JP6856919B2 - Vector system, gene expression method, target gene knockout method, target gene knockdown method, target gene editing method, and gene expression kit - Google Patents

Description

The present invention relates to a vector system, a gene expression method, a target gene knockout method, a target gene knockdown method, a target gene editing method, and a gene expression kit.

The mammalian brain is a complex tissue consisting of dense nerve cells that are interconnected and form complex neural circuits that are responsible for advanced brain functions. In order to understand the detailed molecular cytological mechanism of neural circuit development and function, it is essential to analyze one cell excluding the influence of individual cell connections and the molecular mechanism in these cells. ..
In this way, there is a high need for a system that sparsely manipulates intracellular genes in tissues with high cell density, especially a system that sparsely labels cells in tissues with high cell density, and various methods are used all over the world. Has been developed.

As such a system, two transgenic / knock-in mice based on two transgenic / knock-in mice, a MADM (masic analysis with double markers) system and a SLICK (Single-neuron Labeling with Inducible Cre-mediated Knockout) system, which are two transgenic / knock-in mouse-based systems. (See, for example, Non-Patent Documents 1 and 2).

Espinosa J.S., Wheeler D.G., Tsien R.W., Luo L .. Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron. 2009 Apr 30; 62 (2): 205-17. Young P., Qiu L., Wang D., Zhao S., Gross J., Feng G. Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice. Nat. Neurosci. 2008 Jun; 11 (6): 721 -8.

Systems based only on mouse genetics, such as the MADM system and SLICK system described in Non-Patent Documents 1 and 2, require a large amount of cost, place, and time for bleeding mice. It is hindered.

Specifically, in order to use the MADM system described in Non-Patent Document 1, it is necessary to prepare four types of transgenic mice, and it is necessary to carry out a large amount of mating until a desired mouse is obtained. Yes, it is very troublesome.

Further, the SLICK system described in Non-Patent Document 2 can be used only in a part of the brain due to the nature of the promoter used. There is room for improvement from the perspective of versatility.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a vector system capable of easily performing individual cell-specific gene manipulation in a dense cell group.

We focused on the leakage of tetracycline-regulated transactivator (tTA) -independent tetracycline-responsive factor (TRE), and by combining a positive feedback system of tTA / TRE with a site-specific recombination system. The present invention has been completed by finding that individual cell-specific genetic manipulation is possible in a dense cell group.
That is, the present invention is as follows.

(1) A vector system containing at least one vector, a first regulator containing a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE. A cassette containing the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side, and a cassette located downstream of the cassette, said exogenous. A vector system comprising a second regulator, including a first gene of interest and a gene encoding a tetracycline-regulated transactivator (tTA), under the control of an expression promoter.
(2) The vector system according to (1) above, which comprises a first vector containing the first regulator and a second vector containing the second regulator.
(3) The vector system according to (1) or (2) above, wherein the exogenous expression promoter is at least one selected from the group consisting of a CAG promoter, a CMV promoter, and an EF1α promoter.
(4) Further, the above (1) to (1) to include the cassette and a third regulator containing the cassette and the second target gene located downstream of the cassette and under the control of the exogenous expression promoter. The vector system according to any one of 3).
(5) The vector system according to (4) above, wherein the third regulator further comprises a gene encoding tTA under the control of the exogenous expression promoter.
(6) The vector system according to (4) or (5) above, which comprises a third vector containing the third regulator.
(7) Further, the above (4) to (4) to include the cassette and a fourth regulator containing the cassette and a third target gene located downstream of the cassette and under the control of the exogenous expression promoter. The vector system according to any one of 6) (8) The vector system according to (7) above, which comprises a fourth vector containing the fourth regulator.
(9) The second target gene is a gene encoding a Cas9 protein, and the third regulator is a fifth regulator to which one or more genes including a guide RNA are operably linked. The vector system according to any one of (4) to (8) above.
(10) The vector system according to any one of (4) to (8) above, wherein the second target gene is TALEN left, and the third target gene is TALEN right.
(11) The vector system according to any one of (4) to (8) above, wherein the second target gene is an RNAi-inducible nucleic acid.
(12) The vector system according to any one of (1) to (11) above, wherein the vector is a viral vector.
(13) A method for expressing a target gene specifically for each cell in a dense cell group, which comprises using the vector system according to any one of (1) to (12) above. Gene expression method.
(14) A method for expressing a target gene in a dense cell group in a cell-specific manner, which comprises a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE. The first vector containing the first regulator, including, and the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence are arranged in this order from the 5'side. A second gene containing a first gene of interest and a gene encoding a tetracycline-regulated transactivator (tTA) located downstream of the cassette and under the control of the exogenous expression promoter. A gene characterized in that the number of cells in which the first target gene is expressed is regulated by introducing a second vector containing the regulatory factor of the above into cells and adjusting the amount of the first vector introduced. Expression method.
(15) A method for expressing a target gene in a dense cell group in a cell-specific manner, encoding a tetracycline response factor (TRE) and a first site-specific recombinant enzyme under the control of the TRE. A first vector containing a first regulator containing the gene, an exogenous expression promoter, the first site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the first site-specific recombinant enzyme. The first cassette containing the recognition sequence from the 5'side in this order, and the first target gene and tetracycline-regulated trans-activity located downstream of the first cassette and under the control of the exogenous expression promoter. A second vector containing a gene encoding a chemogenetic factor (tTA) and a second regulator, an exogenous expression promoter, a second site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the second A second cassette containing the site-specific recombinant enzyme recognition sequence of the above in this order from the 5'side, and a second target gene located downstream of the second cassette and under the control of the exogenous expression promoter. A method for expressing a gene, which comprises introducing a third vector containing a third regulator containing, and a second site-specific recombinant enzyme-expressing cell into a second site-specific recombinant enzyme-expressing cell.
(16) A method of knocking out a target gene in a dense cell group in a cell-specific manner, which comprises a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE. The first vector containing the first regulator including, and the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence are arranged in this order from the 5'side. A gene containing a gene encoding a fluorescent protein and a gene encoding a tetracycline-regulated transactivator (tTA) located downstream of the cassette and under the control of the exogenous expression promoter. A second vector containing the two regulators is introduced into a cell having a chromosome containing the site-specific recombinant enzyme recognition sequence, the target gene, and the site-specific recombinant enzyme recognition sequence from the 5'side in this order. A target gene knockout method characterized by that.
(17) A method of knocking down a target gene in a dense cell group in a cell-specific manner, in which a tetracycline response factor (TRE) and a first site-specific recombinant enzyme under the control of the TRE are used. A first vector containing a gene encoding, a first regulator, an exogenous expression promoter, the first site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the first site-specific recombination. The first cassette containing the enzyme recognition sequence from the 5'side in this order, and the gene encoding the fluorescent protein and tetracycline control, which are located downstream of the first cassette and are under the control of the exogenous expression promoter. A second vector containing a gene encoding a trans-activator (tTA) and a second regulator, an exogenous expression promoter, a second site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the above. A second cassette containing the second site-specific recombinant enzyme recognition sequence in this order from the 5'side, and an RNAi-inducible RNAi-inducible located downstream of the second cassette and under the control of the exogenous expression promoter. A target gene knockdown method comprising introducing a nucleic acid and a third vector containing a third regulatory factor into a second site-specific recombinant enzyme-expressing cell.
(18) A method for editing a target gene in a dense cell group in a cell-specific manner, which comprises a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE. The first vector containing the first regulator including, and the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence are arranged in this order from the 5'side. A gene containing a gene encoding a fluorescent protein and a gene encoding a tetracycline-regulated transactivator (tTA) located downstream of the cassette and under the control of the exogenous expression promoter. A second vector containing the two regulators, the cassette, and one or more of the genes encoding the Cas9 protein located downstream of the cassette and under the control of the exogenous expression promoter, and a guide RNA. A method for editing a target gene, which comprises introducing into a cell a third vector containing a fifth regulator to which the gene is operably linked.
(19) A kit for expressing a target gene in a dense cell group in a cell-specific manner, encoding a tetracycline response factor (TRE) and a site-specific recombinant enzyme under the control of the TRE. A first vector containing a gene and a first regulator, an exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the site-specific recombinant enzyme recognition sequence from the 5'side. It contains a cassette contained in this order, and a gene encoding a first target gene and a tetracycline-regulated trans activator (tTA) located downstream of the cassette and under the control of the exogenous expression promoter. A gene expression kit comprising a second vector comprising a second regulator.

