JP6425161B2 - Projection pathway selective gene expression control - Google Patents

Description

  The present invention relates to gene expression regulation. More specifically, the present invention relates to a system that achieves projection pathway selective gene expression control using transsynaptic retrograde transport and its use.

  Neuronal cells have been classified based on various characteristics. At first it was classified anatomically. For example, it is classified into a midbrain substantia nigra pars compacta (SNc) and a new striatum (CPu). Later, in more detail, neurotransmitter classification became possible. For example, it is classified into dopamine nerve and serotonin nerve. The current mainstream is this neurotransmitter classification method. However, even neurons having the same neurotransmitter may have different physiological functions depending on the projection pathway. For example, the projection pathways are different between the dopamine nerve present in SNc and the dopamine nerve present in the ventral tegmental area (VTA), and the physiological functions are also different. If it is possible to selectively control the activity of neurons in the projection pathway, identification of neural circuits and elucidation of the circuit functions will proceed.

  By the way, conventionally, there have been gene expression control systems capable of inducing gene expression only in the presence of Cre recombinase (see, for example, non-patent documents 1 and 2). However, methods for inducing gene expression specifically in specific neural pathways have been limited. Specific neural pathway specific means that gene expression is caused only to neural pathways input to a certain neural cell. Rabies virus has been used for specific neural pathway specific gene expression. However, rabies virus is toxic and kills infected neurons. In addition, because rabies virus requires a special facility (P2A), its use is limited and it has not been widely used.

  Patent Document 1 shows a method of visualizing protein transport and active synapse by retrograde axonal transport using C-terminal fragment of tetanus toxin, and Patent Document 2 shows retrograde axis of rabies virus protein. A means of gene transfer into neurons using cord transport has been shown.

Japanese Patent Application Publication No. 2007-535316 JP, 2012-110290, A

J Neurosci. 2008 Jul 9; 28 (28): 7025-30. Doi: 10.1523 / JNEUROSCI. 1954-08. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. Atasoy D1, Aponte Y, Su HH, Sternson SM. Nat Biotechnol. 2003 May; 21 (5): 562-5. Epub 2003 Mar 31. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Schnutgen F1, Doerflinger N, Calleja C, Wendling O, Chambon P, Ghyselinck NB.

  If gene expression can be induced in a neural pathway specific (in other words, projection pathway selective) input to a certain nerve cell, it becomes possible to identify a neural circuit and elucidate the physiological function that the neural pathway bears. . Then, this invention makes it a subject to provide the system which can make a projection path | route selective express a gene, and its use.

While advancing researches in view of the above problems, the present inventors impart a function of transitioning to transsynaptic retrograde property to a site-specific DNA recombination enzyme (Cre recombinase) and also use an adeno-associated virus vector (AAV vector). We considered a strategy to use and introduce genes. Although Cre recombinase itself does not have a function to shift to transsynaptic retrograde, linking (fusion) a detoxified C-terminal fragment (TTC) of tetanus toxin having a function to shift to transsynaptic retrograde with Cre recombinase By using Cre recombinase (Cre-TTC), which has a function to shift to synaptic retrograde, projection pathway selective gene expression was tried. As a result of this test, according to this strategy, Cre recombinase is transferred to neurons (upstream neurons) that are directly input to neurons infected with the AVV vector carrying Cre-TTC, and there, Cre recombinase specific Gene expression was possible. That is, projection pathway selective gene expression was successful, and it was shown that the above strategy is extremely effective. The following inventions are mainly based on the result.
[1] A first adeno-associated virus vector expressing a gene encoding a ligation product of a site-specific DNA recombination enzyme and a tetanus toxin C-terminal fragment,
A second adeno-associated virus vector that expresses a target gene only in the presence of the site-specific DNA recombination enzyme,
, A gene expression system.
[2] The gene expression system according to [1], wherein the site-specific DNA recombination enzyme is Cre recombinase.
[3] The gene expression system according to [2], wherein the Cre recombinase comprises the sequence shown in SEQ ID NO: 5.
[4] The gene expression system according to any one of [1] to [3], wherein the tetanus toxin C-terminal fragment comprises the sequence shown in SEQ ID NO: 7.
[5] The gene expression system according to any one of [1] to [4], wherein the conjugate contains a nuclear localization signal peptide.
[6] The first adeno-associated virus vector according to any one of [1] to [5], further comprising a reporter gene in an expressible state in addition to the gene encoding the ligation product. Gene expression system.
[7] The gene expression according to [6], wherein the first adeno-associated virus vector has a sequence structure in which the reporter gene and a gene encoding the ligation are linked via a sequence encoding a self-cleaving peptide. system.
[8] The gene expression system according to [6] or [7], wherein the reporter gene is a fluorescent protein gene.
[9] The second adeno-associated virus vector comprises an LSL (LoxP-STOP-LoxP) sequence or an FSF (FRT-STOP-FRT) sequence, and the target gene is disposed downstream of the sequence [1] The gene expression system according to any one of [8].
[10] The gene expression system according to any one of [1] to [8], wherein the second adeno-associated virus vector comprises a FLEX (Flip-Excision) switch incorporating the target gene.
[11] The gene expression system according to any one of [1] to [10], wherein the second adeno-associated virus vector holds a reporter gene in an expressible state in addition to the target gene.
[12] The gene expression system according to [11], wherein the second adeno-associated virus vector comprises a sequence structure in which the reporter gene and the target gene are linked via a sequence encoding a self-cleaving peptide.
[13] The second adeno-associated virus vector comprises an LSL (LoxP-STOP-LoxP) sequence or an FSF (FRT-STOP-FRT) sequence, the sequence structure being disposed downstream of the sequence [12] The gene expression system described in.
[14] The gene expression system according to [12], wherein the second adeno-associated virus vector comprises a FLEX (Flip-Excision) switch incorporating the sequence structure.
[15] The gene expression system according to any one of [11] to [14], wherein the porter gene is a fluorescent protein gene.
[16] A gene expression method using the gene expression system according to any one of [1] to [15],
Infecting neural cells present in the first region with the first adeno-associated virus vector;
Infecting neurons present in a region projecting to the first region with the second adeno-associated virus vector;
Gene expression methods including:
[17] infecting neurons present in the first region with an adeno-associated virus vector expressing a gene encoding a conjugate of Cre recombinase and tetanus toxin C-terminal fragment;
Infecting neurons present in the second region with an adeno-associated virus vector expressing a gene encoding a conjugate of FLP recombinase and tetanus toxin C-terminal fragment;
An adeno-associated virus vector that expresses a first gene of interest only in the presence of Cre recombinase, and an adeno-associated virus vector that expresses a second gene of interest only in the presence of FLP recombinase, said first region and said second region Infecting neurons present in the area to be projected;
Gene expression methods including:
[18] A ligation of the site-specific DNA recombination enzyme with the tetanus toxin C-terminal fragment to a nerve cell present in a specific region of a genetically modified animal that expresses the target gene only in the presence of the site-specific DNA recombination enzyme Infecting an adeno-associated virus vector that expresses a gene encoding a body.
[19] [1] A kit for constructing a system according to
A first adeno-associated virus vector plasmid carrying a gene encoding a conjugate of a site-specific DNA recombination enzyme and a tetanus toxin C-terminal fragment,
A second adeno-associated virus vector plasmid, which retains a target gene in a state capable of expression only in the presence of the site-specific DNA recombination enzyme,
Kit.

