Target gene translation control system

Abstract

PROBLEM TO BE SOLVED: The purpose of the present invention is to provide a novel target gene translation guidance system. SOLUTION: Provided as a translation control system for a target gene in an animal cell is a system that contains a nucleic acid including a translation control region located upstream of the target gene and a predetermined RNA expression vector or a virus that expresses the predetermined RNA in the animal cell. The translation control region has a sequence complementary to the predetermined RNA in the sense strand of the target gene and contains a nucleic acid that forms at least one stem structure.

Description

Translational regulatory systems of target genes

[0001] This invention relates to a translation control system for target genes.

[0002] The gene translation control system is a system that artificially controls the translation of any target gene present within a cell through extracellular stimuli. Existing gene translation regulatory systems include the placement of polynucleotide cassettes (J Nucleic Acids. 2012:2012:748913. doi: 10.1155 / 2012 / 748913. Epub 2012 Oct 24.; Front Chem. 2016 Jun 15:4:25. doi: 10.3389 / fchem.2016.00025. eCollection 2016.) or promoter sequences (Proc Natl Acad Sci US A. 1992 Jun 15;89(12):5547-51. doi: 10.1073 / pnas.89.12.5547.; Science. 1995 Jun 23;268(5218):1766-9. doi:) upstream of any target gene within a cell. 10.1126 / science.7792603.; Nat Biotechnol. 2000 Nov;18(11):1203-8. doi: 10.1038 / 81208.; Methods Mol Biol. 2004;267:451-66. doi: 10.1385 / 1-59259-774-2:451.; Trends Biotechnol. 2003 Feb;21(2):53-5. doi: 10.1016 / s0167-7799(02)00022-7.; Nat Methods. 2009 Jan;6(1):79-81. doi: 10.1038 / nmeth.1278. Epub 2008 Dec 14.; Proc Natl Acad Sci US A. A system is known that incorporates (1994 Aug 16;91(17):8180-4. doi: 10.1073 / pnas.91.17.8180.; Nat Med. 1996 Sep;2(9):1028-32. doi: 10.1038 / nm0996-1028.) to control the translation of target genes.

[0003] An object of the present invention is to provide a novel translational control system for a target gene.

[0004] One embodiment of the present invention is a translational control system for a target gene in an animal cell, including a nucleic acid containing a translational control region disposed upstream of the target gene, an expression vector of a predetermined DNA, or a virus that expresses part or all of the NP gene in the animal cell, wherein the translational control region includes a nucleic acid having a sequence complementary to the predetermined DNA in the sense strand of the target gene and forming at least one stem structure.

[0005] Another embodiment of the present invention is a method for controlling the translation of a target gene in an animal cell, including: (a) introducing an expression vector having the target gene downstream of a translational control region into the animal cell, or inserting a translational control region upstream of the target gene on genomic DNA in the animal cell; and (b) introducing an expression vector of a predetermined DNA into the animal cell, or infecting the animal cell with a virus that expresses a predetermined DNA in the animal cell, wherein the translational control region includes a nucleic acid having a sequence complementary to the predetermined DNA in the sense strand of the target gene and forming at least one stem structure.

[0006] Another embodiment of the present invention is an assay method for investigating the translation-inducing ability of a translation-regulating region located upstream of a target gene in an animal cell, comprising: (a) introducing an expression vector having the target gene downstream of the translation-regulating region into the animal cell, or inserting the translation-regulating region upstream of the target gene on the genomic DNA in the animal cell; (b) introducing an expression vector of a predetermined DNA into the animal cell, or infecting the animal cell with a virus that expresses the predetermined DNA in the animal cell; and (c) examining the difference in the expression level of the protein encoded by the target gene depending on whether or not step (b) is performed, wherein the translation-regulating region includes a nucleic acid having a sequence complementary to the predetermined DNA on the sense strand of the target gene and forming at least one stem structure. Another embodiment of the present invention is a screening method for nucleic acids having translation-inducing ability to a translation-regulating candidate region located upstream of a target gene in an animal cell, comprising: (p) examining the translation-inducing ability of a plurality of translation-regulating candidate regions and / or predetermined DNA using an assay method for examining the translation-inducing ability to the translation-regulating region located upstream of the target gene in the animal cell; and (q) identifying a translation-regulating candidate region and / or predetermined DNA in which the expression level of the protein encoded by the target gene is enhanced by step (p).

[0007] A further embodiment of the present invention is a system for controlling the translation of a target gene in animal cells, comprising a nucleic acid including a translational control region located upstream of the target gene, and an expression vector of a predetermined DNA or a virus that expresses part or all of the NP gene in the animal cell, wherein the translational control region includes a nucleic acid having a sequence complementary to the predetermined DNA on the sense strand of the target gene and forming at least one stem structure. The system may be in the form of a kit.

[0008] The predetermined DNA may be part or all of a virus-derived gene. The virus-derived gene may be the NP gene of influenza B virus or the N gene of SARS-CoV-2. The translational control region may be a nucleic acid containing the base sequence of SEQ ID NO: 5 (5'-TTCACCTCTCTTTTTTAAAAAAGGGT-3') or a nucleic acid containing the base sequence of SEQ ID NO: 6 (5'-TGCAATCCCCCGGATTTTTCTCCAATCAACTTTGAATCAACTAGCCCTTTTTGGGGGC-3').