According to the present invention, individual cell-specific genetic manipulations can be easily performed in a dense cell group.

(A) It is a schematic diagram which shows an example of the vector set used for the vector system of this embodiment. (B) It is a schematic diagram which showed how the vector system of this embodiment works. (A) A low-magnification fluorescence image of a section of a cortical neuron into which a Flpe / FRT-based Supernova GFP (Flpe-SnGFP) vector set has been introduced. (B) It is a high-magnification fluorescence image of a square d in the upper part of FIG. 2 (A). (C) It is a high-magnification fluorescence image of a rectangle e in the upper part of FIG. 2B. (D) is a high-enlarged fluorescence image of a rectangle f in the upper part of FIG. 2 (B). (A) Fluorescent image of L2 / 3 cortical neurons labeled with Flpe-SnGFP. (B) It is a graph which showed the degree of sparseness in the mouse of each age. (A) Fluorescent image of L2 / 3 cortical neurons labeled with Flpe-SnGFP. (B) It is a graph which showed the degree of sparseness in the final concentration of the introduced TRE-Flpe vector. (A) Fluorescent image of the cortex of an Emx1 Cre knock-in (KI) mouse introduced with a Flpe-SnGFP vector. (B) It is a figure which showed the labeling mechanism in the cortex of the Emx1 Crek knock-in (KI) mouse labeled with Flpe-SnGFP vector and CAG-LSL-RFP. (A) Fluorescent images of cortical neurons labeled with Flpe-based SnGFP and SnRFP. (B) Fluorescent images of cortical neurons labeled with Cre-based SnGFP and SnnsRFP. (C) Fluorescent images of cortical neurons labeled with Cre-based SnGFP and PSD95-GFP. (A) It is a schematic diagram of gene knockout via Supernova. (B) Fluorescence image in hippocampal CA1 neurons labeled with SnRFP. (A) It is a graph which shows the ratio of the cell which expresses α2-Chn to the DAPI-stained cell. (B) It is a graph which shows the signal intensity of α2-Chn in an RFP positive cell, and the signal intensity of α2-Chn in an RFP negative cell. (A) Schematic representation of a Supernova vector set designed for α2-Chn gene editing at the single cell level. (B) Fluorescent image of the hippocampus of a wild-type mouse gene-transfected using the Supernova α2-Chn TALEN vector set. (A) An enlarged view of FIG. 9 (B) is shown. (B) An enlarged view of FIG. 9 (B) is shown. (A) It is a graph which shows the ratio of ^{α2-Chn high} , α2-Chn ^low , and α2-Chn ^negative in RFP-negative cells and RFP-positive cells (Supernova α2-Chn TALEN-expressing cells). (B) FIG. 6 is a schematic diagram showing the target positions of left TALEN and light TALEN in the mouse α2-Chn gene. (A) FIG. 6 is a schematic diagram of a Supernova vector set designed for genetic engineering of Creb1 at the single cell level. (B) Fluorescent image showing the hippocampal CA1 region of a wild-type mouse gene-transfected using the Supernova-CRISPR / Cas9 control (not including singleguide RNA (sgRNA)). (C) Fluorescent image showing the hippocampal CA1 region of a wild-type mouse gene-introduced using CRISPR / Cas9 (SnCRISPR / Cas9-Creb1) targeting Supernova-mouse Creb1. (A) It is a graph which shows the ratio of the cell number of CREB positive nerve cell and CREB negative nerve cell in GFP-labeled nerve cell. (B) It is a schematic diagram which shows the typical mutation found in the Creb1 locus. It is a graph which shows the ratio of labeled Cre-SnRFP and Flpe-SnRFP in GFP-positive nerve cells. FIG. 6 is a schematic representation of a Supernova vector set for knockdown of a gene of interest (GOI) in a single cell. (A) It is a fluorescence image in a GFP RNAi-expressing nerve cell. (B) It is a graph which shows the ratio of the GFP-positive cell in the RFP-positive cell. (C) It is a fluorescence image in a LacZRNAi expressing nerve cell. (D) It is a graph which shows the ratio of β-gal activity with respect to SnAmcyan fluorescence intensity. (A) It is a schematic diagram of the AAV-SnRFP vector set. (B) to (D) are fluorescence images in hippocampal CA1 pyramidal neurons. (E)-(G) Fluorescent images in the 5th layer (L5) pyramidal nerve cells of the cerebral cortex. (H)-(I) Fluorescence images in hippocampal CA1 pyramidal neurons.

≪Vector system≫
The vector system of the present invention (hereinafter, also referred to as Supernova) is a vector system containing at least one vector, and encodes a tetracycline response factor (TRE) and a site-specific recombinant enzyme under the control of the TRE. A cassette containing a gene, a first regulator containing the gene, an exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side. A second regulator, including a first gene of interest and a gene encoding a tetracycline-regulated trans-activator (tTA), located downstream of the cassette and under the control of the exogenous expression promoter. ,including.

The vector system of the present invention may include a kind of vector containing a first regulator and a second regulator, but from the viewpoint of expression efficiency, the first vector containing the first regulator and the above-mentioned Those containing a second vector containing the second regulator are preferred.
The vector is not particularly limited, and conventionally known vectors such as a plasmid vector and a virus vector can be used. In utero electroporation, which is preferably mentioned as a gene transfer method described later, gene transfer is performed by applying an electric pulse to the fetal brain. Since strong gene expression can be obtained in about 2 days after gene transfer, it is ideal for analysis of the developing brain. On the other hand, since gene expression is maintained for a long time, it can be used for analysis of adult animals, but it is inconvenient to carry out experiments because the time from gene transfer to analysis is long. In utero electroporation is optimal for gene transfer into the upper layers of the cerebral cortex and the hippocampus, but there is the inconvenience that gene transfer is not possible or difficult depending on the part of the brain. On the other hand, the AAV virus vector described later can be used in all brain regions by directly introducing a gene into an adult nerve cell. On the other hand, since it takes more than one week from gene transfer to gene expression, it is often inappropriate for analysis of the rapidly changing developmental stage.

Examples of the viral vector include a retrovirus (including lentivirus) vector, an adenovirus vector, an adeno-associated (AAV) virus vector, a herpesvirus vector, a Sindbis virus vector, and the like, from the viewpoint of low toxicity. Adeno-associated (AAV) viral vectors are preferred.
Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[First Embodiment]
The vector system of the present embodiment comprises a TRE, a first vector containing a first regulator comprising a gene encoding a site-specific recombinant enzyme under the control of the TRE (SSR), and exogenous expression. A cassette containing the promoter, the site-specific recombinant enzyme recognition sequence (RT), the transcription termination sequence (stop), and the site-specific recombinant enzyme recognition sequence (RT) in this order from the 5'side, and downstream of the cassette. Includes a second vector containing a second regulator, including a first gene of interest and a gene encoding tTA, located and under the control of said exogenous expression promoter.

