JP2005034008A - New method for inhibiting expression of gene and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for inhibiting the expression of a gene by which operation is simple and the inhibitory efficiency of the gene expression is high, and to provide a method for utilization thereof. <P>SOLUTION: The method for inhibiting the gene expression is to transfer a genetic fragment composed of a partial sequence of a target gene or the genetic fragment and an RNA polymerase into a host cell to thereby inhibit the expression of the target gene. Since construction of a vector as in conventional RNA interference is not required according to this method, functional analysis of the gene can simply be carried out in a short time. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明の遺伝子発現抑制法のメカニズムの一例を示す模式図である。
【図２】本発明の実施例において、ｄｓＲＮＡをプロトプラストに導入した場合の遺伝子発現抑制効果を示すグラフである。
【図３】本発明の実施例において、ＤＮＡ断片とＴ７ＲＮＡポリメラーゼとを同時にプロトプラストに導入した場合の遺伝子発現抑制効果を示すグラフである。
【図４】本発明の実施例において、ＤＮＡ断片のみをプロトプラストに導入した場合のルシフェラーゼ活性の測定結果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel gene expression suppression method and use thereof, and more specifically, a novel gene expression suppression method that suppresses expression of a target gene by introducing a gene fragment consisting of a partial sequence of the target gene into a host cell. , And its use.
[0002]
[Prior art]
Antisense RNA means a transcript whose genetic information is reversed with respect to mRNA (sense RNA) transcribed from DNA. Since this antisense RNA is structurally complementary to the mRNA produced from the target gene, they bind to each other to form an RNA duplex (also referred to as double-stranded RNA or dsRNA). In this manner, mRNA covered with a “lid” is degraded by a specific RNase and cannot be transferred to the field of protein synthesis, and the function of the original gene is suppressed.
[0003]
Further, not only the antisense RNA but also the above-mentioned dsRNA formed by combining the antisense RNA and the sense RNA into each other, the mRNA having a sequence homologous to the dsRNA is degraded, It causes the phenomenon of suppressing gene expression. The phenomenon in which gene expression is suppressed by the introduction of dsRNA is called RNA interference (or RNAi, RNA interference) (for example, Non-Patent Documents 1 to 4).
[0004]
RNA interference, which is a phenomenon in which the expression of a gene having a sequence homologous to dsRNA is suppressed by introduction of double-stranded RNA (dsRNA) into a cell, was reported in 1998 by Caenorhabditis elegans. It was discovered for the first time. And since RNA interference has extremely high sequence specificity, in recent years, it has been widely used as an effective method of reverse genetics, mainly nematodes. In addition, the dsRNA has the potential to be widely used for RNA-targeted pharmaceuticals, agricultural chemicals, breed improvement, microbial industry, bioreactor development, and the like.
[0005]
[Non-Patent Document 1]
Fire, A.M. Xu, S .; Montgomery, M .; K. , Kostas, S .; A. , Driver, S.M. E. and Mello, C.I. C. (1998) Nature, 391, 806-811.
[0006]
[Non-Patent Document 2]
Elbashir, S .; M.M. Harborth, J .; Lendeckel, W.M. Yalcin, A .; Weber, K .; and Tuschl, T .; (2001) Nature, 411, 494-498.
[0007]
[Non-Patent Document 3]
Brummelkamp, T .; R. Bernards, R .; and Agami, R.A. (2002) Science, 296, 550-553.
[0008]
[Non-Patent Document 4]
Paddison, P.M. J. et al. , Caudy, A .; A. Bernstein, E .; Hannon, G .; J. et al. and Conklin, D.C. S. (2002) Genes Dev, 16, 948-958.
[0009]
[Problems to be solved by the invention]
As a method for introducing the dsRNA into the cell, a direct injection method by microinjection is frequently used in nematodes and the like. On the other hand, in plants, since there is a strong cell wall made of cellulose or pectin, it is extremely difficult to directly introduce dsRNA into cells. Therefore, in plants, a method of transforming with a vector having an inverted repeat sequence structure that is expected to form dsRNA after transcription is exclusively used (Non-patent Documents 3 and 4). .
[0010]
However, since it is difficult to construct such a vector, it takes a considerable amount of time to produce the vector, and furthermore, it has a problem that the gene expression suppression efficiency is not necessarily high. In addition, conventional gene expression suppression methods are unsuitable for high-throughput screening required in reverse genetics research.
[0011]
As described above, since the preparation of the vector is complicated, a gene expression suppression method suitable for high-throughput screening required in reverse genetics research has not yet been developed.
[0012]
Therefore, the present invention has been made in view of the above-described conventional problems, and an object of the present invention is to provide a novel gene expression suppression method and a method for using the same that can easily and efficiently suppress the expression of a target gene. There is to do.
[0013]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventor has intensively studied a gene expression suppression method that is simple in operation and exhibits high gene expression suppression efficiency. As a result, as in the conventional RNA interference method, the gene fragment consisting of a partial sequence of the target gene is introduced into the host instead of dsRNA or a vector that expresses it, thereby exhibiting suppression efficiency substantially equivalent to RNA interference. As a result, the present invention has been completed.
[0014]
That is, the gene expression suppression method of the present invention is characterized by suppressing the expression of a target gene by introducing a gene fragment consisting of a partial sequence of the target gene into a host.