[0009] As one embodiment of the present invention, the mechanisms of translation inhibition and translation induction of a gene that controls translation (hereinafter referred to as a target gene) are shown. In an animal cell containing a target gene and a translation control region located upstream of it, (A) when the mRNA region corresponding to the translation control region (hereinafter referred to as the translation control region on mRNA) forms a stem structure, even if a ribosome binds to the mRNA, the movement of the ribosome is inhibited, and the mRNA of the target gene is not translated. (B) When a second mRNA exists having an mRNA region corresponding to a translation induction sequence (hereinafter referred to as the translation induction sequence on the second mRNA), which is a nucleotide sequence complementary to the nucleotide sequence of the translation control region, the nucleotide sequence of the translation control region on mRNA and the nucleotide sequence of the translation induction sequence on the second mRNA bind, destabilizing the stem structure, allowing the ribosome to move along the mRNA and translate the target gene. As an example of translational regulatory regions on mRNA, the secondary structures predicted by the RNAfold web server (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) for the nucleotide sequences of (C) SEQ ID NO: 1, (D) SEQ ID NO: 2, (E) SEQ ID NO: 3, (F) SEQ ID NO: 16, and (G) SEQ ID NO: 17 described in the Examples are shown. As one embodiment of the present invention, fluorescence images are shown of 293T cells (upper panel) into which a control EGFP gene expression vector or an EGFP gene expression vector containing the translational regulatory regions of SEQ ID NOs: 4-6 has been introduced, and of 293T cells (lower panel) into which an EGFP gene expression vector containing the translational regulatory regions of SEQ ID NOs: 5-6 and an NP gene expression vector derived from influenza B virus (B / Iwate / 34 / 2017 strain) have been introduced, after being cultured at 37°C. As a comparative example of the present invention, fluorescence and bright-field images are shown of 293T cells cultured at 37°C, in which 293T cells were introduced with either a control EGFP gene expression vector or an EGFP gene expression vector containing the translational regulatory regions of SEQ ID NOs. 5-6, respectively, and in which 293T cells were introduced with a mixture of an EGFP gene expression vector containing the translational regulatory regions of SEQ ID NOs. 5-6 and an HA gene expression vector derived from influenza B virus (B / Iwate / 34 / 2017 strain), respectively.The upper left shows cells into which a control EGFP gene expression vector has been introduced, the upper right shows cells into which an EGFP gene expression vector containing the translational regulatory region of SEQ ID NO: 5 has been introduced, and the lower panel shows cells into which an EGFP gene expression vector containing the translational regulatory region of SEQ ID NO: 6 has been introduced. Cells without the HA gene are indicated by a minus sign (-), and cells with the HA gene have been introduced are indicated by a plus sign (+). As a comparative example of the present invention, fluorescence and bright-field images are shown after introducing either a control EGFP gene expression vector or an EGFP gene expression vector containing the translational regulatory regions of SEQ ID NOs: 18 and 19 (indicated as SEQ ID NOs: 18 and 19 in the figure) into 293T cells and culturing them at 37°C. As an embodiment of the present invention, fluorescence images are shown after culturing 293T cells (upper panel) into which a control EGFP gene expression vector or an EGFP gene expression vector containing the translational regulatory regions of SEQ ID NOs: 5-6 has been introduced into 293T cells (lower panel) and then infecting them with influenza B virus (B / Iwate / 34 / 2017 strain). As one embodiment of the present invention, the stem sequences of SEQ ID NO: 4 (stem sequence is 7 nucleotides) and SEQ ID NO: 5 (stem sequence is 8 nucleotides), which have a stem-loop structure, are subjected to a single nucleotide substitution in the stem sequence, resulting in translational regulatory regions (A) having the stem sequences of SEQ ID NO: 22 (stem sequence is 8 nucleotides) and SEQ ID NO: 23 (stem sequence is 7 nucleotides), respectively. The fluorescence images (B) shown are obtained after introducing an EGFP gene expression vector containing these translational regulatory regions into COS1 cells and culturing them at 37°C. As an example of translational regulatory regions, the secondary structures predicted by the RNAfold web server (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) are shown for the stem sequences of SEQ ID NO: 26 and SEQ ID NO: 27 described in the example. This figure shows the results of measuring luciferase activity after introducing a plasmid containing a luciferase gene having sequence number 28 or 29 upstream into 293T cells and culturing them at 37°C, as one embodiment of the present invention. This figure shows the results of measuring luciferase activity after introducing a plasmid containing a luciferase gene having sequence number 28 or 29 upstream and a plasmid expressing the N gene into 293T cells and culturing them at 37°C, as one embodiment of the present invention.

[0010] The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and those skilled in the art will be able to easily reproduce the present invention from the description herein. The embodiments and specific examples of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only, and do not limit the present invention thereto. It will be apparent to those skilled in the art that various modifications and alterations can be made based on the description herein, within the intent and scope of the present invention as disclosed herein. When embodiments and examples are not specifically described, the methods described in standard protocol sets, or modified or altered methods thereof, should be used. Furthermore, when using commercially available reagent kits or measuring devices, the protocols provided with them should be used unless otherwise specified.

[0011] ==Target Gene Translation Control System== The target gene translation control system in animal cells disclosed herein includes a translation inhibition system and a translation induction system. The translation inhibition system includes a nucleic acid containing a translation control region. This translation control region is not particularly limited, as long as it contains a nucleic acid that forms at least one stem structure. By using such a system, it becomes possible to inhibit the translation of a desired target gene in animal cells.

[0012] The genes that can be applied to the translation inhibition system are not particularly limited and may be endogenous or exogenous genes. For example, they may be suicide genes such as caspase-9 or Bax, which induce cell death. Alternatively, they may be fluorescent proteins such as EGFP or mCherry, or luminescent proteins such as luciferase, which can be used as research reagents, or drug resistance genes such as neomycin resistance genes or hydroxylocyte resistance genes.

[0013] Nucleic acids containing a translational regulatory region used in a translation inhibition system may be present on an expression vector. Expression vectors containing a translational regulatory region can be manufactured using vectors that can be introduced into animal cells to express genes. These vectors may be plasmids such as pcDNA3, pEF, pGL3, pCMV-Script, pCAGGS, pEGFP, or pIRES, or viral vectors such as adeno-associated (AAV) virus vectors, retroviral vectors, or lentiviral vectors. In this case, translation inhibition of the target gene can be achieved by creating an expression vector in which an exogenous gene, as the target gene for translation inhibition, is inserted downstream of the translational regulatory region. An expression vector having a translational regulatory region into which the target gene is inserted may, for example, exist in cells in the form of a plasmid. Alternatively, nucleic acids containing a translational regulatory region can also be inserted upstream of the target gene on the genomic DNA of animal cells by homologous recombination or genome editing. In this way, translation inhibition of endogenous genes can be achieved.

[0014] The stem structure formed by the translational regulatory region is a secondary structure that arises when a single nucleic acid has complementary sequences in opposite directions, and these sequences form a double helix within the molecule. If the complementary sequence contains one or more non-complementary bases, a gap (when there is only one non-complementary base) or a loop (when there are multiple consecutive non-complementary bases) occurs in the non-complementary sequence. The translational regulatory region may form a stem structure with such gaps or loops, i.e., a stem-loop structure. If the formed stem structure is 7 bases or less in length, the formed stem structure becomes unstable and the mRNA translation inhibitory effect is insufficient; therefore, it is preferable that the stem structure is 8 bases or longer. Even if the stem structure is discontinuous due to the presence of one or two gaps or loops, it is sufficient if there are 8 or more paired bases; however, it is preferable that there is a portion of the stem structure that is 8 or more consecutive bases. The total number of paired bases in the stem structure may be 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more.