As the exogenous promoter, at least one selected from the group consisting of the CAG promoter, the CMV promoter, and the EF1α promoter is preferable, and the CAG promoter is more preferable from the viewpoint of more strongly driving the expression of downstream genes. As described above, since there are no restrictions on the exogenous promoter used, the vector system of the present embodiment can be applied not only to nerve cells but also to all cell types.
Moreover, as a transcription termination sequence, a poly A addition signal sequence can be mentioned.

Examples of the site-specific recombinant enzyme include Cre, Flpe, Dre and the like. Flpe is preferable from the viewpoint of performing individual cell-specific genetic manipulation more sparsely in a dense cell group. Since it is not necessary to use only Cre as a site-specific recombinant enzyme, the vector system of this embodiment can be used in combination with Cre-expressing mice. Moreover, since the system does not depend on the vector system Cre of the present embodiment, gene expression can be controlled without using a Flox mouse.
Examples of the site-specific enzyme recognition sequence recognized by the site-specific recombinant enzyme include loxP, FRT, and rox.

The first target gene is not particularly limited, but when a gene encoding a fluorescent protein is used, fluorescent labeling can be sparsely performed at the level of one cell in a dense cell group. This makes it possible to easily and accurately perform morphological analysis of cells and the like.

FIG. 1A is a schematic diagram showing an example of a vector set used in the vector system of the present embodiment. As shown in FIG. 1 (A), the vector system of the present embodiment is, for example, TRE-SSR-WPRE-pA (TRE-SSR) and CAG-RT-stop-RT-XFP-ires-tTA-WPRE. -PA (CAG-RT-stop-RT-XFP-tTA) is included.
Here, TRE indicates a tetracycline response element, SSR indicates a site specific recombination, WPRE indicates a WHP post-trascombination ribosome, RT indicates a recovery, and RT indicates a recompression. ires indicates an internal ribosome entry site, and tTA indicates a tetracycline transactivator.
The vector system of this embodiment enables sparse and bright cell labeling and labeled cell-specific genetic manipulation in the mammalian brain.

Examples of XFP (fluorescent protein) include green fluorescent proteins (Green Fluorescent Protein, GFP) such as TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, and HyPer; and blue fluorescent proteins (GFP) such as EBFP. , BFP); Cyan Fluorescent Protein (CFP) such as ECFP, TagCFP, AmCyan, MidoriishiCyan; TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed RFP); Yellow fluorescent protein (Yellou Fluorescent Protein, YFP) such as TagYFP, EYFP, Venus, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mbannana, KusabiraOrange, Morrange, Orange Examples thereof include fluorescent proteins that emit infrared light such as mCherry, HcRed, KeimaRed mRasbury, and mPlum.

FIG. 1B is a schematic diagram showing how the vector system of the present embodiment works. First, of the many cells into which TRE-SSR and CAG-RT-stop-RT-XFP-tTA have been introduced, only a small number of cells have very weak TRE leakage site-specific recombinant enzymes (hereinafter referred to as “)”. It also drives the expression of SSR). Second, this low level SSR cuts out an RT-stop-RT cassette in several copies of the CAG-RT-stop-RT-XFP-tTA vector, to a lesser extent, XFP (also referred to as fluorescent protein). .) And tTA have started to be transferred. Third, through binding to TRE, tTA promotes SSR expression. Fourth, the RT-stop-RT cassette is excised from the CAG-RT-stop-RT-XFP-tTA vector with a large copy number, and the expression of XFP and tTA is increased. Promotion of this positive tTA / TRE loop induces very high levels of expression of SSR and XFP only in a small number of cells with TRE leaks above the initial threshold.

[Second Embodiment]
The vector system of the present embodiment includes the above-mentioned first vector, the above-mentioned second vector, a cassette containing an exogenous expression promoter, RT, stop, and RT in this order from the 5'side, and the cassette. It contains a second vector located downstream and containing a second gene of interest and a gene encoding tTA under the control of the exogenous expression promoter, and a third vector containing a third regulator.

In the present embodiment, the second vector and the third vector differ only in the target gene under the control of the exogenous expression promoter, and have the same other configurations. The second target gene is not particularly limited. When genes encoding different fluorescent proteins are used for the first target gene and the second target gene, multiple fluorescent labels can be sparsely applied at the level of one cell in a dense cell group.
For example, TRE-Flppe is used as the first vector, CAG-FRT-stop-FRT-GFP-tTA is used as the second vector, and CAG-FRT-stop-FRT-RFP- is used as the third vector. When tTA is used, GFP and RFP multiple labeling can be sparsely performed at the single cell level in a dense cell population.

[Third Embodiment]
The vector system of the present embodiment includes the above-mentioned first vector, the above-mentioned second vector, a cassette containing an exogenous expression promoter, RT, stop, and RT in this order from the 5'side, and the cassette. It contains a third A vector containing a third A regulator, including a second A target gene, located downstream and under the control of the exogenous expression promoter.

In this embodiment, the vector of the third A is the same as the second vector except that it does not have tTA. For example, TRE-Cre is used as the first vector, CAG-loxP-stop-loxP-GFP-tTA is used as the second vector, and CAG-loxP-stop-loxP-th is used as the third A vector. When the target gene of 2A is used, GFP labeling can be sparsely performed at the level of one cell in a dense cell group, and the expression of the target gene of 2A can be confirmed only in GFP-expressing cells. can do.

The target gene of the second A is not particularly limited, and may be, for example, a gene encoding a factor related to genome editing. In such a case, in a dense cell group, fluorescent labeling can be performed sparsely at the level of one cell, and phenotype analysis by genome editing can be performed only in the fluorescently labeled cells. ..

As the genome editing method, for example, a TALEN system, a Zn finger nuclease system, and a CRISPR-Cas9 system can be used. Of these, it is more preferable to use the CRISPR-Cas9 system.

The TALEN system is a system using a transcript activator-like effector nucleoase (TALEN), which is a fusion of a customized DNA binding domain and a non-specific FokI nuclease domain. The DNA-binding domain consists of conserved repeats derived from transcrition activator-like effects (TALE), a protein secreted by Xanthomonas proteobacteria that alters gene transcription in host plants.
To knock in a gene by the TALEN system, TALEN is prepared for the insertion site in the genome, and a donor vector having homology around the insertion site is prepared. By co-introducing these, the target site is cleaved, and then the target foreign DNA is introduced by homologous recombination repair using a donor vector.
To knock out a gene with the TALEN system, the donor vector need not be co-introduced.

The CRISPR-Cas9 system is a system that utilizes a locus of bacteria and archaea that functions as a kind of acquired immunity against nucleic acids (viral DNA, viral RNA, plasmid DNA) that have invaded from the outside. To knock in a gene with the CRISPR-Cas9 system, a CRISPR-Cas9 vector is prepared to cleave the insertion site in the genome. When a gene encoding the Cas9 protein is used as the target gene of the second A, the regulator of the third A is a fifth gene in which one or more genes including a guide RNA that induces Cas9 at the target site are operably linked. Control factors are needed. The fifth regulator may, for example, be integrated into the vector of third A with the gene encoding the Cas9 protein (see FIG. 6 (A)) or in a separate vector. Good. By co-introducing these vectors with the donor vector, the target site is cleaved, and then the target foreign DNA is introduced by homologous recombination repair using the donor vector.
To knock out a gene with the CRISPR-Cas9 system, the donor vector need not be co-introduced.

