRNA To find the optimal conditions for electroporation in scallop cardiomyocytes, poring pulse voltages of 75–200V were investigated at osmotic pressures of 300 mOsmol / kg and 1050 mOsmol / kg (without mRNA). Cells that were not electroporated, or cells to which distilled water was added instead of DNA or mRNA, were used as controls. The results are shown in Figure 3. At an osmotic pressure of 300 mOsmol / kg, cell viability was low even under conditions with low voltage, whereas at an osmotic pressure of 1050 mOsmol / kg, cell viability improved by an average of 36.4%.

[0056] (2) Examination of electroporation conditions in the presence of luciferase mRNA Cells treated with luciferase mRNA via electroporation under conditions of osmotic pressure of 1050 mOsmol / kg and poreing pulse voltage of 75–200 V were seeded into 24-well plates in 500 μl of growth medium, and studies were conducted in the presence of mRNA. Cell viability was determined immediately after electroporation by trypan blue exclusion. Luciferase assays were performed on mRNA-treated cells 3 days after electroporation. The results are shown in Figures 4–6.

[0057] <Example 2: Preparation of a luciferase vector> (1) Selection of promoter To identify promoters with strong activity, we selected and examined a total of 11 promoters, including the Ostreid herpesvirus-1 (OsHV-1) promoter (a bivalve-infecting virus), the endogenous promoter of the scallop *M. yessoensis*, and the leukoplakia syndrome virus (WSSV) promoter (a shrimp-infecting virus). The selected promoters are listed below. As controls, we also examined the CMV IE (Cytomegalovirus immediate early) promoter, which has been conventionally used in bivalves, and the control without a promoter. • Promoters considered Ostreid herpesvirus-1 (OsHV-1) promoter Poshv027, Poshv029, Poshv072, Poshv080, Poshv088, Poshv117 Endogenous promoter of the scallop *M. yessoensis* Pmy-actβ, Pmy-ef1α, Pmy-ef1β Vitiligo syndrome virus (WSSV) promoter Pwsv-ie1, Pwsv465

[0058] (2) Preparation of luciferase vector A vector was prepared using the promoter described above (Figure 7). The OsHV-1 promoter was extracted from the larvae of OsHV-1-infected Pacific oysters (Crassostrea gigas). The 5' upstream regions of the start codons -1201, -1162, -1017, -1038, -1101, and -1069 of six genes selected from OsHV-1 (Poshv027, Poshv029, Poshv072, Poshv080, Poshv088, and Poshv117, respectively) were cloned into a PCR-linearized pNL1.1 vector.

[0059] For the endogenous promoter of the scallop *M. yessoensis*, genomic DNA from scallops in Mutsu Bay was extracted from the adductor muscle using the DNeasy Blood & Tissue Kit (QIAGE NV). The 5'-upstream regions of *M. yessoensis* actβ, ef1α, and the start codons -4706, -3007, and -2221 of ef1β were cloned into a SpeedCut HindIII (Rikaken Holdings Co., Ltd.) linearized pNL1.1 vector.

[0060] For the WSSV promoter, the DNA sequences of the wsv-ie1 promoter and wsv465 promoter were synthesized based on the NCBI database (commissioned to Eurofins Genomics), and the 5'-upstream regions of the start codons -502 and -472 were cloned into the PCR-linearized pNL1.1 vector.

[0061] For the control, the CMV-IE promoter, I purchased the pNL1.1.CMV[Nluc / CMV] Vector (manufactured by Promega Corporation).

[0062] For vector amplification and extraction, the bacterial strain DH-5α (manufactured by Nippon Gene Co., Ltd.) and the Plasmid DNA Extraction Mini / MIDI Kit (manufactured by Favorgen Biotech Corp.) were used. For PCR clean-up and gel extraction, the NuceloSpin Gel and PCR Clean-up Kit (manufactured by Takara Bio Inc.) and the QIAquick Gel Extraction Kit (manufactured by Qiagen N.V.) were used. All molecular cloning was performed using In-Fusion Snap Assembly (manufactured by Takara Bio Inc.).

[0063] The primers used for vector preparation are shown in Table 2.