[0015] When a target gene is transcribed, a cap structure is added to the 5' end of the transcript. Since ribosomes bind to the cap structure and begin to move along the mRNA, it is preferable that the translational regulatory region be located downstream of the transcription start site in order to inhibit ribosome movement through the stem structure of the transcribed mRNA region. Furthermore, it is preferable that the target gene be located downstream of the translational regulatory region.

[0016] In contrast to such translation inhibition systems, translation induction systems include a translation induction vector whose sense strand contains a translation induction sequence that is complementary to the base sequence of the sense strand of the translation control region. Due to the complementarity between the translation control region and the translation induction sequence, the translation control region on mRNA produced by the transcription of a predetermined DNA containing the translation control region and the translation induction sequence on mRNA (also referred to as the second RNA) produced by the transcription of a predetermined DNA (also referred to as the second DNA) containing the translation induction sequence are complementary to each other. This complementarity is preferably high in sequence homology, with a sequence homology of 80% or more being preferable, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 100%. The translation induction vector is not particularly limited as long as it contains part or all of the translation induction sequence that is complementary to the base sequence of the sense strand of the translation control region on its sense strand. The translation induction vector may be a plasmid, a viral vector, or may be contained in a translation induction virus. The translation induction vector contained in a translation induction virus may be an artificially created viral vector or the genome naturally present in the virus. In the case of an artificially created viral vector, a translation induction virus can be produced, for example, by packaging it with a virus. Furthermore, the translation control system, translation inhibition system, and translation induction system can be made into kits to facilitate their use.

[0017] The base sequence of the specified DNA is not particularly limited, but the specified DNA may be part or all of a virus-derived gene. In this case, the virus-based translational control system is a translational control system for a target gene in an animal cell, and includes a nucleic acid containing a translational control region, and an expression vector for part or all of a virus-derived gene, or a virus that expresses part or all of a virus-derived gene in an animal cell, wherein the translational control region includes a nucleic acid having a sequence complementary to part or all of the virus-derived gene in the sense strand of the target gene and forming at least one stem structure. The translational control region may be located upstream of the target gene. Below, examples using part or all of two types of virus-derived genes as the specified DNA are described.

[0018] ==Influenza Virus-Based Translation Control System== The influenza virus-based translation control system disclosed herein is a translation control system for a target gene in an animal cell, comprising a nucleic acid including a translation control region, and an expression vector for part or all of the NP gene of the influenza virus or a virus that expresses part or all of the NP gene in an animal cell, wherein the translation control region includes a nucleic acid having a sequence complementary to part or all of the NP gene on the sense strand of the target gene and forming at least one stem structure. The translation control region may be located upstream of the target gene. The translation control system for a target gene using an influenza virus system will be described in detail below. In the examples, the EGFP gene was used as the target gene, but as described in detail in paragraph

[0013] , the target gene used is not particularly limited. The influenza virus may be influenza B virus.

[0019] The translational regulatory region may have a sequence complementary to part or all of the NP gene of influenza B virus on the sense strand of the target gene, and may include the nucleotide sequence of SEQ ID NO: 5 (5'-TTCACCTCTCTTTTTTAAAAAAGGGT-3'), or may include the nucleotide sequence of SEQ ID NO: 6 (5'-TGCAATCCCCCGGAATTTTCTGCTTCCAATCAACTTTGAATCAACTAGAGCCCTTTTTGTGGGGGC-3'). The translational induction vector may be an expression vector for part or all of the NP gene of influenza B virus, or the translational induction virus may be a virus that expresses part or all of the NP gene in animal cells. The translational induction virus may be a natural influenza B virus, or a virus packaged with an expression viral vector for part or all of the NP gene. Part or all of the NP gene introduced into animal cells is transcribed as a second mRNA.

[0020] The NP gene is the gene that codes for the nucleoprotein (NP) of the influenza B virus. The NP gene contained in the translation induction vector may be part or all of it, and its sequence is not particularly limited as long as the translational regulatory region has a sequence complementary to the transcribed mRNA region. If the translational regulatory region has the nucleotide sequence of SEQ ID NO: 5 or 6, the NP gene may be completely identical to the nucleotide sequence of SEQ ID NO: 2 or 3, and may contain a single nucleotide mutation, a double nucleotide mutation, or a triple nucleotide mutation.Also, for example, B / Iwate / 34 / 2017, B / Lee / 40, B / Victoria / 02 / 1987, B / Yamagata / 16 / 88, B / Paris / 986 / 2018, B / YAMAGATA / 162 / 2019, B / Texas / 24 / 2019, B / S ydney / 704 / 2019, B / Philippines / 1490 / 2019, B / Paris / 2038 / 2019, B / NAG ANO / 2773 / 2019, B / Myanmar / 18M348 / 2018, B / Moscow / 10 / 2019, B / Michiga n / 37 / 2019, B / Kenya / 125 / 2019, B / Chile / 6380 / 2019, B / OSAKA / 65 / 2018, B / Maryland / 15 / 2016, B / Laos / F1664 / 2017, B / Hong_Kong / 269 / 2017, B / AIC HI / 117 / 2018, B / YOKOHAMA / 13 / 2015, B / Victoria / 502 / 2015, B / Texas / 02 / 2013, B / Taiwan / 91061 / 2005, B / Phuket / 3073 / 2013, B / PHILIPPINES / 3701 / 2011, B / Paraguay / 8730 / 2018, B / Ontario / RV3160 / 2018, B / Odessa / 3886 / 2010, B / New_Zealand / 537 / 2015, B / NAGANO / 2272 / 2017, B / Michigan / 35 / 2 016, B / Massachusetts / 02 / 2012, B / Malaysia / 2506 / 2004, B / Kyoto / 13SK0 45 / 2014, B / Iwate / 34 / 2017, B / Hong_Kong / 6 / 2013, B / Florida / 06 / 2017, B / The nucleotide sequence may be completely identical to the nucleotide sequence corresponding to sequence number 2 or 3 in the NP gene of each strain of Florida / 4 / 2006, B / Finland / 292 / 2012, B / England / 393 / 2007, B / Congo / 352 / 2017, B / Brisbane / 60 / 2008, B / California / 44 / 2024, B / France / GES-RELAB-IPP11503 / 2024, and B / YOKOHAMA / 7 / 2024, and may contain a single nucleotide mutation, a double nucleotide mutation, or a triple nucleotide mutation.