The Zn finger nuclease (ZFN) system is a genome editing using an artificial restriction enzyme consisting of a Zn finger domain and a DNA cleavage domain. One Zn finger domain recognizes 3 bases, but by linking 3 to 5 Zn finger domains, a DNA-binding protein that recognizes a specific 9 to 15 base sequence can be designed. By fusing the FokI nuclease domain with this DNA-binding protein, an artificial nuclease ZFN that cleaves the target site is designed.
To knock in a gene by the ZFN system, a ZFN is prepared for the insertion site in the genome, and a donor vector having homology around the insertion site is prepared. By co-introducing these, the target site is cleaved, and subsequently, the target foreign DNA is introduced by H homologous recombination repair using a donor vector.
Donor vectors need not be co-introduced to knock out genes by the ZFN system.

Further, the target gene of the second A may be an RNAi-inducible nucleic acid. RNAi-inducible nucleic acid means a nucleic acid capable of inducing RNA interference by being introduced into a cell. RNA interference refers to the effect that RNA containing a base sequence complementary to mRNA (or a partial sequence thereof) suppresses the expression of the mRNA.
The mRNA targeted by the RNAi-inducible nucleic acid may be a coding region or a non-coding region. Examples of RNAi-inducible nucleic acids include siRNA and miRNA. An example of a vector that is introduced into a cell and causes RNAi in the same manner as siRNA is a shRNA (short hairpin RNA / small hairpin RNA) expression vector.
In such cases, fluorescent labeling can be sparsely performed at the single cell level in a dense cell population, and phenotypic analysis due to RNA interference can be performed only within fluorescently labeled cells. it can.

[Fourth Embodiment]
The vector system of the present embodiment comprises the above-mentioned first vector, the above-mentioned second vector, the above-mentioned third A vector, and the exogenous expression promoter, RT, stop, and RT from the 5'side. It comprises a cassette containing in order, a third vector located downstream of the cassette and under the control of the exogenous expression promoter, and a fourth vector containing a fourth regulator, including a third gene of interest.

In the present embodiment, the vector of the third A and the vector of the fourth A differ only in the target gene under the control of the exogenous expression promoter, and have the same other configurations. The third target gene is not particularly limited. When a gene encoding a fluorescent protein is used as the first target gene and a gene encoding a different protein is used as the second A target gene and the third target gene, in a dense cell group, 1 At the individual cell level, fluorescent labeling can be performed sparsely, and phenotypic analysis can be performed by expressing two different genes only in fluorescently labeled cells.
Further, the above-mentioned TALEN needs to bind to the opposite strand of the target DNA as a pair of TALEN left and TALEN light. For example, TRE-Flpe is used as the first vector, CAG-FRT-stop-FRT-RFP-tTA is used as the second vector, and CAG-FRT-stop-FRT-TALEN is used as the third A vector. When left is used and CAG-FRT-stop-FRT-TALENlight is used as the fourth vector, fluorescent labeling can be performed sparsely in a dense cell group, and the genome can be administered only in fluorescently labeled cells. It is possible to analyze the phenotype by receiving the editing.

≪Composition or gene expression kit≫
The present invention also provides a composition or gene expression kit containing the first vector and the second vector described in [First Embodiment] of the vector system.

≪Gene expression method≫
The gene expression method of the present invention is a method for expressing a target gene specifically for each individual cell in a dense cell group using the above-mentioned vector system.
The gene transfer method is not particularly limited, and examples thereof include a calcium phosphate method, a lipofection method, an electroporation method, and a microinjection method. In vivo electroporation is preferable when the gene transfer target is not only cultured cells but also non-human animals, and in utero electroporation (hereinafter, also referred to as IUE) is preferable when the gene transfer target is a mouse. In utero electroporation is a method of injecting DNA or the like from the outside of the uterus and sandwiching the outside of the uterus with electrodes to perform electroporation, which is generally used as an in vivo electroporation method for mouse embryos. The IUE-based method has made rapid experimental progress. IUE is applicable to many brain regions and all nerve cells. Therefore, the IUE-based approach has many advantages over the pure mouse genetic approach. Although IUEs are generally too tightly labeled in the transgenic brain region, the genetic position expression method of the present invention allows gene expression in a single cell.

The target for gene transfer is not particularly limited to cultured cells, non-human animals, and the like. The non-human animal is not particularly limited as long as it is a non-human animal, but is preferably a mammal, and more preferably a rodentia. Examples of animals classified as mammals include goats, pigs, dogs, cats, mice, rats, guinea pigs, hamsters, and rabbits. Examples of animals classified as rodents include mice, rats, guinea pigs, hamsters and the like.
The gene expression method of the present invention is suitably used for morphological analysis of nerve cells because the target gene can be expressed specifically for each individual cell in a dense cell group.

In addition, the gene expression method of the present invention is a method for expressing a target gene in an individual cell-specific manner in a dense cell group, and is specific to a tetracycline response factor (TRE) and a site-specific control of the TRE. A first vector containing a gene encoding a recombinant enzyme, a first regulator containing, an exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the site-specific recombinant enzyme recognition sequence. The first gene of interest and the tetracycline-regulated trans activator (tTA), which are located downstream of the cassette and are under the control of the exogenous expression promoter, are encoded. By introducing a gene and a second vector containing a second regulator containing the gene into cells and adjusting the amount of the first vector introduced, the number of cells expressing the first target gene is regulated. How to do it.

According to the present invention, as will be described later in Examples, the degree of sparseness of cells expressing the target gene can be adjusted in a dense cell group by adjusting the amount of the vector to be introduced.

In addition, the gene expression method of the present invention is a method for expressing a target gene in a dense cell group in a cell-specific manner, and is a tetracycline response factor (TRE) and a first method under the control of the TRE. A first vector containing a gene encoding a site-specific recombinant enzyme, a first regulator containing, an exogenous expression promoter, the first site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the first. A first cassette containing a site-specific recombinant enzyme recognition sequence in this order from the 5'side, and a first objective located downstream of the first cassette and under the control of the exogenous expression promoter. A second vector containing a gene and a gene encoding a tetracycline-regulated trans-activator (tTA), and a second regulator, an exogenous expression promoter, a second site-specific recombinant enzyme recognition sequence, transcription. A second cassette containing the termination sequence and the second site-specific recombinant enzyme recognition sequence in this order from the 5'side, and located downstream of the second cassette, under the control of the exogenous expression promoter. It is a method of introducing a second target gene and a third vector containing a third regulator containing the second target gene into a second site-specific recombinant enzyme-expressing cell.

For example, by introducing the CAG-loxP-stop-loxP-RFP vector, TRE-Flpe vector, and CAG-FRT-stop-GFP-FRT-GFP-tTA into Cre-expressing Tg mice, RFP was introduced into all the dense cells. GFP can be sparsely expressed while GFP is expressed.

≪Target gene knockout method≫
The target gene knockout method of the present invention is a method of knocking out a target gene in a dense cell group in a cell-specific manner, and is a tetracycline response factor (TRE) and a site-specific recombination under the control of the TRE. A first vector containing a gene encoding an enzyme, a first regulator containing, an exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the site-specific recombinant enzyme recognition sequence. It encodes a cassette containing in this order from the 5'side, a gene encoding a fluorescent protein and a tetracycline-regulated transactivator (tTA) located downstream of the cassette and under the control of the exogenous expression promoter. A gene containing a gene and a second vector containing a second regulator, and a chromosome containing the site-specific recombinant enzyme recognition sequence, a target gene, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side. It is a method of introducing into a cell having.