[0064]

Table 2

[0065] <Example 3: Normalization of Luminescence> To normalize the luminescence of cells transfected with the luciferase gene, the number of adherent cells was counted, and a calculation process was performed to divide the luminescence value of luciferase by the number of adherent cells. For counting, the autofluorescence of scallop cardiomyocytes was utilized. Using the Pmy-ef1α luciferase vector, the Poshv088 luciferase vector, or the Poshv117 luciferase vector, 1×10 6 cells of primary scallop cardiomyocytes were subjected to electroporation at a poring pulse voltage of 100 V (other conditions were the same as above). Subsequently, 5×10 4 cells, 1×10 5 cells, and 2×10 5 cells were seeded in a 24-well plate and cultured in 500 μl of growth medium for 2 days. Then, each well was washed twice with growth medium and observed under a GFP filter at a magnification of 40 times with long exposure (0.67 seconds in the case of Keyence BZ-X800, manufactured by Keyence Corporation). The area of autofluorescence released from adherent cells was 0.77 cm 2Cell counting was performed using supervised machine learning with the ilastik(1.3.3) pixel classification and object classification pipelines, with each well photographed. The results are shown in Figure 8.

[0066] In Poshv088 and Poshv117, R 2 The values ​​were 0.95 and 0.54, indicating a positive correlation between the number of adherent cells and the expression level of luciferase. For Pmy-ef1α, R 2 The value was 0.3, but the luciferase expression level (relative luminescence unit (RLU) value) was normalized by the number of cells.

[0067] <Example 4: Promoter Activity Evaluation - Luciferase Assay> Primary scallop cardiomyocytes 1×10 6 Cells were electroporated with 10 μg of the luciferase vector prepared above using a poreing pulse voltage of 100 V (other conditions were the same as above). After culturing for 2 days, the growth medium was removed, and the cells were washed twice with growth medium without FBS to leave only adherent cells. Then, 100 μl of 1% PBS-TX was added to each well and incubated for 10 minutes to lyse the cells. The collected cell lysates were then centrifuged at 12000 × g for 5 minutes to remove undissolved material. Subsequently, a luciferase assay was performed using the Nano-Glo luciferase assay system (Promega Corporation). Luminescence was measured using a Glomax plate reader (Promega Corporation) and normalized by the number of adherent cells in each well. The results are shown in Figure 9.

[0068] Poshv117 showed the highest expression level, followed by Poshv088. While the CMV IE promoter and Pmy-ef1α showed some activity, it was significantly lower than that of Poshv117 and Poshv088. The expression level of Poshv117 was 16.1 times that of Pmy-ef1α and 24.7 times that of the CMV IE promoter (a promoter conventionally used in bivalves).

[0069] <Example 5: Further optimization of electroporation conditions> Further optimization of electroporation conditions for scallop cardiomyocytes was performed using Poshv117. Poring pulse voltage (100V, 125V, 150V, 175V, 200V), impedance (60Ω, 54Ω, 49Ω, 42Ω, 38Ω), plasmid vector concentration (1μg, 2μg, 5μg, 10μg, 20μg), and culture period after electroporation (1 day, 2 days, 3 days, 4 days, 5 days, 6 days) were investigated. Each condition was examined using a poring pulse voltage of 200V, impedance of 60Ω, plasmid vector concentration of 10μg, and culture period of 2 days after electroporation as a baseline. For impedances of 60-38Ω, the investigation was performed by adding +20μl, +40μl, +60μl, +80μl, and +100μl of Opti-MEM adjusted to an osmotic pressure of 1050mOsmol / kg. The results are shown in Figure 10.

[0070] <Example 6: Promoter Activity Evaluation GFP Assay> (1) Preparation of EGFP vector The EGFP vector was prepared by replacing the luciferase translation region of the above luciferase vector with the EGFP translation region (EGFP CDS) (Figure 11). The vector was amplified and extracted using the same method as described above.

[0071] Table 3 shows the primers used to prepare the vector.

[0072] [Table 3]

[0073] (2) GFP assay in scallop cardiomyocytes Primary scallop cardiomyocytes 1×10 6 Cells were electroporated with 10 μg of EGFP vector at a poring pulse voltage of 150 V (other conditions were the same as above). Poshv117-EGFP vector, Poshv088-EGFP vector, Pmy-ef1α-EGFP vector, and a CMV IE promoter-mediated pCS2-GFP vector were used as EGFP vectors. Two days after electroporation, the cells were observed using a fluorescence microscope (BZ-X800, Keyence Corporation). The results are shown in Figure 12. Fluorescence was observed only under the conditions using the Poshv117-EGFP vector.