[0021] ==Coronavirus-based Translation Control System== The coronavirus-based translation control system disclosed herein is a translation control system for a target gene in an animal cell, comprising a nucleic acid including a translation control region, and an expression vector for part or all of the N gene of a coronavirus or a virus expressing part or all of the N gene in an animal cell, wherein the translation control region includes a nucleic acid having a sequence complementary to part or all of the N gene in the sense strand of the target gene and forming at least one stem structure. The translation control region may be located upstream of the target gene. The coronavirus-based translation control system for a target gene will be described in detail below. In the examples, the EGFP gene was used as the target gene, but as described in detail in paragraph

[0013] , the target gene used is not particularly limited. The coronavirus may be SARS-CoV-2.

[0022] The translational control region may have a sequence complementary to part or all of the N gene of the SARS-CoV-2 virus in the sense strand of the target gene, and may include the nucleotide sequence of SEQ ID NO: 28, or may include the nucleotide sequence of SEQ ID NO: 29.

[0023] The translation induction vector may be an expression vector for part or all of the N gene of the SARS-CoV-2 virus, or the translation induction virus may be a virus that expresses part or all of the N gene in animal cells. The translation induction virus may be a natural SARS-CoV-2 virus, or a virus packaged with an expression viral vector for part or all of the N gene. Part or all of the N gene introduced into the animal cell is transcribed as a second mRNA.

[0024] The N gene is the gene that encodes the nucleocapsid of the SARS-CoV-2 virus. The N gene present in the translation induction vector may be part or all of it, and its sequence is not particularly limited as long as the translational regulatory region has a sequence complementary to the transcribed mRNA region. If the translational regulatory region has the nucleotide sequence of SEQ ID NO: 28 or 29, the N gene may be completely identical to the nucleotide sequence of SEQ ID NO: 26 or 27, and may contain a single nucleotide mutation, a double nucleotide mutation, or a triple nucleotide mutation.

[0025] ==Method for controlling the translation of a target gene== A method for controlling the translation of a target gene in an animal cell disclosed herein includes the steps of introducing an expression vector having a target gene downstream of a translational control region into an animal cell, or inserting a translational control region upstream of a target gene on the genomic DNA of an animal cell, and introducing a translation-inducing vector into the animal cell. The translational control region, expression vector having a target gene downstream of the translational control region, and translation-inducing vector used herein are described in detail in "Target gene translation control system," "Translation control system using influenza B virus," and "Translation control system using SARS virus," respectively.

[0026] The type of animal cell in which the translational control of the target gene takes place is not particularly limited. It may be muscular cells such as smooth muscle cells, cardiomyocytes, and skeletal muscle cells; visceral cells such as cardiac cells, hepatocytes, stomach-derived cells, intestinal-derived cells, and kidney-derived cells; or nervous system cells such as nerve cells and glial cells. The animal species from which the cells originate is also not particularly limited, as long as it is an animal, and may be human, mouse, rat, monkey, pig, etc.

[0027] The method for introducing expression vectors into animal cells and translation induction vectors is not particularly limited and can be any method known to those skilled in the art. For example, it may be lipofection using cationic reagents such as Lipofectamine® (Thermo Fisher Scientific) or PEI (polyethyleneimine), transfection using transfection reagents such as calcium phosphate, or introduction methods using gold nanoparticles, or electroporation or microinjection, or introduction methods using viral vectors.

[0028] In the case of translation-inducing vectors, some or all of the NP gene may be expressed by infecting animal cells with a virus capable of expressing genes within animal cells.

[0029] ==An assay method for investigating the translation-inducing ability of a translation-regulating region located upstream of a target gene==The assay method disclosed herein for investigating the translation-inducing ability of a translation-regulating region located upstream of a target gene in an animal cell comprises: (a) introducing an expression vector having a target gene downstream of a translation-regulating region into an animal cell, or inserting a translation-regulating region upstream of a target gene on genomic DNA in an animal cell; (b) introducing an expression vector of part or all of a virus-derived gene into an animal cell, or infecting an animal cell with a virus that expresses part or all of a virus-derived gene in the animal cell; and (c) examining the difference in the expression level of the protein encoded by the target gene with or without step (b), wherein the translation-regulating region includes a nucleic acid having a sequence complementary to part or all of the virus-derived gene in the sense strand of the target gene and forming at least one stem structure.

[0030] The target gene is not particularly limited, but it is preferably a gene that codes for an easily detectable protein, and may be a gene that codes for a fluorescent protein or a luminescent protein. In that case, the expression level of the protein coded by the target gene can be measured by observing the fluorescence amount with a fluorescence microscope or by examining it with a flow cytometer, or by examining the luminescence amount with a plate reader. Alternatively, the protein produced from the target gene may be examined by Western blotting or ELISA.

[0031] The type of virus is not particularly limited, but influenza viruses or coronaviruses are preferred, and influenza B viruses or SARS-CoV-2 viruses are more preferred. The gene is not particularly limited as long as it has a region with the stem-loop structure described above, but the NP gene of the influenza virus or the N gene of the coronavirus is preferred.

[0032] In this way, by examining the difference in the expression level of the protein encoded by the target gene with and without step (b), it can be seen that the greater the difference, the greater the ability of some or all of the translational regulatory region and / or virus-derived genes used to induce translation of the target gene. Therefore, this assay method is an effective way to determine whether some or all of the translational regulatory region and / or virus-derived genes used function as a translation induction system for the target gene.

[0033] ==Screening method for nucleic acids having translation-inducing ability to a candidate translation-regulating region located upstream of a target gene==The screening method for nucleic acids having translation-inducing ability to a candidate translation-regulating region located upstream of a target gene in an animal cell disclosed herein is a method comprising: (p) examining the translation-inducing ability of some or all of a plurality of candidate translation-regulating regions and / or viral genes using an assay method for examining the translation-inducing ability to a candidate translation-regulating region located upstream of a target gene in an animal cell; and (q) identifying some or all of the candidate translation-regulating regions and / or viral genes in which the expression level of the protein encoded by the target gene is enhanced by step (p).This screening method makes it possible to identify some or all of the candidate translation-regulating regions and / or viral genes in which the translation induction system of the target gene functions effectively.The assay method for examining the translation-inducing ability of a candidate translation-regulating region located upstream of a target gene is as described above.