[0015]
Another gene expression suppression method of the present invention is characterized by suppressing the expression of a target gene by introducing a gene fragment consisting of a partial sequence of the target gene and RNA polymerase into a host.
[0016]
In the gene expression suppression method of the present invention, the gene fragment may have a promoter sequence at the 5 ′ end. The gene fragment is preferably double-stranded. The gene fragment is preferably DNA. The gene fragment may be composed of at least 21 bases or base pairs.
[0017]
According to the above invention, the expression of the target gene can be suppressed only by introducing a gene fragment consisting of a partial sequence of the target gene, or the gene fragment and RNA polymerase into the host. That is, the construction of a dsRNA expression vector and the production of dsRNA in vitro, which are indispensable for the conventional RNA interference method and require a lot of time, are not required. Moreover, the gene expression suppression method of the present invention shows suppression efficiency substantially the same as conventional RNA interference by dsRNA. Therefore, the operation is dramatically simpler than in the past, and the time can be greatly shortened. Furthermore, it is possible to obtain a gene expression suppression effect comparable to that of RNA interference.
[0018]
Furthermore, according to the above invention, since it is an experimental system that only produces the gene fragment, a large amount of samples can be comprehensively analyzed in a short time. That is, the gene expression suppression method of the present invention can be applied to high-throughput screening.
[0019]
The transformant of the present invention is a transformant produced by using the above gene expression suppression method, in which the expression of the target gene is suppressed. Since the expression of the target gene is suppressed, this transformant can be used as a disease model animal lacking a part (or all) of the function of the gene or a plant with improved breed.
[0020]
The gene function analysis method of the present invention is a method for analyzing the function of a target gene using any one of the above gene expression suppression methods. Preferably, the target gene is a function-unknown gene comprising a known base sequence. The method includes a step of introducing a gene fragment composed of a partial sequence of a target gene into a host and a step of comparing the functions of the host before and after the introduction of the gene fragment.
[0021]
In other words, the gene functional analysis method of the present invention suppresses the function of a function unknown gene by introducing a gene fragment consisting of a partial sequence of a function unknown gene with a clear base sequence into a host. It is preferable to analyze the function.
[0022]
According to said invention, the function of the gene is suppressed by introduce | transducing into a host the gene fragment which consists of a partial sequence of a target gene (preferably function unknown gene). Then, by analyzing the function of the host before and after the introduction of the gene fragment, the function of the target gene (preferably a function unknown gene) can be clarified. That is, a function-unknown gene can be identified as having a suppressed function by suppressing the expression of the gene.
[0023]
Thus, if the function (physiological role) of a function-unknown gene becomes clear, the gene can be applied to pharmaceuticals, agricultural chemicals, breed improvement, microbial industry, and bioreactors.
[0024]
As described above, the gene expression suppression method of the present invention does not require the construction of a vector, and is simple and can be performed in a short time. Since the gene function analysis method of the present invention is also an experimental system that only amplifies gene fragments, a large amount of function unknown gene samples can be comprehensively analyzed in a short time. That is, the gene function analysis method of the present invention is suitable for high-throughput screening for elucidating the functions of a large number of function-unknown genes and selecting genes having the desired functions from the genes.
[0025]
The gene expression inhibitor of the present invention contains a gene fragment consisting of a partial sequence of a target gene and suppresses the expression of the target gene. The therapeutic agent for a genetic disease of the present invention comprises a gene whose function has been clarified by the functional analysis method for the above gene or the above gene expression inhibitor as an active ingredient. Thereby, the novel gene expression inhibitor and gene disease therapeutic agent which can suppress the expression of a target gene can be provided.
[0026]
The kit of the present invention is a kit used for any of the above gene expression suppression methods or the above-mentioned gene functional analysis method, and comprises a gene fragment consisting of a partial sequence of a target gene, the above gene expression inhibitor, or the above gene Including any of the therapeutic drugs. Thereby, the gene expression suppression method and gene analysis method of the present invention can be performed more simply, and the versatility of the present invention can be improved.
[0027]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in more detail below, but the present invention is not limited to this description.
[0028]
(1) Gene expression suppression method of the present invention
The gene expression suppression method of the present invention is a method shown in the following (a) or (b).
(A) A method for suppressing the expression of a target gene by introducing a gene fragment comprising a partial sequence of the target gene into a host.
(B) A method for suppressing the expression of a target gene by introducing a gene fragment comprising a partial sequence of the target gene and RNA polymerase into a host.
[0029]
That is, the above (a) is a method of introducing only a gene fragment consisting of a partial sequence of a target gene into a host cell. The above (b) is a method of introducing RNA polymerase into the host cell in addition to the gene fragment consisting of the partial sequence of the target gene.
[0030]
In the present invention, the “target gene” refers to a gene whose function should be suppressed, and includes DNA and RNA. DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA serving as a sense strand or an anti-coding strand serving as an antisense strand. Further, the “target gene” may include an untranslated region (UTR) sequence. In addition, the target gene has an unknown or known function, an individual or a cell of another organism, or a function in a cell, but is unknown in a cell to be introduced, or a plurality of types. Those whose functions are unknown are listed. In addition, any of those endogenous to the cell, exogenous, chromosomal, non-chromosomal, etc. may be used, and its origin is not particularly limited.