For example, in cells in which RFP is sparsely expressed by introducing the CAG-loxP-stop-loxP-RFP-tTA vector and the TRE-Cre vector into a mouse having a chromosome containing a target gene sandwiched between loxP. The target gene can be knocked out only in.

≪Target gene editing method≫
The target gene editing method of the present invention is a method for editing a target gene in a dense cell group in a cell-specific manner, and is a tetracycline response factor (TRE) and a site-specific recombination under the control of the TRE. A first vector containing a gene encoding an enzyme, a first regulator containing, an exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, a transcription termination sequence, and the site-specific recombinant enzyme recognition sequence. It encodes a cassette containing in this order from the 5'side, a gene encoding a fluorescent protein and a tetracycline-regulated transactivator (tTA) located downstream of the cassette and under the control of the exogenous expression promoter. A guide with a second vector containing the gene and a second regulator, the cassette, and a gene encoding the Cas9 protein located downstream of the cassette and under the control of the exogenous expression promoter. A method of introducing into a cell a third vector containing a fifth regulator in which one or more genes including RNA are operably linked.

For example, cells in which GFP was sparsely expressed by introducing TRE-Cre vector, CAG-loxP-stop-loxP-GFP-tTA vector, and U6-sgRNA-CAG-loxP-stop-loxP-Cas9 into cells. The target gene can only be edited inside.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1]
To enable widespread use of the Supernova system, the inventors systematically and comprehensively evaluated the properties of Supernova labeling. To estimate the sparseness and lightness of Supernova labeling, we found Flpe / FRT-based Supernova GFP (Flpe-SnGFP) vector sets (TRE-Flpe) in cortical neurons of layer 2/3 (L2 / 3). And CAG-FRT-stop-FRT-GFP-tTA) were introduced into the embryonic period 15.5 (E15.5) by inventor electroporation (hereinafter, also referred to as IUE) (see FIG. 1 (A)). A coronary section was prepared from the brain of a mouse having 22 days after birth (P22), and fluorescence observation was performed. The upper part of FIG. 2A shows a low-magnification fluorescence image. Among the transgenic neurons, a sparse small population was GFP positive (1.2% ± 0.2%, n = 5 mice were used). The upper part of FIG. 2B shows a highly magnified fluorescence image of the square d in the upper part of FIG. 2A. The Scale bar in the lower part of FIG. 2 (A) shows 50 μm. The Scale bar in the lower part of FIG. 2B shows 100 μm. 2 (C) and 2 (D) show high-magnification fluorescence images of rectangles e and f in the upper part of FIG. 2 (B). The Scale bar in FIG. 2C shows 10 μm. The Scale bar in the upper part of FIG. 2 (D) shows 50 μm. The Scale bar in the lower part of FIG. 2 (D) shows 10 μm.
Labeled L2 / 3 pyramidal neurons shown in FIG. 2 (B) (dendritic spines (see FIG. 2 (C)), axon branches (see upper row of FIG. 2 (D)), axon buttons. It was confirmed that the label by Supernova was bright enough to sufficiently visualize the morphology (including (see the lower part of FIG. 2D)).
In addition, CAG-RFP introduced together with Flpe-SnGFP was confirmed to be expressed in many dense cells, while Flpe-SnGFP was confirmed to be sparsely expressed at the single cell level.

[Example 2]
The inventors quantitatively evaluated the sparseness of labeling by Supernova by transfecting both Flpe-SnGFP and CAG-RFP at different developmental and adult stages. CAG-RFP was introduced with the TRE-Flpe vector to label all transgenic cells. Coronal sections were prepared from the brains of P8, P22, 2-month-old (2M), and 4M mice, and fluorescence observation was performed to evaluate the ratio of Flpe-SnGFP-positive neurons to RFP-positive neurons. FIG. 3 (A) shows a fluorescence image of L2 / 3 cortical neurons labeled with Flpe-SnGFP. The final concentration of the TRE-Flpe vector used for the introduction was 5 ng / μL. The number below the fluorescence image indicates the sparseness of Flpe-SnGFP labeling at each age (calculated at n = 5 in P8, P22, 2M mice, n = 3 in 4M mice). Calculated in.). The degree of sparseness was calculated as the ratio of the number of Flpe-SnGFP positive cells to the number of RFP positive cells which are transgenic cells. FIG. 3B is a graph showing the degree of sparseness in mice of each age. Each rhombus represents an individual. The bar shows the average value. It was confirmed that the sparseness and brightness of Supernova labeling were constant from the early postnatal stage to adulthood. The Scale bar in FIG. 3 indicates 100 μm.

By varying the concentration of the TRE-SSR vector in the Supernova vector composition, it may be possible to control the sparseness of the label. To confirm this possibility, Flpe-SnGFP vector compositions containing different concentrations of TRE-Flpe vectors (5, 50, 500 ng / μL) were prepared and each composition was L2 / using IUE at E15.5. It was introduced into 3 cortical neurons. CAG-RFP was co-introduced to label the transgenic neurons. FIG. 4 (A) shows a fluorescence image of L2 / 3 cortical neurons labeled with Flpe-SnGFP. The final concentrations of the introduced TRE-Flpe vector were 5 ng / μL (left in FIG. 4 (A)), 50 ng / μL (center in FIG. 4 (A)), and 500 ng / μL (right in FIG. 4 (A)), respectively. .. Coronary sections were prepared from the brains of P8 mice. FIG. 4B is a graph showing the degree of sparseness at the final concentration of the introduced TRE-Flpe vector (calculated at n = 5 in each group of mice). Each rhombus represents an individual. The bar shows the average value. Very few RFP-positive neurons were labeled with SnGFP when a 5 ng / μL TRE-Flpe vector was used (1.4 ± 0.1%, n = 5 mice were used). When a 50 ng / μL TRE-Flpe vector was used, approximately half of the RFP-positive neurons were labeled with SnGFP (48.0 ± 5.4%, n = 5 mice were used). When a 500 ng / μL TRE-Flpe vector was used, almost all RFP-positive neurons were labeled with SnGFP (98.7 ± 2.5%, n = 5 mice were used).
From these data, it was confirmed that the sparseness of nerve cells labeled by Supernova can be adjusted by changing the concentration of the TRE-SSR vector. The Scale bar in FIG. 4 indicates 100 μm.

[Example 3]
Many Cre-expressing mouse strains have been reported today and contribute to brain comprehension by producing region-specific or cell-type-specific knockout mice. To investigate whether Supernova is applicable to Cre-expressing brain regions, the Flpe-SnGFP vector was introduced into the cortex of Emx1 Cre knock-in (KI) mice. When the CAG-loxP-stop-loxP-RFP-pA (CAG-LSL-RFP) vector was co-introduced into the cortex of Emx1 Cre knock-in (KI) mice as an indicator of recombination, the neurons were densely labeled. It was confirmed that it would be done. On the other hand, when the Flpe-SnGFP vector was introduced together with CAG-LSL-RFP, it was confirmed that only a small number of cortical neurons were labeled with GFP and the morphology of the dendrites of SnGFP-labeled neurons was clearly visualized ( See FIG. 5 (A).). Similar results were shown with the Dre-based Supernova GFP (Dre-SnGFP) vector set (TRE-Dre and CAG-rox-stop-rox-tTA). IUE was performed at E14.5 and the brain was disassembled at P8 to produce coronary sections. FIG. 5B is a diagram showing a labeling mechanism in neurons labeled with Flpe-SnGFP vector and CAG-LSL-RFP. It was confirmed that the Supernova system based on Flpe / FTR and Dre / Rox is capable of sparse labeling of nerve cells in Cre-expressing mice. The Scale bar in FIG. 5 indicates 100 μm.