[0034] [Example 1] Inhibition of EGFP mRNA translation by complementary sequence of influenza B virus-derived NP gene, and induction of EGFP translation by NP gene introduction. In this example, fluorescence when the target gene, the EGFP gene, is expressed in cells is used as an indicator to demonstrate the inhibition of EGFP translation by a translation regulatory region having a sequence complementary to the base sequence of the influenza B virus-derived NP gene, and the induction of EGFP translation by NP gene introduction.

[0035] [1] Translational regulatory region An expression vector for the target gene was constructed such that the sense strand of the translational regulatory region contains complementary sequences (Sequence IDs 4-6) of Sequence IDs 1-3 (Table 1 below), which form at least one stem-loop structure, from the nucleotide sequence of the NP gene (Accession No. EPI1147765) of influenza B virus (B / Iwate / 34 / 2017 strain). Of the nucleotide sequence of the NP gene of B / Iwate / 34 / 2017, Sequence ID 1 corresponds to the nucleotide sequence of 51-75 bp, Sequence ID 2 to 514-536 bp, and Sequence ID 3 to 870-927 bp. It was predicted that Sequence ID 1 would form a stem structure of 7 nucleotides as shown in Figure 1(C), Sequence ID 2 to 8 nucleotides as shown in Figure 1(D), and Sequence ID 3 to a total of 14 nucleotides as shown in Figure 1(E).

[0036] [2] Construction of Target Gene Expression Vectors Target gene expression vectors were constructed using the green fluorescent protein EGFP (Accession No. U55761.1) gene as the target gene. Specifically, PCR was performed using the EGFP gene as a template, with forward primers of SEQ ID NOs. 7, 8, and 9 containing the recognition sequence of the restriction enzyme EcoRI (GAATTC), the nucleotide sequence with GCCGCC, the translational regulatory sequences of SEQ ID NOs. 4-6, and the 5' terminal sequence of EGFP (ATGGTGAGCAAGGGGC), and the reverse primer of SEQ ID NO. 11 containing the recognition sequence of the restriction enzyme XhoI (CTCGAG), the stop codon (TTA), and the 3' terminal sequence of EGFP (CTTTGTACAGCTCGTCCA). The PCR conditions involved processing at 94°C for 3 minutes, followed by 35 cycles of 98°C for 10 seconds, 57°C for 15 seconds, and 68°C for 40 seconds. After processing the amplified PCR product with EcoRI and XhoI, it was inserted between the EcoRI and XhoI sites of pcDNA3.1 (invitrogen, catalog number: V79020) using Ligation High Ver. 2 (Toyobo Co., Ltd., catalog number: LGK-201). The inserted plasmid was transformed into competent cells DH5α (Toyobo Co., Ltd., catalog number: DNA-901), and the plasmid was amplified. The amplified plasmid was purified using LaboPass Mini (Hokkaido System Science Co., Ltd., catalog number: CMP0111). Each obtained plasmid was used as an expression vector for the EGFP gene, with the nucleotide sequences of SEQ ID NOs. 4-6 as translational regulatory regions. Furthermore, an EGFP gene expression vector without the translational regulatory region was prepared as a control expression vector, using the Forward primer of Sequence ID No. 10, which contains the recognition sequence of the restriction enzyme EcoRI (GAATTC), the nucleotide sequence with GCCGCC, and the 5' terminal sequence of EGFP (ATGGTGAGCAAGGGGC), in all other similar ways.

[0037] [3] Preparation of NP gene expression vector The viral RNA gene was extracted from influenza B virus (B / Iwate / 34 / 2017 strain) using the RNeasy Mini kit (Qiagen, catalog number: 74104). This was reverse transcribed using ReverTra Ace (Toyobo, catalog number: TRT-101) and oligo dT primers to synthesize cDNA. To clone into the pCAGGS-MCS vector (inhouse), the NP gene was amplified by PCR using cDNA as a template with primers (SEQ ID NOs: 12, 13) that had the restriction enzyme SacI (GAGCTC) sequenced at the 5' end and the restriction enzyme XhoI (CTCGAG) sequenced at the 3' end. The PCR conditions involved processing at 94°C for 3 minutes, followed by 35 cycles of 98°C for 10 seconds, 57°C for 15 seconds, and 68°C for 1 minute and 15 seconds. After treating the amplified NP gene with SacI and XhoI, it was inserted between SacI and XhoI in the pCAGGS-MCS vector. The inserted plasmid was transformed into competent cells DH5α (Toyobo Co., Ltd., catalog number: DNA-901), and the plasmid was amplified. The amplified plasmid was purified using LaboPass Mini (Hokkaido System Science Co., Ltd., catalog number: CMP0111).

[0038] [4] Introduction of target gene expression vectors and NP gene expression vectors into cells 250 ng each plasmid of the EGFP gene expression vector containing the nucleotide sequences of SEQ ID NOs. 4-6 prepared in [2] as translational regulatory regions and 250 ng of a pCAGGS empty vector that does not contain the NP gene, or a mixture of 250 ng each plasmid of the EGFP gene expression vector containing the nucleotide sequences of SEQ ID NOs. 4-6 as translational regulatory regions and 250 ng of the plasmid of the NP gene expression vector prepared in [3], were introduced into 293T cells using TransIT-LT1 Translation Reagent and cultured at 37°C. After 2 days, observation with a fluorescence microscope revealed that cells introduced with the EGFP gene expression vector containing the nucleotide sequences of SEQ ID NOs. 5-6 as translational regulatory regions showed weaker EGFP expression compared to control cells, while cells introduced with the expression vector containing the nucleotide sequence of SEQ ID NO. 4 showed expression at approximately the same level as control cells (Figure 2, top panel). In cells introduced with a mixture of plasmids containing each of the EGFP gene expression vectors with the nucleotide sequences of SEQ ID NOs. 5-6 as translational regulatory regions and plasmids containing the NP gene expression vector, EGFP expression was stronger compared to cells introduced with only the EGFP gene expression vector containing each of the nucleotide sequences of SEQ ID NOs. 5-6 as translational regulatory regions (Figure 2, bottom panel). This indicates that when the stem structure of the translational regulatory region has a nucleotide length of 7 nucleotides or less, the resulting stem structure is unstable, while when the stem structure has a nucleotide length of 8 nucleotides or more, the stem structure stabilizes, making ribosome movement impossible and thus inhibiting EGFP translation. Furthermore, it is thought that the binding of NP gene mRNA transcribed from the NP gene expression vector to the translational regulatory region, which takes the form of a stem-loop structure on the EGFP gene mRNA, destabilizes the stem-loop structure of the translational regulatory region, allowing ribosome movement and thus releasing the inhibition of EGFP translation.