Description of site-specific DNA recombination enzyme (Cre) dependent gene expression. LSL: LoxP-STOP-LoxP (left) and FLEX: Flip-Excision-Excision (right) are used to express the target gene (channelrhodopsin (ChR2) in this figure) in a Cre-dependent manner. In LSL, site-specific recombination by Cre recombinase occurs, a translational stop signal (STOP) is cut out, and a target gene is expressed. In FLEX, when loxP and lox2272 are in the opposite direction, the inserted sequence is reversed (reversible reaction. (1), (2)), and when loxP and lox2272 are in the same direction, the inserted sequence is cut out (Irreversible reaction. (3) (4)). Example of projection pathway selective gene expression by multiple selection. By using two types of site-specific DNA recombination enzymes (Cre recombinase, FLP recombinase (FLPe)), cell-specific gene expression that is input to the A region and B region becomes possible. Transsynaptic retrograde transport of Cre recombinase using tetanus toxin C-terminal fragment (TTC). Cre recombinase (Cre-TTC) linked to TTC is transported to the upstream neurons by the transsynaptic retrograde transport function of tetanus toxin. Cre-dependent gene expression using the FLEX switch. Site-specific recombination occurs only in the presence of Cre recombinase (the destination of Cre-TTC), and hrGFP is expressed. Structure of Cre-TTC expressing adeno-associated virus vector (AAV-CMV-mCherry-2A-Cre-TTC). A sequence encoding mCherry (SEQ ID NO: 1) (SEQ ID NO: 1), a sequence encoding 2A peptide (SEQ ID NO: 3) under the control of CMV promoter (SEQ ID NO: 4), a sequence encoding Cre recombinase (SEQ ID NO: 5) (SEQ ID NO: 6), a sequence (SEQ ID NO: 8) encoding tetanus toxin C-terminal fragment (TTC) (SEQ ID NO: 7) is arranged in order from the upstream side. Structure of Cre-dependently expressed adeno-associated virus vector (see AAV-CMV-FLEX-hrGFP). Under the control of the CMV promoter, FLEX composed of a combination of the loxP sequence (SEQ ID NO: 9) and the lox 2272 sequence (SEQ ID NO: 10) is arranged. In the FLEX, a sequence (SEQ ID NO: 12) encoding hrGFP (SEQ ID NO: 11) is incorporated in an inverted state. Manipulation of viral vectors. A region for local injection of AAV-CMV-mCherry-2A-Cre-TTC (new striatum (CPu)) and a region for local injection of AAV-CMV-FLEX-hrGFP (midbrain substantia nigra pars compacta (SNc)) Indicates Manipulation of viral vectors. Indicate the region for local injection of AAV-CMV-mCherry-2A-Cre-TTC (the nucleus accumbens nucleus (NAc)) and the region for local injection of AAV-CMV-FLEX-hrGFP (the ventral tegmental area (VTA)) . Manipulation of viral vectors. The region for local injection of AAV-CMV-mCherry-2A-Cre-TTC (lateral hypothalamus (LHA)) and the region for local injection of AAV-CMV-FLEX-hrGFP (amygdala (CeA)) are shown. Results of fluorescence detection. In sections where neostriatum (CPu) is present, red fluorescence is observed (upper left). The red fluorescent area shows the infected area of AAV-CMV-mCherry-2A-Cre-TTC, in which Cre-TTC is expressed (coronary, scale bar is 1 mm). On the other hand, it is observed that green fluorescence is emitted in the section in which the mesenteric substantia nigra pars compacta (SNc) is present (lower right). hrGFP is selectively expressed in neural pathways that project to CPu (coronary, scale bar is 1 mm). Results of fluorescence detection. In sections where the nucleus accumbens (NAc) are present, it is observed that red fluorescence is emitted (upper left). The red fluorescent area shows the infected area of AAV-CMV-mCherry-2A-Cre-TTC, in which Cre-TTC is expressed (coronary, scale bar is 1 mm). On the other hand, in the section in which the ventral tegmental area (VTA) is present, it is observed that green fluorescence is emitted (lower right). hrGFP is selectively expressed in neural pathways that project to NAc (coronary, scale bar is 1 mm). Results of fluorescence detection. In sections where the lateral hypothalamic area (LHA) is present, red fluorescence is observed (upper left). The red fluorescent area shows the infected area of AAV-CMV-mCherry-2A-Cre-TTC, in which Cre-TTC is expressed (coronary, scale bar is 1 mm). On the other hand, it is observed that green fluorescence is emitted in the section in which the amygdala (CeA) is present (lower right). hrGFP is selectively expressed in the neural pathway that projects to LHA (coronary, scale bar is 1 mm).