[Example 4]
To see if co-multiple expression of genes is possible in a sparse cell population, neurons were sparsely labeled by expressing RFP and GFP with high co-expression efficiency using Flpe-based Supernova. Specifically, SnGFP and SnRFP were both introduced into cortical neurons at E15.5 using IUE (TRE-Flpe, CAG-FSF-GFP-tTA, and CAG-FSF-RFP-tTA). The brain was disassembled at P8, coronal sections were prepared, and fluorescence observation was performed (see FIG. 6 (A)). At P8, most labeled neurons were found to express both GFP and RFP (RFP / GFP = 51/53 cells, GFP / RFP = 51/51 cells, n = 5 mice). There was.).
Using the Cre-based Supernova system, RFPs (SnnlsRFPs) and GFPs (SnGFPs) fused with nuclear localization signals were sparsely co-expressed in cortical neurons. The brain was disassembled at P4, coronal sections were prepared, and fluorescence observation was performed (see FIG. 6 (B)).
In addition, using the Cre-based Supernova system, SnRFP and Supernova PSD (postsynaptic thickening protein) 95-GFP (SnPSD95-GFP) were gene-inserted into the P16 somatosensory cortex by IUE and tangent to E14.5. Sections in the direction were prepared and fluorescence observation was performed. The upper part of FIG. 6C shows a low-magnification fluorescence image, and the lower part of FIG. 6C shows a high-magnification magnified image of a quadrangle on FIG. 6C. Dendritic spines were co-labeled with RFP and PSD95-GFP. The Scale bar of FIGS. 6A and 6B shows 100 μm, and the Scale bar of FIG. 6C shows 25 μm.
According to Example 4, it was confirmed that the use of Supernova enables multiple staining of one cell with a plurality of fluorescent proteins.

[Example 5]
The inventors systematically and quantitatively analyzed the efficiency and specificity of Cre-based Supernova in knockout of loxP-introduced endogenous genes in mice. In mice into which loxP was introduced, genes were knocked out in a labeled cell-specific manner via Cre-based Supernova. FIG. 7 (A) shows a schematic diagram of gene knockout via Supernova.
In one of the SnRFP-labeled hippocampal CA1 neurons, α2-Chimaerin (hereinafter, also referred to as α2-Chn) sandwiched between loxP was knocked out (see FIG. 7 (B)). The Cre-SnRFP vector set was introduced into the hippocampal CA1 region ^{of α2-Chnflox / flox mice by IUE at E14.5.} The brain was disassembled at P14, coronal sections were prepared, immunohistological analysis of α2-Chn and DAPI staining were performed, and fluorescence observation was performed. The upper part of FIG. 7B shows the hippocampus of ^{α2-Chnflox / flox mice.} The enlarged fluorescence image is shown in the middle of FIG. 7 (B). Arrows indicate Cre-SnRFP labeled CA1 neurons. FIG. 7B, the upper Scale bar indicates 500 μm, and the lower Scale bar indicates 100 μm.
The Supernova-dependent α2-Chn knockout efficiency was then quantified. FIG. 8 (A) shows the ratio of cells expressing α2-Chn to DAPI-stained cells. Expression of α2-Chn was detected in almost all SnRFP-negative CA1 hippocampal cells (97.7% ± 0.1%, n = 3 mice). On the other hand, the proportion in SnRFP-positive CA1 hippocampal cells was 5.9% ± 3.0% (n = 3 mice). All values are shown as mean ± standard error. ( ^** ) indicates 0.001 <P <0.01 by Welch's t-test.
Next, the signal intensity of α2-Chn in RFP-positive cells and the signal intensity of α2-Chn in RFP-negative cells were measured (see FIG. 8 (B)) (n = 14 cells. 3 mice were used in each group. .). The bar shows the average value. ( ^*** ) indicates P <0.001 by Welch's t-test. The expression level of α2-Chn was also very low in the α2-Chn-positive neurons labeled with SnRFP as compared with the RFP-negative cells.
From these facts, it was confirmed that the specificity and efficiency of gene knockout induced by Supernova is extremely high, and that Supernova is optimal for conditional gene knockout using mice into which loxP has been introduced.

[Example 6]
In order to efficiently suppress gene expression, labeled cell-specific gene editing was performed in wild-type mice by TALEN via Supernova. FIG. 9A shows a schematic representation of a Supernova vector set designed for α2-Chn gene editing at the single cell level. FIG. 9B shows a fluorescence image of the hippocampus of a wild-type mouse gene-introduced using the Supernova α2-Chn TALEN vector set. Further, the enlarged image is shown in FIG. The Scale bar in FIG. 9B shows 250 μm.
In hippocampal CA1 on P14, most RFP-negative neurons express high levels of α2-Chn (see Cell 3 in the Exclusive image set 1 in FIG. 10 (A)). On the other hand, expression of α2-Chn protein was not detected in most RFP-positive neurons (Supernova α2-Chn TALEN-expressing cells) (see Cell 1 in the Example set 1 in FIG. 10 (A)). However, very few RFP-positive neurons showed expression of the α2-Chn protein, albeit at very low levels (see Cell 2 in the Exclusive image set 2 of FIG. 10 (B)). The Scale bar in FIGS. 10A and 10B shows 20 μm.
The inventors classified DAPI-stained cells in the hippocampal CA1 region into three groups ^{, α2-Chn high} , α2-Chn ^low , and α2-Chn ^negative , according to the intensity of the staining signal by the anti-α2-Chn antibody. FIG. 11 (A) shows the proportions of ^{α2-Chn high} , α2-Chn ^low , and α2-Chn ^negative in RFP-negative cells and RFP-positive cells (Supernova α2-Chn TALEN-expressing cells). 76.0% ± 6.0% (n = 3 mice) of SnRFP-labeled cells were 2-Chn ^negative cells. There were no α2-Chn ^high cells, and the rest were α2-Chn ^low cells. On the other hand, among RFP-positive cells, α2-Chn ^high cells and α2-Chn ^low cells were 95.1% ± 0.5% and 3.2% ± 0.1% (n = 3 mice), respectively. .. In TALEN-expressing cells targeting α2-Chn, α2-Chn protein levels were dramatically reduced. From this, it was confirmed that Supernova-mediated TALEN can successfully inhibit the expression of α2-Chn in CA1 pyramidal neurons of wild-type mice.
To confirm TALEN-mediated genome editing targeting α2-Chn, the inventors introduced a Supernova-mediated α2-ChnTALEN vector into E14.5 from a P1 brain introduced by IUE into a fluorosensitive-active cell sorting (FACS). ) Fluorescently labeled cells were collected. Then, the target locus was amplified by PCR from a pool of 200 cells sorted by FACS, introduced into a plasmid, and cloned. Of the 13 clones, 12 had mutations (3 patterns) and 1 clone was a wild-type sequence. By repeating the experiment, a total of 9 mutation patterns were identified (see FIG. 11 (B)).
FIG. 11B is a schematic diagram showing the target positions of left TALEN and light TALEN in the mouse α2-Chn gene. In FIG. 11B, the broken line indicates the spacer region (17bp). The mutation pattern at the α2-Chn locus is shown at the bottom.
From these data, it was confirmed that genome editing of endogenous genes induced by TALEN via Supernova is very efficient.