[0039] Thus, an expression vector containing a translation regulatory region positioned upstream of the target gene can be suitably used as a translation inhibition system for the target gene, and an NP gene expression vector can be suitably used as a translation induction system that can induce translation of the target gene by expressing NP gene-derived mRNA in the introduced cells. Together, these two can be suitably used as a translation regulatory system for the target gene in animal cells.

[0040] [Comparative Example 1] Effect of EGFP Translation Induction by HA Gene Derived from Influenza B Virus This comparative example demonstrates that when the HA gene derived from influenza B virus is used instead of the NP gene, no translation induction of the target gene occurs.

[0041] [1] Preparation of an HA gene expression vector Using the cDNA of influenza B virus synthesized in Example 1[3] as a template, the HA gene (Accession No. EPI1147772) was amplified by PCR using primers (Sequence IDs 14 and 15 in the sequence listing) with the restriction enzyme SacI (GAGCTC) sequenced at the 5' end and the restriction enzyme XhoI (CTCGAG) sequenced at the 3' end. The PCR conditions were 35 cycles of treatment at 94°C for 3 minutes, followed by 10 seconds at 98°C, 15 seconds at 57°C, and 1 minute 15 seconds at 68°C. After treating the amplified NP gene with SacI and XhoI, it was inserted between SacI and XhoI in the pCAGGS-MCS vector. The inserted plasmid was transformed into competent cells DH5α (Toyobo Co., Ltd., catalog number: DNA-901), and the plasmid was amplified. The amplified plasmid was purified using LaboPass Mini (Hokkaido System Science Co., Ltd., catalog number: CMP0111).

[0042] [2] Introduction of target gene expression vectors and HA gene expression vectors into cells Each plasmid of the EGFP gene expression vector containing the nucleotide sequences of SEQ ID NOs. 4-6 prepared in Example 1[2] as a translational regulatory region and 250 ng of pCAGGS empty vector, or a mixture of each plasmid of the EGFP gene expression vector containing the nucleotide sequences of SEQ ID NOs. 4-6 prepared in Example 1[2] and 250 ng of the plasmid of the HA gene expression vector prepared in Comparative Example 1[1], was introduced into 293T cells using TransIT-LT1 Translation Reagent and cultured at 37°C. After 3 days, observation with a fluorescence microscope revealed that, unlike the results for the NP gene expression vector in Example 1[4] (Figure 2), EGFP expression was not induced in cells introduced with the mixture of each plasmid of the EGFP gene expression vector containing the nucleotide sequences of SEQ ID NOs. 5-6 as a translational regulatory region and the plasmid of the HA gene expression vector (Figure 3). This result suggests that when the HA gene, which does not have a sequence complementary to the translational regulatory region of the EGFP gene expression vector, is introduced, the stem-loop structure of the translational regulatory region remains unchanged, inhibiting ribosome progression and thus preventing the release of EGFP translation inhibition.

[0043] [Comparative Example 2] Effect of complementary sequences of NP genes that do not adopt or stably form a stem-loop structure on EGFP translation This comparative example shows that when complementary sequences of NP genes that do not adopt or stably form a stem-loop structure are used instead of complementary sequences of NP genes that adopt a stem-loop structure, the translation of the target gene is not inhibited.

[0044] [1] Complementary sequences of the NP gene that do not form or stably form a stem-loop structure Using the complementary sequences (SEQ ID NO: 18, 19) of the sequences (SEQ ID NO: 16, 17) in the nucleotide sequence of the NP gene (Accession No. EPI1147765) of influenza B virus (B / Iwate / 34 / 2017 strain) that do not form or stably form a stem-loop structure, an expression vector for the target gene was constructed. Among the nucleotide sequences of the NP gene of B / Iwate / 34 / 2017, SEQ ID NO: 16 corresponds to the nucleotide sequence of 242 - 266 bp, and SEQ ID NO: 17 corresponds to the nucleotide sequence of 1063 - 1087 bp. As shown in Figure 1(F), the stem structure of SEQ ID NO: 16 does not form a stem structure, and although the stem structure of SEQ ID NO: 17 was predicted to form a stem structure with a length of 3 bases as shown in Figure 1(G), considering the experimental results of Example 1, the stem structure of SEQ ID NO: 17 was predicted to be very unstable.

[0045] [2] Preparation of an expression vector for the target gene containing the complementary sequence of the NP gene that does not form or stably form a stem-loop structure Using the green fluorescent protein EGFP (Accession No. U55761.1) gene as the target gene, an expression vector for the target gene containing the sequences of SEQ ID NO: 18, 19 was prepared. Specifically, using the EGFP gene as a template, each Forward primer of SEQ ID NO: 20, 21 containing the recognition sequence (GAATTC) of restriction enzyme EcoRI, the nucleotide sequence with GCCGCC, the complementary sequences of SEQ ID NO: 18, 19, and the sequence (ATGGGTGAGCAAGGGGC) at the 5'-end of EGFP, and the Reverse primer of SEQ ID NO: 11, PCR was performed respectively. The PCR conditions and subsequent insertion into pcDNA3.1 and plasmid preparation were carried out in the same manner as in Example 1[2].