[0031]
The “gene fragment” is a polynucleotide including at least a partial sequence of the target gene, and may include at least a partial sequence of the target gene or all of the target gene. In other words, the gene fragment is not particularly limited as long as the base sequence of at least a partial region of the target gene is continuous. Details of the gene fragment will be described later.
[0032]
The “host” is an organism (individual) or cell into which the gene fragment is introduced. Examples of the host include single cells, multicells, plants, animals, and the like, and are not particularly limited as long as the gene fragment can be introduced in the same manner as the transformant described later.
[0033]
The above-mentioned “suppression of target gene expression” indicates a decrease in the functional level of at least a part of the target gene by introduction of the gene fragment, and the target gene function may be lost. Specifically, for example, loss of protein and / or mRNA derived from the target gene, inhibition of transcription of mRNA from the target gene, or reduction in the level thereof is shown.
[0034]
One mechanism of the gene expression suppression method of the present invention will be described with reference to FIG. 1 using plant cells as hosts. That is, the mechanism shown in FIG. 1 suppresses the expression of a target gene at a post-transcriptional stage by introducing a gene fragment into a plant cell.
[0035]
Specifically, first, a double-stranded DNA fragment containing a target gene partial sequence is amplified by a PCR method using a DNA fragment containing a partial sequence of the target gene to be suppressed as a template and a primer to which a T7 promoter is added. . As a result, a double-stranded DNA fragment having a T7 promoter sequence at both ends is prepared. When this DNA fragment is introduced into a plant cell together with T7 RNA polymerase, the polymerase acts to form double-stranded RNA corresponding to the sequence of the introduced DNA fragment in the plant cell. As a result, this double-stranded RNA suppresses the expression of the target gene by inducing RNA interference.
[0036]
As described above, the mechanism of FIG. 1 causes RNA interference in a plant cell by introducing a double-stranded DNA fragment having a T7 promoter sequence into the plant cell together with T7 RNA polymerase.
[0037]
However, as shown also in the examples described later, even when only a double-stranded DNA fragment is introduced into a host cell, the mechanism other than the above is also conceivable because the expression of the target gene is similarly suppressed. For example, the mechanism that the DNA fragment itself suppresses the expression of the target gene cannot be denied, and the details of the mechanism are not clear so far.
[0038]
However, by introducing a gene fragment consisting of at least a partial sequence of the target gene into the host cell, the expression of the target gene is reliably suppressed. The gene expression suppression method of the present invention does not require complicated operations such as construction of a vector or production of dsRNA in vitro, as in conventional RNA interference. Therefore, it is extremely simpler than the conventional RNA interference method, and can greatly reduce the time, and can suppress the expression of the target gene with a gene suppression efficiency comparable to that of the conventional RNA interference method. An expression suppression method can be provided.
[0039]
In the gene expression suppression method of the present invention, a promoter sequence may be linked to the 5 ′ end of the gene fragment. Thereby, as shown in the Example mentioned later, the suppression efficiency of a target gene can be made high.
[0040]
Further, if the gene fragment has a promoter sequence, the efficiency of transcription of the gene fragment into RNA can be improved. Therefore, as in the mechanism of FIG. 1 described above, if the dsRNA transcribed from the gene fragment suppresses the expression of the target gene, the target gene can be recovered in a shorter time than when it does not have a promoter sequence. Expression can be suppressed.
[0041]
In the gene expression suppression method of the present invention, the gene fragment may be single-stranded or double-stranded. Similar to normal RNA interference, in order to efficiently suppress the expression of the target gene, the gene fragment is preferably double-stranded. Moreover, if the gene fragment is double-stranded, the expression of the target gene can be suppressed at a lower concentration than in the case of single-stranded. Furthermore, it is preferable that the gene fragment is double-stranded because degradation of intracellular nuclease can be suppressed as compared with a single-stranded case.
[0042]
In the gene expression suppression method of the present invention, the gene fragment consists of a partial sequence of the target gene. In the present invention, the types of nucleic acids of the target gene and gene fragment are not necessarily the same, and may be different. For example, when the target gene is DNA, the gene fragment may be a DNA fragment or an RNA fragment. That is, “a gene fragment consisting of a partial sequence of a target gene” means at least a DNA fragment consisting of a partial sequence of the target gene or an RNA fragment corresponding to the partial sequence. Considering the stability of the gene fragment, the gene fragment is preferably DNA. Thereby, it becomes possible to continuously suppress the expression of the target gene.
[0043]
The partial sequence of the target gene serving as the gene fragment is not particularly limited. For example, when only a specific gene in a gene family having very similar base sequences is desired as a target gene, the gene fragment includes an untranslated region such as 3′-UTR as a partial sequence of the target gene. It is preferable to do. Since this region has low sequence homology even between gene families, the expression of only the target gene can be specifically suppressed from the gene families.
[0044]
If the mechanism of the gene expression suppression method of the present invention requires the formation of dsRNA, the gene fragment preferably contains an exon part, more preferably an ORF part, as a partial sequence of the target gene. preferable. Since the dsRNA fragment transcribed from the gene fragment containing the exon portion of the target gene is homologous to the target mRNA transcribed from the target gene, siRNA (small-interfering RNA) generated by the degradation of dsRNA by dicer is the target mRNA. Hybridize to. As a result, the target mRNA is degraded, so that the expression of the target gene can be suppressed.