1. Projection pathway selective gene expression system The first aspect of the present invention relates to a gene expression system. According to the gene expression system of the present invention, it is possible to selectively express a specific gene (target gene) in a projection pathway. Specifically, neural cells (one-stage upstream neural cells) specific gene expression that allows direct input to specific neural cells is possible. The gene expression system of the present invention is roughly divided into two elements, ie, a first adeno-associated virus vector that expresses a gene encoding a combination of a site-specific DNA recombination enzyme and a tetanus toxin C-terminal fragment, and the site-specific And a second adeno-associated virus vector that expresses the target gene only in the presence of the DNA recombination enzyme.

(1) First Adeno-Associated Viral Vector The first Adeno-Associated Viral Vector supplies a site-specific DNA recombination enzyme that transtranslates retrogradely. When gene expression is performed using the system of the present invention, the neural cells that receive input (neurons present in the projection area) are infected with the vector. The first adeno-associated virus vector retains a gene encoding a ligation product of a site-specific DNA recombination enzyme and a tetanus toxin C-terminal fragment (hereinafter referred to as "a ligation gene") in an expressible state. The “site-specific DNA recombination enzyme” in the present invention is foreign to the target cell (ie, an enzyme that is not originally present in the target cell), recognizes a specific DNA sequence, and is recombined Site specific recombination reaction). The site-specific DNA recombination enzyme is not particularly limited, and for example, Cre recombinase or FLP recombinase (flipase) can be used. It is recommended to use a site-specific DNA recombination enzyme whose sequence has been modified to function well in mammals. As an example of such a site-specific DNA recombination enzyme subjected to sequence optimization, Cre recombinase having the amino acid sequence shown in SEQ ID NO: 5 can be mentioned.

  Tetanus toxin C-terminal fragment (TTC) is part of the heavy chain of tetanus toxin and is non-toxic. The tetanus toxin C-terminal fragment is transported from the postsynaptic cell over the synapse to the presynaptic cell. That is, it is transported in the opposite direction (transsynaptic retrograde) to the projection of the nerve. An example of the amino acid sequence of TTC is shown in SEQ ID NO: 7. The amino acid sequence is subjected to sequence modification so as to function well in mammals.

  In the ligated gene, a sequence encoding a site-specific DNA recombination enzyme and a sequence encoding a tetanus toxin C-terminal fragment are linked in frame. Preferably, the two sequences are linked such that the site-specific DNA recombination enzyme is located upstream (5 '). Another sequence (typically, a spacer) may be interposed between the two sequences.

  The first adeno-associated virus vector contains a promoter to retain the ligation gene in an expressible manner. The ligation gene is under the control of a promoter. Examples of promoters are CMV promoter, SV40 late promoter, retrovirus LTR (long terminal repeat element), CAG promoter, EF1a promoter, synapsin promoter and the like. The expression may be regulated using a Tet operator (TetO) sequence. In addition, the first adeno-associated virus vector may carry regulatory elements (enhancer, terminator, etc.) other than the promoter.

  In a preferred embodiment, the conjugate comprises a nuclear localization signal peptide. The nuclear localization signal peptide is responsible for the specificity (selectivity) of the system of the present invention. That is, by using a nuclear localization signal peptide, since Cre is transferred to the nucleus and localized, the uptake into the secondary nerve is extremely suppressed, and only the primary nerve acts. The nuclear localization signal peptide is, for example, incorporated into the amino acid sequence of Cre recombinase.

  In order to increase the stability of the transcript, an intron (eg, β-globin intron) may be placed upstream of the ligation gene, or a WCH (woodchuck hepatitis post-transcriptional regulatory element) sequence may be placed downstream of the ligation gene. . Also, usually, a poly A addition signal sequence is arranged downstream of the ligation gene. Transcription is terminated by the use of a poly A addition signal sequence.

The first adeno-associated viral vector may hold the reporter gene in an expressible state, in addition to the ligation gene. When the first adeno-associated virus vector having such a configuration is used, infection and gene transfer of the first adeno-associated virus vector to the target cell can be confirmed using the expression of the reporter gene as an index. Examples of reporter genes include fluorescent protein gene, luciferase (LUC) gene, β-galactosidase (lacZ) gene and the like . Among them, the fluorescent protein gene is particularly preferable because it has advantages such as high sensitivity detection, live imaging, and simultaneous labeling with multiple wavelengths. Various fluorescent protein genes are available, and specific examples thereof are GFP, hrGFP (Agilent), mCherry (Clontech), mKate 2 (Wako).