[0046] [3] Introduction of target gene expression vectors containing complementary sequences of NP genes that do not adopt or stably form a stem-loop structure into cells 250 ng each plasmid of the target gene expression vector containing sequences of SEQ ID NOs. 18 and 19 and 250 ng of a pCAGGS empty vector that does not contain the NP gene, or a mixture of 250 ng each plasmid of the target gene expression vector containing sequences of SEQ ID NOs. 18 and 19 and 250 ng of the plasmid of the NP gene expression vector, were introduced into 293T cells using TransIT-LT1 Transfer Reagent and cultured at 37°C. After 3 days, observation with a fluorescence microscope revealed that cells introduced with the target gene expression vector containing sequences of SEQ ID NOs. 18 and 19 showed EGFP expression similar to that of control cells (Figure 4). In Example 1[4], when the stem-loop structure in the translational regulatory region located upstream of the EGFP gene was 8 bases or more, ribosome progression was inhibited, and EGFP translation was inhibited. In contrast, sequences 18 and 19, which do not adopt a stem-loop structure or do not stably form one, did not inhibit EGFP translation. Thus, the stem-loop structure in the translational regulatory region upstream of the target gene is important for the regulation of EGFP translation.

[0047] [Example 2] Translation induction of EGFP by influenza B virus infection In this example, the fluorescence of the target gene EGFP when it is expressed in cells is used as an indicator to demonstrate the translation induction of EGFP by infecting cells into which a target gene expression vector has been introduced with influenza B virus.

[0048] The plasmids of the expression vectors of the EGFP gene containing each of the base sequences of SEQ ID NOs: 4 to 6 created in Example 1[2] as translation control regions were each introduced into 293T cells in the same manner as in Example 1[4] and cultured at 37°C. When observed with a fluorescence microscope 6 hours later, the expression of EGFP was suppressed in the cells into which the expression vectors of the EGFP gene containing each of the base sequences of SEQ ID NOs: 5 to 6 as translation control regions were introduced (upper row of FIG. 5). These cells were infected with type B influenza virus (B / Iwate / 34 / 2017 strain) at a condition of MOI (Multiplicity of Infection) = 1 (plaque forming unit / cell) and cultured. One day later, when observed with a fluorescence microscope, it was confirmed that the expression of EGFP was induced in the type B influenza virus-infected cells (lower row of FIG. 5).

[0049] Thus, for the induction of translation of the target gene, it is only necessary to introduce the mRNA derived from the NP gene, and the introduction method is not affected by it.

[0050] [Example 3] Effect of modified secondary structure sequences on EGFP expression suppression The base sequences of SEQ ID NO: 4 (stem portion sequence is 7 bases) and SEQ ID NO: 5 (stem portion sequence is 8 bases), which have a stem-loop structure, were subjected to a single base substitution with the stem structure sequence (referred to as the stem sequence herein), and the base sequences of SEQ ID NO: 22 (stem sequence is 8 bases) and SEQ ID NO: 23 (stem sequence is 7 bases) were designed, respectively (Figure 6A). Then, EGFP genes with these base sequences upstream were constructed as follows. First, a forward primer (SEQ ID NO: 24 or 25) and a reverse primer (SEQ ID NO: 11) were combined to amplify the EGFP genes with the base sequences of SEQ ID NO: 22 and SEQ ID NO: 23 upstream, respectively, by PCR. These primers were conjugated with the restriction enzyme EcoRI (GAATTC) at the 5′ end and the restriction enzyme XhoI (CTCGAG) at the 3′ end for cloning into pcDNA3.1. The PCR conditions consisted of 35 cycles of treatment at 94°C for 1 minute, followed by 10 seconds at 98°C and 1 minute 45 seconds at 68°C. After treating the amplified PCR product with EcoRI and XhoI, it was inserted between the EcoRI and XhoI sites of pcDNA3.1 using Ligation High Ver. 2. The inserted plasmid was transformed into competent cells DH5α, and the plasmid was amplified. The amplified plasmid was purified using LaboPass Mini. 500 ng of the plasmid prepared in this manner was introduced into African green monkey kidney-derived COS1 cells using TransIT-LT1 Transfection Reagent and cultured at 37°C. Plasmid introduction was performed according to the method described in the reagent's package insert. After one day, EGFP fluorescence was observed using a fluorescence microscope. Cells into which the EGFP gene with sequence number 22 (stem sequence of 8 nucleotides) upstream was introduced showed suppression of EGFP expression compared to cells into which sequence number 4 (stem sequence of 7 nucleotides) was introduced. Cells into which the EGFP gene with sequence number 23 (stem sequence of 7 nucleotides) upstream was introduced showed recovery of EGFP expression (sequence number 23) compared to cells into which the EGFP gene with sequence number 5 (stem sequence of 8 nucleotides) upstream was introduced (Figure 6B).As demonstrated in this example, EGFP expression was significantly enhanced with the 8-base stem sequence compared to the 7-base stem sequence in all sequences. Thus, a stem sequence of at least 8 bases is necessary to suppress the expression of the target gene.