[0045]
The length (number) of the base sequence is not particularly limited as long as the gene fragment consists of a partial sequence of the target gene and the expression of the target gene can be suppressed by introduction into the host. In general, the length of dsRNA used for RNA interference is a polynucleotide of about 200 bases. Therefore, the gene fragment may also be a polynucleotide of about 200 bases.
[0046]
In addition, in RNA interference, a short-chain RNA (siRNA) of about 20 to 30 bases decomposed from a dsRNA of about 200 bases is said to suppress the expression of the target gene.
[0047]
Therefore, the gene fragment only needs to be a partial sequence of at least 21 bases in the target gene. Thereby, the expression of a target gene can be suppressed efficiently.
[0048]
The gene fragment may be a modified form in which modification is performed so that one or more bases are substituted, deleted, inserted, and / or added as long as the expression of the target gene is suppressed. However, in the present invention, the homology between the gene fragment and the target gene is a very important factor. For this reason, when the said modification is used as said gene fragment, the expression suppression efficiency of a target gene may fall.
[0049]
Such a variant can be obtained by, for example, using a site-directed mutagenesis method, a method of introducing a point mutation into a base sequence using a PCR method, or the like in the base sequence of the target gene. It can be made by making modifications such that a base is substituted, deleted, inserted, and / or added.
[0050]
Further, the gene fragment may be chemically modified, for example, by labeling in order to easily confirm the suppression of expression of the target gene. However, care must be taken when chemically modifying the above gene fragment. This is because, in the case of conventional RNAi, chemical modification to the 3 ′ end on the antisense strand side of dsRNA does not affect the gene expression suppression effect, whereas the 5 ′ end on the antisense strand side of dsRNA is chemically modified. This is because, if modified, the gene expression suppression effect may be lost. It should be noted that chemical modification on the sense strand side of dsRNA does not affect the gene expression suppressing effect for both 5 ′ and 3 ′.
[0051]
The method for obtaining the gene fragment according to the present invention is not particularly limited, and a gene fragment comprising a partial sequence of the target gene can be obtained by various methods based on the sequence information of the target gene.
[0052]
For example, the gene fragment can be easily synthesized by the PCR method (or its modification method) using a partial sequence of the target gene as a primer. For example, in the base sequence of the target gene, primers are prepared from the 5 ′ side and 3 ′ side sequences, and using these primers, a gene containing a partial sequence of the target gene to be amplified (for example, target A large amount of the gene fragment according to the present invention can be obtained by performing PCR or the like using genomic DNA (or cDNA) having a gene or a fragment thereof as a template and amplifying a DNA region sandwiched between both primers.
[0053]
In other words, the starting material for obtaining the gene fragment is not necessarily double-stranded as long as it is DNA or RNA containing a partial sequence of the target gene, and may be single-stranded. This is because a double-stranded DNA (gene fragment) can be amplified using the single-stranded or double-stranded DNA or RNA as a template. If the template is RNA, PCR may be performed after reverse transcription to DNA (so-called RT-PCR).
[0054]
A gene fragment having a promoter sequence at the 5 ′ end can be obtained by ligating a promoter sequence to the 5 ′ end of the primer. When the gene fragment has a promoter sequence as in the examples described later, the efficiency of suppression of the target gene can be improved by introducing RNA polymerase into the host together with the gene fragment. Here, the “RNA polymerase” is not particularly limited, and various polymerases such as T7 RNA polymerase used in Examples described later can be used.
[0055]
The gene fragment can be obtained not only by chemically synthesizing by PCR but also by fragmenting genomic DNA or cDNA purified from cells or tissues of an organism by enzymatic treatment.
[0056]
The method for introducing the gene fragment into the host cell is not particularly limited, and can be introduced into the host cell by a known genetic engineering technique (gene manipulation technique). Specifically, it can be performed by, for example, a liposome method, an electroporation method (electroporation method), microinjection, particle gun, Agrobacterium, etc. used for RNA interference method.
[0057]
For example, if the host is a nematode, the gene fragment can also be introduced by mixing the gene fragment with food or immersing the nematode in a solution containing the gene fragment.
[0058]
The method for confirming suppression of expression of the target gene by introducing the gene fragment is not particularly limited. For example, as shown in the Examples described later, when a gene involved in chemiluminescence (luciferase) is suppressed, it can be confirmed by comparing the luciferase activity before and after the introduction of the gene fragment. In addition, for example, confirmation can also be made by the form of host cells, the amount of intracellular substances, the amount of substances secreted by cells, the dynamics of intracellular substances, cell-cell adhesion, cell movement, or cell behavior such as cell life. Is possible. In addition, when the function of the target gene is known in other cells, it is preferable to perform the trait related to the function. As a method for analyzing a change in a character, for example, when analyzing a change in a character of a cell, a method of detecting with a microscope or macroscopically can be used. In addition, when the substance to be confirmed is a gene, it can be confirmed by Northern Blot analysis, PCR, or in situ hybridization, and in the case of a protein, a method for measuring reactivity with an antibody or enzyme activity, etc. Is mentioned.
[0059]
As described above, the gene expression suppression method of the present invention introduces a gene fragment consisting of a partial sequence of a target gene, or the gene fragment and RNA polymerase into a host cell.