  The reporter gene is linked to the linked gene, for example, via a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is a 2A peptide derived from Threea asigna virus, but is not limited thereto. As self-cleavage peptides, 2A peptide (F2A) derived from epidemic virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), 2A peptide (P2A) derived from porcine teschovirus (PTV-1), etc. are known. It is done.

(2) Second Adeno-Associated Virus Vector The second adeno-associated virus vector expresses a target gene only in the presence of the site-specific DNA recombination enzyme. In order to exert this function, the second adeno-associated virus vector retains a target gene as well as a recognition sequence corresponding to the site-specific DNA recombination enzyme to be used. For example, when Cre recombinase is employed as a site-specific DNA recombination enzyme, loxP sequences or mutant lox sequences (lox7, lox66, lox511, lox2272, etc.) are used. Similarly, when FLP recombinase is employed, FRT sequences or mutant FRT sequences (FRTG, FRTH, FRTF3, etc.) are used.

  In order to express the target gene only in the presence of a site-specific DNA recombination enzyme, for example, LSL (LoxP-STOP-LoxP) sequence or FSF (FRT-STOP-FRT) sequence can be used. The former is used when Cre recombinase is adopted as a site-specific DNA recombination enzyme, and the latter is used when FLP recombinase is adopted as a site-specific DNA recombination enzyme. When these sequences are used, the gene of interest will be located downstream (3 ') of the LSL or FSF sequence. Using these sequences, as shown in FIG. 1 (in the case of LSL sequence as a representative), the sequence (STOP) flanked by LoxP sequences is excised only when a site-specific recombination enzyme (Cre recombinase) is present. The target gene (ChR2) is expressed.

  In another aspect, a FLEX (Flip-Excision) switch is used. The FLEX (Flip-Excision) switch is a combination of two recognition sequences, and the target gene is integrated in the reverse direction (see FIG. 1). FIG. 1 shows the configuration of a FLEX switch when Cre recombinase is employed as a site-specific DNA recombination enzyme. In this example, two types of lox sequences (loxP and lox2272) are used, and from the 5 'upstream side to the 3' downstream side, forward loxP, forward lox2272, inverted target gene (ChR2), reverse Direction loxP and reverse direction lox 2272 are arranged in order. The presence of a site-specific recombination enzyme (Cre recombinase) results in two steps of recombination, which results in reverse expression of the gene of interest. Note that, instead of loxP and lox2272, a dFRT switch (the target gene is inverted and expressed) using FRT and F3 sequences that are recognition sequences of FLP recombinase can also be used.

  The target gene is a gene to be expressed in cells infected with the second adeno-associated virus vector (neurons directly input to cells infected with the first adeno-associated virus vector). Various genes can be adopted as target genes. For example, genes that enable live imaging (eg, GFP, luciferase, cameleon, GCaMP), genes useful for histological analysis (eg, β-galactosidase, alkaline phosphatase), genes that control cellular activity (eg, DREADD, ChR2, Arch, tetanus toxin (TeNT), diphtheria toxin A fragment (DTA)), genes of unknown function can be used as target genes.

  The second adeno-associated virus vector contains a promoter necessary for expression of the gene of interest. When recombination with a site-specific DNA recombination enzyme occurs, the target gene is under the control of a promoter and is expressed. Examples of promoters are the same as in the case of the first adeno-associated virus vector. In addition, the second adeno-associated virus vector may carry regulatory elements other than the promoter (eg, an enhancer, a terminator, etc.).

  As in the case of the first adeno-associated virus vector, an intron (eg, β-globin intron) is placed upstream of the gene of interest, or a WPRE sequence is placed downstream of the gene of interest to increase the stability of the transcript. It is good to do. In addition, usually, a poly A addition signal sequence necessary for the termination of transcription is disposed downstream of the target gene.

  In addition to the target gene, the second adeno-associated virus vector may hold the reporter gene in an expressible state. Using the second adeno-associated virus vector of such a configuration, infection and gene transfer of the second adeno-associated virus vector to the target cell can be confirmed using the expression of the reporter gene as an index. Examples of reporter genes are the same as in the first adeno-associated virus vector. If a reporter gene different from the reporter gene used for the first adeno-associated virus vector is used for the second adeno-associated virus vector, cells infected with the first adeno-associated virus vector and cells infected with the second adeno-associated virus vector It can be detected while being distinguished. For example, if a fluorescent protein gene of a specific fluorescent color is used for the first adeno-associated virus vector and a fluorescent protein gene of a different fluorescent color is used for the second adeno-associated virus vector, the infected cells are distinguished by two-color fluorescence Can be labeled.

  The reporter gene is linked to the gene of interest via, for example, a sequence encoding a self-cleaving peptide (such as 2A peptide). The sequence structure in which the reporter gene and the target gene are linked is, for example, located downstream of the LSL sequence or FSF sequence, or incorporated into the FLEX switch, and can be expressed only in the presence of a site-specific DNA recombination enzyme It will be in the state.

The first adeno-associated virus vector and the second adeno-associated virus vector can be prepared according to a method described in the art or using a commercially available dedicated kit or the like. A typical preparation method is as follows. First, a plasmid in which an expression cassette is disposed so as to be sandwiched by ITRs (terminal inverted sequences) derived from adeno-associated virus (AAV vector plasmid) is prepared. On the other hand, a plasmid (capsid plasmid) that expresses a Rep gene (a gene encoding a replication protein) and a Cap gene (a gene encoding a virus capsid protein), and each gene of adenovirus genes E2A, E4 and VA are expressed. Prepare a plasmid (helper plasmid) to Next, these three plasmids are cotransfected into packaging cells (for example, HEK 293 cells) expressing the E1 gene. Adeno-associated virus particles (the first adeno-associated virus vector or the second adeno-associated virus vector) are recovered from the cells after transfection. Such a method can be performed using a dedicated kit (eg, AAV helper free system (Agilent Technology), AAV helper free expression system (Cell Biolabs), etc. ).