[0074] Furthermore, to confirm that the fluorescence expressed from electroporated scallop cells was a GFP signal, scallop cardiomyocytes were stained with an anti-GFP antibody using immunofluorescence. (Scallop primary cardiomyocytes 1×10⁶) 6Cells were electroporated with 10 μg of Poshv117-EGFP vector using a poreing pulse voltage of 150 V (other conditions were the same as above). Two days later, the cells were fixed with 4% formaldehyde at room temperature for 15 minutes. Subsequently, they were permeabilized with 0.1% PBS-TX for 20 minutes and blocked with 3% BSA at room temperature for 30 minutes. Then, the cells were reacted overnight at 4°C under a 200-fold dilution of anti-GFP antibody (rabbit IgG, Invitrogen, Thermo Fisher Scientific, Inc.) (10 μg / ml) in 0.1% PBS-TX. The following day, the cells were reacted for 1 hour at room temperature under a 200-fold dilution of secondary antibody (AlexaFlour Plus 594 donkey anti-rabbit IgG, Invitrogen, Thermo Fisher Scientific, Inc.) (10 μg / ml) in 0.1% PBS-TX. The cells were then observed using a fluorescence microscope (BZ-X800, Keyence Corporation). The results are shown in Figure 12. Under a fluorescence microscope, signals for GFP and red fluorescent protein (RFP) were observed in the same scallop cardiomyocytes. This result clearly indicates that the fluorescence expressed from electroporated scallop cells is the GFP signal.

[0075] (3) GFP assay in scallop hematopoietic cells In scallop hematopoiesis, cell mortality was high after electroporation, so EGFP vectors were introduced by lipofection. Hematopoiesis were cultured for 1 day in 500 μl of growth medium containing 500 ng of EGFP vector using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Inc.). Poshv117-EGFP vector, Poshv088-EGFP vector, Pmy-ef1α-EGFP vector, and the CMV IE promoter pCS2-GFP vector were used as EGFP vectors. After removing the vector-containing medium, the cells were observed using a fluorescence microscope (BZ-X800, Keyence Corporation). The results are shown in Figure 13. GFP fluorescence was observed in scallop hematopoiesis only under the conditions using the Poshv117-EGFP vector.

[0076] (4) GFP assay in HEK293 cells HEK293 cells 3 x 10 5 Cells were suspended in 100 μl of Opti-MEM (300 mOsmol / kg) and electroporated with 10 μg of EGFP vector at a poring pulse voltage of 155 V. Poshv117-EGFP vector, Poshv088-EGFP vector, and a CMV IE promoter-mediated pCS2-GFP vector were used as EGFP vectors. Two days after electroporation, cells were observed using a fluorescence microscope (BZ-X800, Keyence Corporation). Immunofluorescence staining with anti-GFP antibody was also performed, similar to the scallop cardiomyocytes. The results are shown in Figure 14. GFP fluorescence was observed in HEK293 cells only under the conditions using the Poshv117-EGFP vector.

[0077] (5) GFP assay in zebrafish embryos EGFP vectors, prepared at 100 ng / μl, were microinjected into the blastodisk (cell body) of zebrafish at the one-cell or two-cell stage. Poshv117-EGFP vector, Poshv088-EGFP vector, and the CMV IE promoter-promoted pCS2-GFP vector were used as EGFP vectors. The embryos were then cultured at 28°C and observed 24 hours after microinjection using a fluorescence microscope (BZ-X800, Keyence Corporation). The results are shown in Figure 15. GFP fluorescence was observed in zebrafish embryos only under the conditions using the Poshv117-EGFP vector.

[0078] <Example 7: Sequence Analysis> The DNA sequences of the Poshv117 and oshv117 translation regions were analyzed using NNPP, NSite, CDD programs, and manual methods.

[0079] Figure 16 shows the arrangement of oshv117 and its adjacent genes. The OsHV-1 gene contains a 4600 bp overlapping region consisting of oshv116, oshv117, oshv118, and oshv119. This region has 100% identical DNA sequences and is complementary (i.e., one is located in the 5'-3' direction and the other in the 3'-5' direction).