[0060]
In contrast, in the conventional RNA interference, by introducing double-stranded RNA or introducing a vector and expressing double-stranded RNA in the host cell, mRNA homologous to the RNA sequence is degraded, and the target gene is expressed. It suppresses. In other words, conventional RNA interference is one in which a double-stranded RNA homologous to mRNA transcribed from a target gene is introduced into a host cell or expressed from a vector.
[0061]
Therefore, the gene expression suppression method of the present invention is completely different from the conventional RNA interference method in that a gene fragment consisting of a partial sequence of a target gene is used. Since the gene expression suppression method of the present invention does not require the construction of a vector, it is a very simple gene expression suppression method that replaces the conventional RNA interference method.
[0062]
(2) Use of the gene expression method of the present invention
(1) Transformant of the present invention
The transformant of the present invention is a transformant in which the expression of a target gene is suppressed by the above gene expression suppression method. That is, the gene expression suppression method of the present invention can also be referred to as a method for producing the transformant of the present invention. In the transformant of the present invention, at least a part of the function of the target gene is suppressed by introducing the gene fragment according to the present invention.
[0063]
Here, the “transformant” is meant to include not only cells / tissues / organs into which the gene fragment has been introduced, but also animals / plant individuals. The target animal is not particularly limited, and examples thereof include mammals including humans such as cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice and rats. The individual also includes its progeny and clones, as well as propagation material for the progeny and clones.
[0064]
In particular, if humans are to be transformed, gene fragments with the gene as a target gene are administered to patients with various diseases caused by gene abnormalities such as gene overexpression and gene mutations. This makes it possible to treat these diseases. More specifically, since the gene fragment can suppress the expression of only the target gene (specific gene), for example, inhibition of virus growth, treatment of persistently infected virus, disease caused by point mutation of gene ( For example, medical applications such as treatment of cancer suppressor gene abnormality) are possible.
[0065]
If animals other than humans are targeted, experimental animals and pathological animals in which the expression of the target gene is suppressed can be produced. Rodents such as mice and rats are widely used as experimental animals and pathological model animals, and many inbred strains have been created, and mice that have techniques for culturing fertilized eggs, in vitro fertilization, etc. It is preferable as an experimental animal / pathological model animal, and knockout mice and the like are useful for further functional analysis of target genes and their homologues, development of diagnostic methods for diseases involving these target genes, and development of therapeutic methods thereof. .
[0066]
In the examples described below, transformants into which the above gene fragment has been introduced by the polyethylene glycol method are prepared using protoplasts of Arabidopsis mesophyll cells as hosts. The gene fragment introduced into the transformant is a partial sequence of a firefly-derived luciferase gene as a target sequence (positions 93 to 597 in the ORF of the firefly luciferase shown in SEQ ID NO: 1 (positions 93 to 597 downstream of the start codon). 505 bp)). In addition, it was confirmed that the transformant was introduced with an expression vector for the full-length gene of T7 RNA polymerase and firefly luciferase together with the gene fragment, and that the luciferase activity was significantly suppressed as compared with the control. Has been. This result shows that the transformant introduced with the gene fragment can suppress the expression of the target gene with high efficiency.
[0067]
The category of the plant includes not only a complete plant but also a part thereof, for example, a leaf, a seed, a tuber, a cutting, and the like. Furthermore, the above-mentioned plants include plant tissues, protoplasts, cells, calli, organs, plant seeds, germs, pollen, egg cells, zygotes, etc. that originate from genetically modified plants and their progeny that have been transformed beforehand. Materials; parts of plants including flowers, stems, fruits, leaves, roots, etc .; suspended cultured cells;
[0068]
Once a transformed plant having the gene fragment introduced thereinto is obtained, offspring can be obtained from the transformed plant by sexual reproduction or asexual reproduction. It is also possible to obtain propagation materials (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from transformed plants and their descendants or clones, and mass-produce transformed plants based on them. Is possible.
[0069]
In this way, a transformed plant can be produced in which the expression of the target gene is remarkably suppressed as compared with the wild-type plant.
[0070]
In addition, about the method of producing the said transformant, as shown in the method of introduce | transducing the gene fragment mentioned above, conventionally well-known various transformation methods can be used. For example, liposome method, polyethylene glycol method, electroporation method (electroporation method), microinjection, particle gun, Agrobacterium-mediated method, ballistic particle acceleration method, and protoplast transformation / regeneration method can be used. . These transformation methods are preferably selected as appropriate according to the type of host.
[0071]
The transformant contains a gene fragment in which the expression of the target gene is suppressed and the partial sequence of the target gene. On the other hand, the above gene fragment does not exist in the transformant produced by the conventional RNA interference method. Therefore, each transformant can be distinguished by confirming the presence or absence of the gene fragment.
[0072]
(2) Gene expression inhibitor and gene disease therapeutic agent of the present invention
As described above, the gene expression suppression method of the present invention can be used to produce a transformant in which the expression of a target gene is suppressed. In other words, the gene fragment consisting of the partial sequence of the target gene is a gene expression inhibitor for suppressing the expression of the target gene. This gene expression inhibitor only needs to contain at least the above gene fragment.
[0073]
When the gene expression inhibitor is administered to cells, tissues, and individuals of humans, mice, and rats and transformed, it is effective for diagnosis of a disease involving a target gene and development of a therapeutic method.