  The serotypes of the first and second adeno-associated virus vectors are not particularly limited. For example, AAV type 1 (Sr1), AAV type 2 (Sr2), AAV type 5 (Sr5), AAV type 6 (Sr6), AAV type 7 Each vector can be constructed as (Sr7), AAV8 (Sr8), AAV9 (Sr9), AAV10 (Sr10), AAVDJ (SrDJ), and AAVDJ8 (SrDJ8). On the other hand, new serotypes of adeno-associated virus vectors provided with hybrid cupsids can be prepared by DNA family shuffle (for example, AAV helper free expression system (Cell Biolabs) can be used). It is also possible to construct a first adeno-associated virus vector and a second adeno-associated virus vector as such new serotypes of adeno-associated virus vectors. The serotype of the adeno-associated virus vector is determined by the type of Cap gene.

2. Gene Expression Method The second aspect of the present invention provides a gene expression method using the gene expression system of the present invention. In the gene expression method of the present invention, the first adeno-associated virus vector is used to infect nerve cells present in a specific region (also referred to as “first region” or “projected region” for convenience of explanation) (step 1) Infect the nerve cells present in the area projecting to the first area (ie, the nerve cells directly input to the nerve cells infected with the first adeno-related virus vector) (step 2) ).

  As a result of step 1, in the infected cells present in the first region, a ligation product of a site-specific DNA recombination enzyme and a tetanus toxin C fragment is expressed from the first adeno-associated virus vector. When the first adeno-associated virus vector carries a reporter gene, the reporter gene is also expressed, and the presence or absence of infection and the presence or absence of gene expression can be confirmed. The above-mentioned connector is transported transversally retrogradely by the function of the tetanus toxin C fragment, and is present in the area projecting to the nerve cell (upstream nerve cell) directly inputting to the infected cell (ie, the first area) It is taken into cells. As a result, a cell-specific site-specific DNA recombination enzyme is directly expressed in the cells infected with the first adeno-associated virus vector.

  On the other hand, the second adeno-associated virus vector infects the nerve cells present in the area projected to the first area by the step 2, and the nerve cells express the target gene only in the presence of the site-specific DNA recombination enzyme. It will be in the state of Therefore, among the neurons infected with the second adeno-associated virus vector, those directly inputted to the cells infected with the first adeno-associated virus vector cause recombination by the action of the site-specific DNA recombination enzyme. , The target gene is expressed. Thus, by performing steps 1 and 2, the target gene can be expressed specifically in nerve cells directly input to the nerve cells present in the first region. That is, it becomes possible to induce gene expression selectively in the projection pathway. For example, if a gene that activates nerve cells is adopted as a target gene, only a specific neural pathway can be activated. Conversely, if a gene that promotes inactivation (suppression) of nerve cells is the target gene, a specific neural pathway can be inactivated. In addition, when a fluorescent protein gene or the like that is useful for labeling is employed, a specific neural pathway can be labeled, which enables identification or identification of the neural pathway.

  In a cell directly input to a cell infected with the first adeno-associated virus vector, the target gene is expressed only in the state where the site-specific DNA recombination enzyme is incorporated and in the presence of the site-specific DNA recombination enzyme The order of step 1 and step 2 does not matter as long as the states are simultaneously formed. However, in consideration of the time required for transport of a conjugate of a site-specific DNA recombinant enzyme and a tetanus toxin detoxified C fragment, it is preferable to precede the infection of the first adeno-associated virus vector, ie, step 1. Steps 1 and 2 can be carried out by injection, dropping, etc. of the vector solution to the target region (first region in step 1, region projected onto the first region in step 2).

  Examples of the first region infecting the first adeno-associated virus vector are neostriatum (CPu), nucleus accumbens (NAc), and the lateral hypothalamic area (LHA). The area infected with the second adeno-associated virus vector is, as described above, the area projected onto the first area, which depends on the first area to be adopted. When the first region is a neostriatum (CPu), for example, a midbrain substantia nigra pars compacta (SNc) is infected with a second adeno-associated virus vector. Similarly, when the first region is the nucleus accumbens (NAc), for example, the ventral tegmental area (VTA), and when the first region is the hypothalamic lateral area (LHA), for example, the amygdala (CeA) To infect a second adeno-associated virus vector.

  Basically, the gene expression method of the present invention is applied to a living body, but it is also possible to apply the gene expression method of the present invention to tissues or organs in a state of being removed from the living body. Examples of living organisms here are non-human mammals (mice, rats, guinea pigs, hamsters, small primates (marmosets etc.), monkeys, chimpanzees).