[0051] [Example 4] Suppression of luciferase expression by complementary sequence of SARS-CoV-2-derived N gene, and restoration of luciferase expression by N gene introduction From the nucleotide sequence of the N gene (Accession No. LC528232.2) of SARS-CoV-2 (SARS-CoV-2 / Hu / DP / Kng / 19-020 strain), sequences 226-255 (SEQ ID NO. 26) and sequences 1047-1075 (SEQ ID NO. 27), which are regions that take on a characteristic secondary structure, were selected, and complementary sequences for SEQ ID NOs. 26 and 27 (SEQ ID NOs. 28 and 29, respectively) were designed. Figure 7 shows their secondary structures, and in both cases the stem structure has a length of 8 nucleotides or more. Luciferase genes (Promega Inc.) having SEQ ID NO. 28 or 29 upstream, or luciferase genes (control) without the insertion of the complementary sequence were prepared as follows. First, PCR was performed using a combination of Forward primer (SEQ ID NO: 30) and Reverse primer (SEQ ID NO: 32), or Forward primer (SEQ ID NO: 31) and Reverse primer (SEQ ID NO: 32), to amplify the luciferase gene having SEQ ID NO: 28 or 29 upstream. For cloning to pcDNA 3.1, the restriction enzyme EcoRI (GAATTC) was added to the 5′ end and the restriction enzyme XhoI (CTCGAG) to the 3′ end of these primers. The PCR conditions consisted of 1 minute of treatment at 94°C, followed by 35 cycles of 10 seconds at 98°C, 15 seconds at 63°C, and 1 minute 30 seconds at 68°C. After treating the amplified PCR products with EcoRI and XhoI, Ligation high Ver. Using 2, the plasmid was inserted between the EcoRI and XhoI sites of pcDNA3.1. The inserted plasmid was transformed into competent cells DH5α, and the plasmid was amplified. The amplified plasmid was purified using LaboPass Mini.Meanwhile, to clone the N gene of SARS-CoV-2 (SARS-CoV-2 / Hu / DP / Kng / 19-020 strain) into the pCAGGS-MCS vector, PCR was performed using forward primers (SEQ ID NO: 33) and reverse primers (SEQ ID NO: 34) with the restriction enzyme SacI (GAGCTC) sequenced at the 5′ end and the restriction enzyme XhoI (CTCGAG) sequenced at the 3′ end. The N gene was amplified using cDNA (provided by the research group Torii, Shiho, et al. Cell reports 35.3 (2021)) as a template. The PCR conditions consisted of 1 minute of processing at 94°C, followed by 35 cycles of 10 seconds at 98°C, 15 seconds at 61°C, and 1 minute 30 seconds at 68°C. The amplified N gene was treated with SacI and XhoI, and then inserted between SacI and XhoI in the pCAGGS-MCS vector. Competent cells DH5α were transformed with this plasmid, and the plasmid was amplified. The amplified plasmid was purified using LaboPass Mini. 100 ng of plasmid containing a luciferase gene with sequence number 28 or 29 upstream, or a luciferase gene without a complementary sequence insertion (control), was introduced into human embryonic kidney cell line 293T cells using TransIT-LT1 Transfection Reagent and cultured at 37°C. Plasmid introduction was performed according to the reagent's package insert. One day later, ONE-Glo™ EX Luciferase Assay System (Promega, catalog number: E8110) was added according to the instructions in the package insert, and luciferase activity was measured using a microplate reader (TECAN Spark), with the results graphed (Figure 8). As shown in Figure 8, cells into which the luciferase gene with sequence number 28 or 29 upstream was introduced showed significantly lower luciferase activity compared to the control. Thus, the insertion of a sequence complementary to the N gene upstream of the luciferase gene suppresses luciferase translation and reduces luciferase expression.Furthermore, 10 ng of plasmids expressing a luciferase gene with sequence number 28 or 29 upstream, or a luciferase gene without a complementary sequence insertion (control), and 10 ng of plasmids expressing the N gene were introduced into human embryonic kidney cell line 293T cells using TransIT-LT1 Transfection Reagent and cultured at 37°C. The introduction was performed according to the reagent's package insert. After 1 day, ONE-Glo™ EX Luciferase Assay System was added according to the package insert, and luciferase activity was measured using a microplate reader. The results were graphed (Figure 9). As shown in Figure 9, luciferase expression was restored in cells supplied with N gene mRNA (Figure 9). In this way, when mRNA transcribed from the N gene binds to the complementary strand of the N gene, which is inserted upstream of the luciferase gene, the secondary structure of the complementary strand changes, enabling ribosome movement and releasing the translational repression of luciferase.

[0052] The sequences used in the above examples and comparative examples are shown in Table 1 below.

[0053]

[0054] This invention makes it possible to provide a novel translation control system for target genes.

Claims

1. A translation control system for a target gene in an animal cell, comprising: a nucleic acid including a translation control region located upstream of the target gene; an expression vector for a predetermined DNA, or a virus that expresses the predetermined DNA in the animal cell, wherein the translation control region includes a nucleic acid having a sequence complementary to the predetermined DNA on the sense strand of the target gene and forming at least one stem structure.

2. The system according to claim 1, wherein at least one stem structure has a length of 8 bases or more.

3. The translation control system according to claim 1 or 2, wherein the predetermined DNA is part or all of a virus-derived gene.

4. The system according to claim 3, wherein the virus-derived gene is the NP gene of influenza B virus.

5. The system according to claim 3, wherein the virus-derived gene is the N gene of SARS-CoV-2.

6. The translation control system according to claim 1 or 2, wherein the translation control region is a nucleic acid containing the base sequence of SEQ ID NO: 5 (5'-TTCACCTCTCTCTTTTTAAAAAGGT-3').

7. The translation control system according to claim 1 or 2, wherein the translation control region is a nucleic acid containing the base sequence of Sequence ID No. 6 (5'-TGCAATCCCCCGGATTTCCTGCTCCAATCAACTTGAATCAACTAGCCCTTTTTGTGGGGGC-3').

8. A method for controlling the translation of a target gene in an animal cell, comprising: (a) introducing an expression vector having the target gene downstream of a translational control region into the animal cell, or inserting a translational control region upstream of the target gene on genomic DNA in the animal cell; and (b) introducing an expression vector of a predetermined DNA into the animal cell, or infecting the animal cell with a virus that expresses the predetermined DNA in the animal cell, wherein the translational control region comprises a nucleic acid having a sequence complementary to part or all of the virus-derived gene in the sense strand of the target gene and forming at least one stem structure.

9. An assay method for investigating the translation-inducing ability of a translation-regulating region located upstream of a target gene in an animal cell, comprising: (a) introducing an expression vector having the target gene downstream of the translation-regulating region into the animal cell, or inserting the translation-regulating region upstream of the target gene on the genomic DNA in the animal cell; (b) introducing an expression vector of a predetermined DNA into the animal cell, or infecting the animal cell with a virus that expresses the predetermined DNA in the animal cell; and (c) examining the difference in the expression level of the protein encoded by the target gene depending on whether or not step (b) is performed, wherein the translation-regulating region includes a nucleic acid having a sequence complementary to the predetermined DNA on the sense strand of the target gene and forming at least one stem structure.

10. A screening method for nucleic acids having translation-inducing ability to a translation-regulating candidate region located upstream of a target gene in an animal cell, comprising: (p) examining the translation-inducing ability of a plurality of translation-regulating candidate regions and / or predetermined DNA using the assay method of claim 9; and (q) identifying a translation-regulating candidate region and / or predetermined DNA in which the expression level of the protein encoded by the target gene is enhanced by step (p).

11. A system for controlling the translation of a target gene in animal cells, comprising: a nucleic acid including a translational control region located upstream of the target gene; and an expression vector for a predetermined DNA or a virus that expresses the predetermined DNA in the animal cell, wherein the translational control region includes a nucleic acid having a sequence complementary to the predetermined DNA on the sense strand of the target gene and forming at least one stem structure.

12. The system according to claim 11, in the form of a kit.

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