[0074]
Moreover, the medicine which uses the said gene expression inhibitor as an active ingredient can be utilized as a gene disease therapeutic agent. As described in (1) above, if this gene disease therapeutic agent is administered to a patient who has a genetic abnormality, the expression of the gene can be suppressed. For example, it is possible to suppress the expression of genes that are highly sensitive to environmental factors and cancer suppressor genes that have undergone mutation. Therefore, it is effective as a novel therapeutic agent (medicine) for diseases caused by genetic abnormalities such as allergic diseases and cancer. In addition, the administration method in using as a pharmaceutical is not specifically limited, such as oral and intravenous injection.
[0075]
(3) Functional analysis method of the gene of the present invention
The gene function analysis method of the present invention is a method of analyzing the function of a target gene using the gene expression suppression method of the present invention described above.
[0076]
Both proteins and RNA, which are biopolymers, are responsible for various functions in life activities as end products of DNA. Although it is said that there are about 40,000 human genes by genome analysis, less than half of them are known to function, and many genes are unknown.
[0077]
Therefore, it is important to clarify the function of such a function-unknown gene in order to understand the principle of life activity and to elucidate the relationship between the gene and the disease.
[0078]
As described above, when the gene fragment is introduced into a cell, the expression of the target gene is suppressed. Therefore, if a gene fragment is prepared using a gene whose base sequence is known but whose function is unknown as a target gene and then introduced into an appropriate host cell, only the expression of that gene is artificial. It is possible to produce a transformant that is suppressed in the above manner. As a result, the function of the function unknown gene can be identified.
[0079]
That is, the gene functional analysis method of the present invention is particularly effective for performing functional analysis of such a function-unknown gene. Since the gene function analysis method of the present invention uses the gene expression suppression method of the present invention, it can be carried out simply and in a short time. For this reason, it is also suitable for high-throughput screening for rapidly and comprehensively analyzing unknown genes. Therefore, the gene functional analysis method of the present invention is an effective approach for understanding life.
[0080]
Hereinafter, the method for analyzing the function of the gene of the present invention will be described with reference to an example of a method for analyzing the function of a gene whose function is unknown.
[0081]
The gene functional analysis method of the present invention comprises a step of introducing a gene fragment consisting of a partial sequence of a gene with unknown function (gene fragment introduction step) and a step of comparing the functions of the host before and after introduction of the gene fragment (comparison). Process).
[0082]
The gene fragment introduction step is a step of introducing a gene fragment into a host in the same manner as described above. Thereby, the host suppresses the function of the function unknown gene. In the gene fragment introduction step, it is preferable to suppress the function of the unknown function gene as much as possible, and it is more preferable to completely delete the function. For example, as the gene fragment, this makes it easy to estimate the function of a function-unknown gene in the subsequent comparison step.
[0083]
Subsequently, the comparison step is a step of comparing the degree of suppression of expression of the target gene before and after the gene fragment is introduced into the host. That is, the comparison step is a step of identifying the function of a function unknown gene.
[0084]
When the gene fragment is introduced in the gene fragment introduction step, the function of the unknown function gene in the host is suppressed. Therefore, after the gene fragment introduction step, in the same manner as the method for confirming the suppression of the expression of the target gene in the gene expression suppression method of the present invention described above, the expression of the unknown function gene is suppressed by a molecular biological or biochemical method. By confirming the above, the function of the function-unknown gene can be identified. That is, since a trait that appears only in a host into which a gene fragment of an unknown function gene has been introduced is associated with suppression of expression of the unknown function gene (target gene), the function of the target gene can be identified.
[0085]
The gene analysis method of the present invention is applied to a library of unknown functions whose base sequence is determined, such as a library consisting of exon fragments having ORFs contained in genomic DNA, a cDNA library derived from a specific tissue, or a By applying to a mixture, high-throughput screening that comprehensively analyzes all such function-unknown genes can be performed. Thereby, the gene which has the target function can be selected from many function unknown genes. Furthermore, the function of a function-unknown gene can also be identified.
[0086]
Thus, the function unknown gene whose function has been identified is a novel gene and may be used as the aforementioned gene expression inhibitor or gene disease therapeutic agent. The present invention includes genes whose functions have been newly clarified by the functional analysis method of the present invention. A gene whose function has been clarified is effective for diagnosis and treatment of a disease associated with the gene.
[0087]
(4) Kit of the present invention
The kit of the present invention is a kit for carrying out the method described in (1) or (2) above (1) or (3), and includes at least a gene fragment consisting of a partial sequence of a target gene and the gene expression suppression. Any one of these agents, the above-mentioned gene disease therapeutic agent, or a mixture thereof may be contained. Such a kit can be used for easily performing gene expression suppression methods, diagnosis of genetic diseases, prediction of therapeutic effects of therapeutic agents for genetic diseases, and the like. Therefore, the versatility of the present invention is improved by making a kit.
[0088]
The kit further includes reagents for cultured cells, media, enzymes, primers, buffers, etc., reagents for transformation, selective agents, and nucleic acid introduced from the cells to determine the nucleotide sequence. Primers, reagents and the like may be included.
[0089]
The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.
[0090]
【Example】
In the following examples, as an example of a method for effectively suppressing the expression of a target gene using a double-stranded DNA fragment, a partial sequence of a firefly luciferase gene or sGFP (S65T) gene is amplified by PCR, Firefly luciferase expression vector (reporter), Renilla luciferase (internal standard), and T7 RNA polymerase were introduced into the host Arabidopsis mesophyll cell protoplasts by the PEG method, and the gene expression inhibitory effect was observed by Dual luciferase assay It is. The present invention is not limited to the following examples.