[Example 7]
The inventors then performed Supernova-mediated labeled cell-specific gene editing in wild-type mice with CRISPR / Cas9 as another genome editing technique. FIG. 12 (A) shows a schematic representation of a Supernova vector set designed for genetic engineering of Creb1 at the single cell level. To reduce the number of vectors used in IUE, the inventors used a pX330 vector expressing hSpCas9 and sgRNA from a single vector. An LSL cassette was inserted into this vector to allow Cre dependence (see FIG. 12 (A); U6-SgRNA-CAG-LSL-Cas9). This vector was introduced into hippocampal CA1 pyramidal neurons using IUE at E14.5 with the TRE-Cre and CAG-LSL-GFP-tTA vectors. Then, the expression of CREB protein in the hippocampus of P8 mice was examined by immunohistological analysis. The brain was disassembled at P8, coronal sections were prepared, CREB immunohistological analysis and DAPI staining were performed, and fluorescence observation was performed. FIG. 13 (A) shows the ratio of the number of CREB-positive neurons to CREB-negative neurons in GFP-labeled neurons.
FIG. 12B shows the hippocampal CA1 region of a wild-type mouse gene-introduced using the Supernova-CRISPR / Cas9 control (not containing singleguide RNA (sgRNA)). FIG. 12C shows the hippocampal CA1 region of wild-type mice gene-transfected with CRISPR / Cas9 (SnCRISPR / Cas9-Creb1) targeting Supernova-mouse Creb1. In the figure, CREB shows the gene product of Creb1 and is widely and strongly expressed in the hippocampal CA1 region of the brain of wild-type mice. As shown in FIG. 12 (B), expression of CREB was confirmed in all GFP-positive cells expressing the Supernova-CRISPR / Cas9 control (111 of 111 cells, 3 mice used. 3 mice used. FIG. 12 (B). ) See the arrow.).
On the other hand, as shown in the upper part of FIG. 12C, it was confirmed that the expression of CREB was specifically deficient in most GFP-positive cells expressing SnCRISPR / Cas9-Cleb1. (83 cells out of 85 cells. 3 mice were used. See the arrow in the upper row of FIG. 12C.).
In addition, as shown in the lower part of FIG. 12C, very few exceptions were confirmed (2 cells out of 85 cells. See the arrow in the lower part of FIG. 12C). The Scale bar in FIGS. 12B and 12C shows 50 μm.
Finally, the inventors performed cloning of Creb1 in GFP-positive cells sorted by FACS by amplifying the target locus by PCR from a pool of 300 cells and introducing it into a plasmid. Sequence analysis identified 7 mutation patterns in 12 clones (see FIG. 13B). No wild-type clones were identified.
FIG. 13 (B) shows the representative mutations found at the Creb1 locus. The target position of sgRNA is shown by a thick line. The PAM sequence is shaded. The number of clones showing the corresponding mutation pattern is shown on the right.
From these data, it was confirmed that CRISPR / Cas9 via Supernova can knock out one cell-specific gene in vivo very efficiently.

[Example 8]
In order to compare the performance of Cre-SnRFP and Flpe-SnRFP, each of Flpe-SnRFP and Cre-SnRFP having a final concentration of 5 ng / μL was gene-introduced into cortical neurons by IUE at E15.5. CAG-GFP was co-expressed to show transgenic cells.
As a result, as shown in FIG. 14, 14.8% ± 3.3% of GFP-positive neurons (n = 4 mice were used) were labeled on P6 by Cre-SnRFP. On the other hand, when Flpe-SnRFP was used for labeling, the labeled ratio was 1.9% ± 0.2% (n = 4 mice were used). All values are shown as mean ± standard error. The bar shows the average value. ( ^** ) indicates 0.001 <P <0.01 by Welch's t-test. It was confirmed that Flpe-SnRFP showed better sparseness in nerve cell labeling compared to Cre-SnRFP.

[Example 9]
In order to achieve sparsely labeled cell-specific gene manipulation without the use of loxP-introduced mice, knockdown of labeled cell-specific genes in wild-type mice with RNAi via Supernova was performed.
FIG. 15 shows a schematic representation of a Supernova vector set for knockdown of a gene of interest (GOI) in a single cell. The shRNA for GOI was expressed by CAG-LSL-mir30, which is a Cre-dependent shRNA expression vector.
At E14.5, CAG-LSL-mir30 (scrambled sequence of GFP RNAi; left panel) or CAG-LSL-mir30 (GFP RNAi; right panel), along with Supernova nlsRFP (Sn-nlsRFP), by IUE, CAG-loxP -CAT-loxP-GFP reporter Introduced into the cortex of mice. Brains were harvested at P8, sections were prepared and stained with GFP antibody (see FIG. 16 (A)). GFP signals in GFP RNAi-expressing neurons are signals in cells expressing the scrambled sequence of GFP RNAi. It was much lower (see Figure 16 (B)). The knockdown efficiency of GFP was determined by the relative fluorescence intensity of GFP corrected by nlsRFP. ( ^** ) indicates 0.001 <P <0.01 by Welch's t-test.
Also, in E14.5, CAG-LSL-mir30 (scramble sequence of LacZ RNAi; left panel) or CAG-LSL-mir30 (LacZ RNAi; right panel) was added to Rosa-loxP-stop-loxP by IUE together with SnAmcyan. -NlsLacZ (RNZ) was introduced into the cortex of reporter mice. Brains were harvested at P8, sections were prepared and stained with anti-β-Gal antibody (see FIG. 16 (C)).
The knockdown efficiency of LacZ was quantified as the ratio of the fluorescence intensity of the anti-β-Gal antibody to Amcyan. The expression level of β-Gal was significantly reduced in SnAmcyan-labeled neurons co-expressing LacZ RNAi as compared to SnAmcyan-labeled neurons co-expressing the LacZ RNAi scramble sequence (Fig.). 16 (D).). ( ^*** ) indicates P <0.001 by Welch's t-test. The Scale bar in FIGS. 16A and 16C shows 50 μm.
From these facts, it was confirmed that gene knockdown is possible in a labeled cell-specific manner.

[Example 10]
A Supernova vector system based on an adeno-associated virus (hereinafter, also referred to as AAV) was constructed in order to enable labeling of one cell using a virus. Due to the limitation of the insertion size of the AAV vector, the double-floxed inverted open reading frame (DIO) strategy (Sohal VS, Zhang F, Yizhar O, Deisseroth K (2009)) to reduce the size of the fluorescent protein expression vector. neurons and gumma rhythms enhance vector performance. Nature. Jun 4; 459 (7247): 698-702.) Was used (see FIG. 17 (A)).
A mixture of AAV-TRE-Cre and AAV-EF1α-DIO-RFP-tTA (also referred to as AAV-Supernova RFP (AAV-SnRFP)) shown in FIG. 17 (A) is used in the hippocampus and deep cerebral cortex of P10-P13 mice. Was injected into. AAV-EF1α-GFP-WPRE was injected together as a control. Cortical sections were prepared 30 days after infection. As shown in FIGS. 17 (B) to 17 (G), hippocampal CA1 neurons (FIGS. 17 (B) to 17 (D)) and cerebral cortex layer 5 (L5) pyramidal neurons (FIGS. 17 (E)). A clear fluorescent image of ~ 17 (G)) could be obtained. The fluorescent image is sparsely labeled and allows a clear view of detailed cell morphology.
From this result, it was confirmed that the AAV-based Supernova vector system can label individual cells with a small background and high fluorescence intensity. In addition, AAV-based Supernova vector systems retain many of the superior features of IUE-based Supernova vector systems, with comparable sparseness and label brightness 10 days after infection. It has been confirmed that it is adjustable only by adjusting the amount of AAV-TRE-Cre and does not significantly affect the fluorescence intensity.