  The present invention further provides a method of specifically expressing a gene in a neural cell input in two regions by using two types of site-specific DNA recombination enzymes in combination. In this method, a neuron associated with an adeno-associated virus vector (referred to as "Cre-first adeno-associated virus vector") expressing a gene encoding a conjugate of Cre recombinase and a tetanus toxin C fragment is present in the first region (Step I), and an adeno-associated virus vector (referred to as "FLP-first adeno-associated virus vector") expressing a gene encoding a conjugate of FLP recombinase and tetanus toxin C fragment is (B) infecting neurons present in the region (Step II), and an adeno-associated virus vector (referred to as "Cre-second adeno-associated virus vector") which expresses the first target gene only in the presence of Cre recombinase, Adeno-associated virus vector ("FLP-second adeno-associated," the second target gene is expressed only in the presence of FLP recombinase The virus is referred to as a vector "), the step of infecting (step III) in neurons present in the region to be projected in the first region and the second region. According to this method (see FIG. 2), the first target gene and the second target gene are expressed in neural cells that are input to two regions (A region and B region in FIG. 2). That is, gene expression can be made selective for the projection pathway by multiple selection. If the second target gene is one that functions in cooperation with the first target gene (for example, a gene encoding an expression product that functions by forming a complex with the expression product of the first target gene), it is more complicated. Or complex genetic manipulation is possible. In addition, by using two types of fluorescent protein genes, it is possible to specifically label neural cells entering two specific regions with two types of fluorescent light, and more detailed information on neural pathways Is brought about. Here, in place of the Cre-second adeno-associated virus vector and the FLP-second adeno-associated virus vector, an expression cassette capable of expressing the first target gene only in the presence of Cre recombinase, and in the presence of the FLP recombinase It is also possible to use an adeno-associated virus vector carrying both of the expression cassettes that allow expression of the second gene of interest only.

  By the way, at present, a genetically modified animal (TG animal) that expresses a target gene only in the presence of a site-specific DNA recombination enzyme is in an available environment. In such TG animals, an expression cassette using a recognition sequence of a site-specific DNA recombination enzyme has been introduced. In a further aspect of the present invention, a gene expression method using the TG animal is provided. Specifically, it corresponds to a specific region of a genetically modified animal that expresses a target gene only in the presence of a DNA recombination enzyme (corresponding to the above-mentioned first region, eg, neostriatum (CPu), nucleus accumbens (NAc) Neurons present in the lateral hypothalamic area (LHA) are infected with an adeno-associated virus vector that expresses a gene encoding a conjugate of a site-specific DNA recombination enzyme and a tetanus toxin C fragment. A genetically modified animal that expresses a target gene only in the presence of a site-specific DNA recombination enzyme can be produced according to a conventional method. Also, corresponding TG animals are commercially available, and commercially available TG animals may be used.

3. Kits The invention also applies to the kits used to construct the system of the invention. The kit of the present invention comprises only a first adeno-associated virus vector plasmid carrying a gene encoding a ligation of a site-specific DNA recombination enzyme and a tetanus toxin C fragment, and only in the presence of a site-specific DNA recombination enzyme A second adeno-associated virus vector carrying the target gene in an expressible state is included. Examples of other elements that can be included in the kit include helper plasmids, capsid plasmids, packaged cells, and various reagents, devices, devices, etc. necessary for each operation and reaction (transfection, culture, recovery, etc.) . In addition, the instruction manual is usually attached to the kit of the present invention.

1. Aim: We aimed to develop a system to realize projection pathway selective gene expression control. Although Cre recombinase itself does not have the function to transfer to transsynaptic retrograde, Cre is transferred to transsynaptic retrograde by binding to a detoxified C-terminal fragment of tetanus toxin having a function to transfer to transsynaptic retrograde. Recombinase (Cre-TTC). Cre-TTC was introduced into neurons in a specific area using an adeno-associated virus vector (AVV). As a result, Cre recombinase is taken up by neurons (upstream neurons) that are directly input to the neurons (FIG. 3). The pathway specific gene expression was attempted by infecting another AAV performing gene expression in a Cre-dependent manner in the region where the cell body of the upstream neural cell exists. In order to distinguish neurons that express Cre by AAV infection and neurons that shift to function (upstream neurons), it was decided to express red fluorescent protein simultaneously with Cre-TTC. Neurons infected with AAV express both Cre-TTC and red fluorescent protein, resulting in red fluorescence. On the other hand, in the nerve cells (upstream nerve cells) in which Cre-TTC has migrated and functioned, red fluorescence is not seen, but fluorescence due to Cre-dependent gene expression (we decided to use green fluorescent protein) was observed (Figure 4).

2. Methods (1) Preparation of Cre-TTC Expressing Adeno-Associated Virus Vector (AAV-CMV-mCherry-2A-Cre-TTC, See FIG. 5)
Adeno-associated virus vectors expressing Cre-TTC and red fluorescent protein mCherry were constructed in the following manner. First, XhoI-koz-mCherry (-STP) -SalI was amplified by PCR using pmCherry-N1 (Clontech) as a template, and subcloned into pCR4 (Invitrogen) (pCR4-mCherry). On the other hand, pAAV-MCS (Agilent) was cleaved with SalI at MCS. In pAAV-MCS, ITR (terminal inverted sequence), CMV promoter, β-globin intron, MCS (multi cloning site), hGH poly A, ITR are arranged in order from the upstream side (5 'side).

  XhoI-koz-mCherry (-STP) -SalI was excised from pCR4-mCherry and ligated to pAAV-MCS after digestion with SalI. The obtained pAAV-CMV-mCherry (-STP) was digested with SalI and BglII, and SalI-2A-Cre-TTC-BglII excised with SalI and BglII from the plasmid into which the artificial gene (2A-Cre-TTC) was inserted By ligation, pAAV-CMV-mCherry-2A-Cre-TTC was obtained.

HEK293 cells were transfected with pAAV-CMV-mCherry-2A-Cre-TTC, helper plasmid (Agilent) and capsid plasmid (U. Penn vector core). After culture, virus particles (AAV-CMV-mCherry-2A-Cre-TTC) were recovered by freeze-thaw treatment (2 × 10 ¹² copies / ml).