[0091]
1. Plant material
Arabidopsis thaliana ecotype Columbia (purchased from Height Culture Co., Ltd.) cultivated at 22 ° C. and 60% humidity meteorological device (light period 12 hours, dark period 12 hours) was used.
[0092]
2. DNA fragment preparation
A DNA fragment containing a target sequence of about 500 bp was prepared by PCR using a primer having a T7 promoter added to the 5 ′ end. The primers used are shown below (underlined: T7 promoter).
Target: firefly luciferase (93 to 597 downstream of the start codon in the base sequence of the ORF of firefly luciferase shown in SEQ ID NO: 1: 505 bp)
Forward primer: 5′-TAATACGACTCACTATAGGGAGATACGCCCCTGGTTC-3 ′ (SEQ ID NO: 3)
Reverse primer: 5′-TAATACGACTCACTATAGGGAGAGGAGTTCATGATCAGTG-3 ′ (SEQ ID NO: 4)
Target: sGFP (S65T) (downstream of start codon 113 to 614 in the base sequence of ORF of sGFP (S65T) shown in SEQ ID NO: 2: 502 bp)
Forward primer: 5′-TAATACGACTCACTATAGGGCCCACCTACGGCAAGCTGAC-3 ′ (SEQ ID NO: 5)
Reverse primer: 5'-TAATACGACTCACTATAGGGGTGGGTGCTCAGGTAGTGTT-3 '(SEQ ID NO: 6)
In the primer sequences shown in SEQ ID NOs: 3 to 6, the 5 'terminal T (from thymine) to the 20th G (guanine) corresponds to the T7 promoter sequence.
[0093]
The PCR conditions were 30 cycles, with 94 ° C for 1 min, 52 ° C for 1 min, and 72 ° C for 1 min for one cycle. Next, the length of the PCR product was confirmed by 1.5% agarose gel electrophoresis, and then purified using phenol and chloroform.
[0094]
3. Preparation of dsRNA
Using the above DNA fragment as a template, dsRNA was prepared by in vitro transcription. In vitro transcription was performed using RiboMAX Large Scale RNA Production Systems-T7 (Promega) or T7 RiboMAX Express Large Scale Production Instruction. The RNA obtained by in vitro transcription was purified using phenol and chloroform, and the length was confirmed by 1.5% agarose gel electrophoresis.
[0095]
4). Preparation of mesophyll cell protoplasts
The mesophyll protoplasts were prepared according to the method developed by Sheen [Shen, 2002 # 511]. That is, Arabidopsis rosette leaves (1-2 cm: 10-20 sheets) 3 to 4 weeks after germination were cut into a width of about 0.5 to 1 mm with a scalpel blade and 10 ml of enzyme solution (1.5 % Cellulase Onozuka R10, 0.4% Macerozyme R10, 0.4 M Mannitol, 20 mM KCl, 10 mM CaCl ₂ , 0.1% BSA, 20 mM MES, pH 5.7). After performing deaeration under reduced pressure for 15 minutes in a desiccator, the mixture was gently shaken at 23 ° C. and 50 rpm for 90 minutes. After confirming that the protoplast was formed with a microscope, the protoplast solution was filtered with a nylon mesh, and the filtrate was collected in a 50 ml centrifuge tube. The supernatant was removed by centrifugation at 100 × g for 3 minutes at room temperature. Ice-cold W5 solution (154 mM NaCl, 125 mM CaCl ₂ , 5 mM KCl, 2 mM MES, pH 5.7) and gently mixed, and then centrifuged again under the above conditions to remove the supernatant. After adding an appropriate amount of ice-cold W5 solution and mixing gently, the cell density is calculated with a hemocytometer and 2 × 10 ⁵ The cells were diluted with an ice-cold W5 solution so as to be cells / ml. This protoplast solution was incubated in ice for 30 minutes and then centrifuged at 100 × g for 3 minutes at room temperature. After removing the supernatant, the cell density is 2 × 10 ⁵ ice-cold MMg solution (0.4 M Mannitol, 15 mM MgCl) ₂ , 4 mM MES, pH 5.7).
[0096]
5. Gene transfer to mesophyll protoplasts using PEG (polyethylene glycol)
Introduction of genes into mesophyll protoplasts using PEG was performed according to a method developed by Sheen [Shen, 2002 # 511]. First, 0.5 μg of firefly luciferase expression vector (reporter) (SEQ ID NO: 7) and 0.5 μg of Renilla luciferase expression vector (internal standard) (SEQ ID NO: 8) were added to a 1.5 ml microcentrifuge tube. . Subsequently, 5 μg of dsRNA or 5 μg of DNA fragment was added. Two samples to which DNA fragments were added were prepared, and 100 units of T7 RNA polymerase (Promega) was further added to one of the samples. To each of these tubes, 200 μL of protoplast solution was added, and PEG solution (40% PEG4000, 0.2 M Mannitol, 100 mM CaCl was added. ₂ 220 μL) and mixed well by gentle pipetting. After incubating at 23 ° C. for 30 minutes, 800 μL of W5 solution was added and gently mixed by inversion. After centrifugation at 100 × g for 3 minutes at room temperature, the supernatant was removed. After adding 100 μL of W5 solution and mixing well by gentle pipetting, it was transferred to a 24-well plate into which 1 ml of W5 solution had been dispensed in advance and mixed gently. Wrapped in aluminum foil and incubated at 22 ° C for 12-15 hours.