[0080] Poshv117 consists of the 5'-UTR (233 bp) of oshv117, the entire translational region of oshv118 (669 bp), and a partial 5'-UTR (167 bp) of oshv118.

[0081] Figure 17 shows the sequence analysis of Poshv117 by NNPP and NSite (SEQ ID NO: 1). NNPP (a neural network-based promoter prediction program) predicted that the transcription start sites were located at -58 and -30 positions upstream from the start codons of oshv117 and oshv118. Additionally, a TATA box (TATATAA) was detected at -30 upstream from the transcription start site of oshv117.

[0082] Furthermore, NSite (a consensus-based search tool for estimated functional motifs) predicted the following combined motifs (the lowercase parts below indicate differences from the consensus sequence). • TACGTGGG is a conserved sequence that binds to hypoxia-inducible factor 1 (HIF-1) in mammals (Homo sapiens, Mus musculus, Rattus norvegicus). • GGATTGGC, the putative binding site for mammalian dihydrofolate reductase (DHFR) (M. musculus); • GAGGGAagGT, the putative binding site for mammalian mammary cell activator (MAF) (H. sapiens, M. musculus); • ACACCatTACATT, near the TATA box of oshv117, is an RPG box-like sequence presumed to bind to Ras-associated protein 1 (RAP1) of Saccharomyces cerevisiae.

[0083] Furthermore, the CAATT box consensus sequence (T / C)GATTGG(T / C)(T / C)(G / A) was found at position -91, 5' upstream of the oshv117 transcription start site. The Kossack consensus sequence (A / G)NNATG(A / G) was conserved around the start codon of oshv117. In addition, consecutive palindromic sequences and discrete palindromic sequences (TTCCCTGGT and GCCAGGGAA, AATGCGT and ACGCATT; AACATGTT, AAATATTT, TCCATATGGA, TGATATCA) and three direct repeat sequences (CAACAACAA, CTGTATCTGTAT, AACAACAACAAC) were found.

[0084] <Example 8: Promoter Deletion Analysis> To identify the core promoter region that controls Poshv117 expression, we performed promoter deletion analysis. The mutant promoters used are as follows. The labels No. 1 to No. 5 in Figure 17 correspond to the deletion sites in the promoter region. • Promoter No. 1: 1~1069. Full-length region (approximately 1kbp upstream of ORF117, Poshv117). (Sequence ID 1) • Promoter No. 2: 1-902. Missing upstream region of ORF118. (Sequence ID 32) • Promoter No. 3: 1-587. Approximately half of the translation region of ORF118 (including the HIF binding motif) is missing. (Sequence ID 33) • Promoter No. 4: 1-233. The entire translation region of ORF118 (including the HIF binding motif) is missing, leaving only the upstream region of ORF117. (Sequence ID 34) • Promoter No. 5: 1-151. Only the region containing the CCAAT box within the upstream region of ORF117. (Sequence ID 35) • Promoter No. 6: No promoter insertion sequence.

[0085] The above promoter was cloned into the pNL1.1 vector to prepare the vector, and then a luciferase assay was performed using the same method as in Example 4. The results are shown in Figure 18.

[0086] The full-length sequence showed the highest promoter activity, and as the length was shortened from the upstream end, the promoter activity decreased in proportion to the length.

Claims

1. A promoter consisting of any of the following DNAs (1) to (4): (1) DNA containing a base sequence represented by any of sequence numbers 1 to 3, (2) DNA having promoter activity, which includes a base sequence in which one or more bases are substituted, added, or deleted in any of the base sequences represented by Sequence ID No. 1 to 3. (3) DNA having promoter activity and containing a base sequence that has 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 3 (4) DNA having promoter activity, which contains a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by any of sequence numbers 1 to 3.

2. An expression vector comprising the promoter described in claim 1.

3. The expression vector according to claim 2, wherein the target gene is incorporated under the control of the promoter according to claim 1.

4. A transformer comprising the expression vector according to claim 2 or 3.

5. The transformant according to claim 4, which is an aquatic invertebrate.

6. A method for producing a target substance encoded by an expression target gene, comprising the step of culturing a transformant containing the expression vector described in claim 3.

7. A method for producing a transformant, comprising introducing the expression vector described in claim 2 or 3 into a host cell.

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