  AAV-CMV-mCherry-2A-Cre-TTC carries a CMV (cytomegalovirus) promoter and can be expressed in a wide variety of cell types. In infected cells, mCherry that emits red fluorescence is expressed. TTC (tetanus toxin C fragment) only has a transsynaptic retrograde transport function of tetanus toxin. By linking TTCs, Cre recombinase (a protein not present in wild type mice) is transsynaptically transported to upstream neurons having synapses directly in infected cells.

(2) Preparation of Cre-dependently Expressed Adeno-Associated Virus Vector (AAV-CMV-FLEX-hrGFP See FIG. 6)
Adeno-associated virus vectors expressing the green fluorescent protein hrGFP in a Cre-dependent manner were constructed in the following manner. First, SalI-koz-hrGFP-XhoI was amplified by PCR using pAAV-hrGFP (Agilent) as a template, and subcloned into pCR4 (Invitrogen) (pCR4-hrGFP). On the other hand, pAAV-CMV is obtained by ligating EcoRI-MCS1-FLEX-MCS2-BglII cut out with EcoRlI and BglII from a plasmid in which an artificial gene (TetO-MCS1-FLEX-MCS2) is inserted into pAAV-MCS (Agilent). -MCS1-FLEX-MCS2 was prepared and cleaved with XhoI at MCS2. In pAAV-CMV-MCS1-FLEX-MCS2, ITR, CMV promoter, β-globin intron, MCS1 (multicloning site 1), MSC2 (multicloning site 2) were incorporated sequentially from the upstream side (5 'side) A FLEX switch, hGH poly A, ITR are arranged.

  SalI-koz-hrGFP-XhoI was excised from pCR4-hrGFP and ligated to pAAV-CMV-MCS1-FLEX-MCS2 after cleavage with XhoI to give pAAV-CMV-FLEX-hrGFP.

HEK 293 cells were transfected with pAAV-CMV-FLEX-hrGFP, helper plasmid (Agilent) and capsid plasmid (Upenn vector core). After culture, virus particles (AAV-CMV-FLEX-hrGFP) were recovered by freeze-thaw treatment (2 × 10 ¹² copies / ml).

  AAV-CMV-FLEX-hrGFP carries a CMV promoter and can be expressed in a wide variety of cell types. In this viral vector, the gene for hrGFP, which emits green fluorescence, is arranged in the opposite direction while being sandwiched between FLEX sequences (combination of loxP and lox2272 sequences) (FIG. 4). Thus, infected cells of wild type mice (in the absence of Cre recombinase) do not express hrGFP. On the other hand, when Cre recombinase is present, gene recombination occurs selectively in the FLEX sequence, the hrGFP sequence is recombined in the correct direction, and the hrGFP gene is expressed (FIG. 4).

(3) Local injection of AAV vector and immunostaining Adeno-associated virus (AAV) vector in a specific region in the brain of 8 to 10 week old wild type mice (C57BL / 6J) using a brain stereotaxic apparatus Was injected locally. The injection site of the AVV vector was as follows.
(a) Local injection of AAV-CMV-mCherry-2A-Cre-TTC into neostriatum (CPu) and local injection of AAV-CMV-FLEX-hrGFP into midbrain substantia nigra pars compacta (SNc) (Figure) 7).
(b) Local injection of AAV-CMV-mCherry-2A-Cre-TTC into the nucleus accumbens (NAc) and local injection of AAV-CMV-FLEX-hrGFP into the ventral tegmental area (VTA) (FIG. 8) .
(c) AAV-CMV-mCherry-2A-Cre-TTC is injected locally into the lateral hypothalamus (LHA) and AAV-CMV-FLEX-hrGFP is injected locally into the amygdala (CeA) (FIG. 9).

  Perfusion fixation was performed about 3 weeks after the local injection and fixed with 10% formalin. The fixed brain was replaced with a 30% sucrose-containing PBS solution, embedded and frozen, and a 40 μm thick brain slice was prepared with a cryostat. Then, brain sections were immunostained to identify the type of the labeled nerve cell.

3. Results First, as a negative control, AAV-CMV-FLEX-hrGFP was locally injected into the lateral hypothalamus (LHA), locus coeruleus (LC) and amygdala (CeA), and no hrGFP was expressed in the absence of Cre recombinase It was confirmed. Then, the projection from SNc, which is the projection pathway of dopamine nerve, to CPu was confirmed using this system. Specifically, AAV-CMV-mCherry-2A-Cre-TTC was locally injected into CPu, and AAV-CMV-FLEX-hrGFP was locally injected into SNc (FIG. 7). The results are shown in FIG. In sections where neostriatum (CPu) was present, red fluorescence indicating infection with AAV-CMV-mCherry-2A-Cre-TTC was observed. Cre-TTC is expressed in the same region. On the other hand, green fluorescence was observed in the section in which the mesenchymal compact area (SNc) was present. That is, it was confirmed that hrGFP was expressed selectively in the neural pathway that projects to CPu.

  Since hrGFP was not expressed in VTA near the substantia nigra pars compacta, it was confirmed that the dopamine nerve of VTA was not projected to CPu. On the other hand, it has been reported that the dopamine nerve of VTA is projected to the nucleus accumbens (NAc), which is very in agreement with this result. Thus, it was shown that the system developed this time works properly.

  On the other hand, when AAV-CMV-mCherry-2A-Cre-TTC is injected locally into the nucleus accumbens (NAc) and AAV-CMV-FLEX-hrGFP is injected locally into the ventral tegmental area (VTA), Red fluorescence indicating infection with AAV-CMV-mCherry-2A-Cre-TTC is observed in sections in which the nucleus accumbens (NAc) are present, and green fluorescence is visible in sections in which the ventral tegmental area (VTA) is present It was observed (FIG. 11).