[0097]
In the firefly luciferase expression vector sequence shown in SEQ ID NO: 7, the 2433 to 2878th is the CaMV promoter, the 2914 to 4566th is the firefly luciferase, the 4656 to 4930th is the NOS terminator, the 2401 to 2406th is HindIII, and the 2427 to 2432 are BamH. I, 2879 to 2884th corresponds to Sal I, 4737 to 4644th corresponds to Not I, and 4931 to 4936th corresponds to EcoR I.
[0098]
In the Renilla luciferase expression vector shown in SEQ ID NO: 8, the 2433-2878th is a CaMV promoter, the 2896-3383th is a Renilla luciferase, the 3858-4132th is a NOS terminator, the 2401-2406th is HindIII, and 2427-2432. BamH I, 2879 to 2884th correspond to Sal I, 3839 to 3846th correspond to Not I, and 4133 to 4138th correspond to EcoR I.
[0099]
6). Dual luciferase assay
The protoplast solution was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 100 × g for 3 minutes at room temperature. After removing the supernatant, 100 ml of 1 × Passive Lysis buffer (Promega) was added. Cells were lysed by gentle vortexing and left at room temperature for 5 minutes. 10 ml of this cell lysate was set in a luminometer (Turner Designs TD-20 / 20 Luminometer), and the firefly and Renilla luciferase activities were measured. The efficiency of plasmid introduction in each assay was normalized by dividing the activity value of firefly luciferase by the activity value of Renilla luciferase.
[0100]
7. Experimental result
First, in order to confirm that RNAi can be observed in this assay system, dsRNA of about 500 bp was introduced. As a result, an expression suppression effect of 80% or more was observed (FIG. 2). From this result, it was shown that this assay system is applicable to the observation of RNAi.
[0101]
Next, when a DNA fragment amplified by PCR and T7 RNA polymerase were simultaneously introduced, an expression suppression effect of about 80% was observed (FIG. 3).
[0102]
On the other hand, an inhibitory effect of about 70% was also observed in the case of only the DNA fragment (without T7 RNA polymerase) (FIG. 4). From these results, it was found that the contribution rate of T7 RNA polymerase to silencing was about 10%, which was considerably smaller than the original expectation. On the other hand, the expression of the target gene was significantly increased only with the DNA fragment (about about 70%).
[0103]
【The invention's effect】
As described above, the gene expression suppression method of the present invention is applied to the conventional RNA interference method by introducing only a gene fragment consisting of a partial sequence of a target gene or the gene fragment and RNA polymerase into a host cell. It is characterized by having a comparable gene expression suppression effect.
[0104]
This method can be applied to, for example, high-throughput screening of genes with unknown functions as a method of reverse genetics that replaces the conventional method because of the ease of operation and high suppression efficiency, and is extremely useful in practice.
[0105]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of the mechanism of the gene expression suppression method of the present invention.
FIG. 2 is a graph showing the gene expression inhibitory effect when dsRNA is introduced into protoplasts in Examples of the present invention.
FIG. 3 is a graph showing the effect of suppressing gene expression when a DNA fragment and T7 RNA polymerase are simultaneously introduced into a protoplast in an example of the present invention.
FIG. 4 is a graph showing measurement results of luciferase activity when only DNA fragments are introduced into protoplasts in Examples of the present invention.

Claims (12)

A gene expression suppression method for suppressing the expression of a target gene by introducing a gene fragment comprising a partial sequence of the target gene into a host. A gene expression suppression method for suppressing expression of a target gene by introducing a gene fragment comprising a partial sequence of the target gene and RNA polymerase into a host. The gene expression suppression method according to claim 1 or 2, wherein the gene fragment has a promoter sequence at the 5 'end. The gene expression suppression method according to any one of claims 1 to 3, wherein the gene fragment is a double strand. The gene expression suppression method according to any one of claims 1 to 4, wherein the gene fragment is DNA. The gene expression suppression method according to any one of claims 1 to 5, wherein the gene fragment comprises at least 21 bases or base pairs. A transformant in which expression of a target gene is suppressed, produced using the gene expression suppression method according to claim 1. A gene function analysis method for analyzing the function of a target gene using the gene expression suppression method according to any one of claims 1 to 6. The target gene is a gene with unknown function consisting of a known base sequence,
Introducing a gene fragment consisting of a partial sequence of a target gene into a host;
The method for analyzing the function of a gene according to claim 8, further comprising a step of comparing the functions of the host before and after the introduction of the gene fragment. A gene expression inhibitor comprising a gene fragment consisting of a partial sequence of a target gene and suppressing the expression of the target gene. The gene disease therapeutic agent which uses the gene by which the function analysis method of the gene of Claim 9 was clarified, or the gene expression inhibitor of Claim 10 as an active ingredient. A kit used for the gene expression suppression method according to any one of claims 1 to 6, or the gene function analysis method according to claim 8 or 9,
A kit comprising any one of a gene fragment comprising a partial sequence of a target gene, the gene expression inhibitor according to claim 10, or the gene disease therapeutic agent according to claim 11.

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