  In addition, when AAV-CMV-mCherry-2A-Cre-TTC is locally injected into the lateral hypothalamus (LHA) and AAV-CMV-FLEX-hrGFP is locally injected into the amygdala (CeA), the lateral hypothalamus In sections where (LHA) was present, red fluorescence indicating infection with AAV-CMV-mCherry-2A-Cre-TTC was observed, and in sections where amygdala (CeA) was present, green fluorescence was observed (FIG. 12). ).

  As described above, projection route selective labeling is possible even for projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and from the amygdala (CeA) to the lateral hypothalamic area (LHA) Met.

4. Summary
By using a viral vector having the sequence of Cre-TTC, we succeeded in specifically labeling upstream neurons directly having synapse in infected cells. In this new system, by incorporating various genes into the FLEX sequence, it is possible to control the neural activity selectively in the projection pathway. If this system is combined with genetically modified mice, it is also possible to limit the neurons that express Cre-TTC.

  By using the system of the present invention, it is possible to express a target gene specifically in nerve cells (one-step upstream nerve cells) to be input to a specific nerve cell. Therefore, the system of the present invention is a very effective tool for identifying a projection pathway involving a specific nerve cell. In addition, if the system of the present invention is used, it becomes possible to express various genes only in neural cells that project to a specific region and control its activity. Therefore, it is expected to contribute to the identification of neural circuits and the elucidation of their circuit functions.

  The present invention is not limited to the description of the embodiments and examples of the above-mentioned invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive of the claims without departing from the scope of the claims. The contents of articles, published patent publications, patent publications, etc. specified in the present specification are incorporated by reference in their entirety.

Claims (14)

Infecting a non-human mammalian neuronal cell present in the first region with a Cre-first adeno-associated virus vector expressing a gene encoding a conjugate of Cre recombinase and a tetanus toxin C-terminal fragment;
Infecting a non-human mammalian neuronal cell present in the second region with an FLP-first adeno-associated virus vector expressing a gene encoding a conjugate of the FLP recombinase and the tetanus toxin C-terminal fragment;
A Cre-second adeno-associated virus vector that expresses the first target gene only in the presence of Cre recombinase, and a FLP-second adeno-associated virus vector that expresses the second target gene only in the presence of FLP recombinase, Infecting a non-human mammalian neuronal cell present in one region and a region projecting to the second region;
Gene expression methods including:   The gene expression method according to claim 1, wherein the Cre recombinase comprises the sequence shown in SEQ ID NO: 5.   The gene expression method according to claim 1 or 2, wherein the tetanus toxin C-terminal fragment comprises the sequence shown in SEQ ID NO: 7.   The gene expression method according to any one of claims 1 to 3, wherein the ligation product comprises a nuclear localization signal peptide. In addition to the gene encoding the ligation product, the Cre-first adeno-associated virus vector holds a reporter gene in an expressible state,
The gene expression method according to any one of claims 1 to 4, wherein the FLP-first adeno-associated virus vector holds a reporter gene in an expressible state in addition to the gene encoding the ligation product. . The Cre-first adeno-associated virus vector comprises a sequence structure in which the reporter gene and a gene encoding the ligation are linked via a sequence encoding a self-cleaving peptide,
The gene expression method according to claim 5, wherein the FLP-first adeno-associated virus vector comprises a sequence structure in which the reporter gene and a gene encoding the ligation are linked via a sequence encoding a self-cleaving peptide. .   The gene expression method according to claim 5 or 6, wherein the reporter gene is a fluorescent protein gene. The Cre-second adeno-associated virus vector comprises an LSL (LoxP-STOP-LoxP) sequence, and the first target gene is disposed downstream of the sequence;
The FLP-second adeno-associated virus vector comprises an FSF (FRT-STOP-FRT) sequence, and the second target gene is placed downstream of the sequence. Gene expression method. The Cre-second adeno-associated virus vector comprises a FLEX (Flip-Excision) switch incorporating the first target gene,
The gene expression method according to any one of claims 1 to 7, wherein the FLP-second adeno-associated virus vector comprises a FLEX (Flip-Excision) switch incorporating the second target gene. In addition to the target gene, the Cre-second adeno-associated virus vector holds a reporter gene in an expressible state,
The gene expression method according to any one of claims 1 to 9, wherein the FLP-second adeno-associated virus vector holds a reporter gene in an expressible state in addition to the target gene. The Cre-second adeno-associated virus vector comprises a sequence structure in which the reporter gene and the target gene are linked via a sequence encoding a self-cleaving peptide,
11. The gene expression method according to claim 10, wherein the FLP-second adeno-associated virus vector comprises a sequence structure in which the reporter gene and the target gene are linked via a sequence encoding a self-cleaving peptide. The Cre-second adeno-associated virus vector comprises an LSL (LoxP-STOP-LoxP) sequence, the sequence structure being located downstream of the sequence;
The gene expression method according to claim 11, wherein the FLP-second adeno-associated virus vector comprises a FSF (FRT-STOP-FRT) sequence, and the sequence structure is disposed downstream of the sequence. The Cre-second adeno-associated virus vector comprises a FLEX (Flip-Excision) switch incorporating the sequence structure,
The gene expression method according to claim 11, wherein the FLP-second adeno-associated virus vector comprises a FLEX (Flip-Excision) switch incorporating the sequence structure. The reporter gene is a fluorescent protein gene, gene expression method according to any one of claims 10 to 13.

Images