[Example 11]
In AAV-based Supernova vector systems, only in sparsely labeled neurons is Cre expressed at very high levels sufficient to induce excision of the loxP fragment of the chromosome. To examine the efficiency and specificity of Cre-mediated recombination, AAV-SnRFP was injected into hippocampal CA1 neurons of P10 Rosa26-loxP-stop-loxP-nlsLacZ (RNZ) reporter mice. When the brain was collected 40 days after infection, it was confirmed that LacZ was expressed in most RFP-labeled neurons (76/80 cells, n = 3 mice). In addition, all LacZ-positive cells were labeled with AAV-SnRFP (76/76 cells, n = 3 mice) (see the upper part of FIG. 17 (H)). Similar results were confirmed in the cortical layer (see the lower part of FIG. 17 (H)). From these facts, it was confirmed that Cre-dependent chromosomal recombination is specific to AAV-SnRFP.
Next, the efficiency of knockout of endogenous genes using ^{α2-Chnflox / flox} mice in an AAV-based Supernova vector system was examined. AAV-SnRFP was injected into hippocampal neurons of P2 α2-Chn ^{flox / flox mice.} When the brain was dissected at P18, RFP-negative cells expressed α2-Chn in the non-injected region of hippocampal CA1 (204/210 cells, n = 3 mice). On the other hand, in the AAV-SnRFP infused region of hippocampal CA1, all RFP-positive cells did not express α2-Chn (0/20 cells, n = 3 mice) (see FIG. 17 (I)). From these facts, it was confirmed that the AAV-based Supernova vector system can perform labeled cell-specific gene knockout by loxP-introduced mice (flox mice) with high efficiency.

The materials used in this example are shown below.
[Plasid construction]
<Supernova based on Cre>
For pK031.TRE-Cre and pK029. CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE, see Mizuno H., Luo W., Tarusawa E., Saito Y.M., Sato Y. ., Itohara S., Iwasato T. NMDAR-regulated dynamics of layer 4 neuronal dendrites daring thalamocortical reorgation in neonates.

pK038.CAG-loxP-STOP-loxP-EGFP-ires-tTA-WPRE: EGFP and ires-tTA fragments from the pEGFP-N1 vector (manufactured by Clontech) and pK029 vector, respectively, from HM050 (SEQ ID NO: 5; HM055 (SEQ ID NO: 6; GGAATCCTTATAATTAACTCCGATCTAGGAATTACTTACTTGTACAGCTCGTCCATCGCCGAGA) and HM054 (SEQ ID NO: 7; TCTCGGCATCGACGAGCTGTACAAGTAAGATATCTACAGATCCGAGTTAGTACGAGTCAGTGCTACGAGTCAGGTAGGTACGTGAGTCAGGTAGGTAGGTACGACT The two obtained PCR products were combined and amplified again by PCR using HM050 / HM035 to prepare EGFP-ires-tTA. Next, EGFP-ires-tTA was inserted into the SalI / NotI site of the pK029 vector to obtain the pK038 vector.

pK039.CAG-loxP-STOP-loxP-AmCyan-ires-tTA-WPRE: AmCyan fragment from pAmCyan vector (manufactured by Clontech) was cut out by SalI / StuI digestion, and the SalI / EcoRV site of the pK029 vector was inserted into the SalI / EcoRV site. Obtained.

pK098.CAG-loxP-STOP-loxP-nlstagRFP-ires-tTA-WPRE: Nuclear localization signal (ns) -tagRFP from pK022TagRFP-N (manufactured by EcoRV), HM079 (SEQ ID NO: 9; 10; CCGATATCTTCAATTAAGTTTGTGCCCCAGTTTGCTAGG) was used to amplify by PCR and the amplification product was inserted into the SalI / EcoRV site of the pK038 vector.

pK102.CAG-loxP-STOP-loxP-PSD95eGFP-ires-tTA-WPRE: PSD95eGFP was cut out from pCMV-PSD95GFP (provided by Dr. Okabe of the University of Tokyo) with EcoRI and NotI, blunted with Crenow fragment, and EGFP. In exchange, it was inserted into the blunted SalI / EcoRV of the pK038 vector.

<Supernova based on Flpe>
pK036.TRE-Flpe-WPRE: Flpe coding sequence from pK016.CAG-Flpe-ires-Puro (manufactured by GeneBridge) to AY101 (SEQ ID NO: 11; Then, the amplification product was inserted into the BamHI / NotI site of pK026.TRE-Tight vector (manufactured by Clontech). WPRE excised from the pK029 vector was inserted into the NotI site of the pTRE-Flpe vector.

The loxP-STOP-loxP sequence between EcoRI / SalI of pK037.CAG-FRT-STOP-FRT-RFP-ires-tTA-WPRE: pK029. Was replaced with the FRT-STOP-FRT sequence. FRT-STOP-FRT sequences from pBS302 vector (Gibco-BRL Co.), HM009 (SEQ ID NO: 14; CCGTCGACATGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTAGGTCCCTCGACCTGCAGCCCAAGCTTA); was amplified using / HM008 (GGGAATTCTAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCATTAAGGGTTCCGGATCCTCGGGGACACCA SEQ ID NO: 13).

pK068.CAG-FRT-STOP-FRT-EGFP-ires-tTA-WPRE: EGFP-ires-tTA was cut out from the pK038 vector with SalI and NotI and inserted into the SalI / NotI site of the pK037 vector to prepare a pK068 vector.

<Supernova based on Dre>
From the pK073.TRE-Dre-WPRE: pK055.CAG-Dre-ires-puro.gcc vector (provided by Dr. Morimoto, National Institute of Genetics), WL006 (SEQ ID NO: 15; CGAATTCGCC ACCATGGGTG CT) / WL007 (SEQ ID NO: The Dre sequence was amplified by PCR using No. 16; TGCGGCCGCT CACACTTTCC TCTT). The amplification product was inserted into the EcoRI / NotI site of pK036.TRE-Flpe-WPRE to prepare a pK073 vector.

pK129.CAG-Rox-STOP-Rox-RFP-ires-tTA-WPRE: pK078.JFRC176-10XUAS-rox-dSTOP-rox-myr :: GFP (provided by Gerald Rubin) in BglII and XhoI STOP-Rox was cut out and a pCAGplay vector (Niwa, H., Yamamura, K., and MIyazaki, J. (1991). S.-y., And Hirano, T. (2006). Internal ribosome entry α3β1 suppresses long-term potentiation at inhibitory synapses on the cerebellar Purkinje neur .. The resulting vector was digested with EcoRI and XhoI and inserted into the pK037 vector to replace FRT-STOP-FRT with Rox-stop-Rox.

pK224.CAG-Rox-STOP-Rox-EGFP-ires-tTA-WPRE: Rox-STOP-Rox was excised from pK078 by BglII and XhoI and inserted into the BamHI / XhoI site of the pCAGplay vector. EGFP-ires-tTA was excised from the pK068 vector by SalI and NotI and inserted into the obtained vector. The WPR excised from pK046.CAG-loxP-STOP-loxP-RFP-WPRE with NotI was inserted into the NotI site of the obtained pCAG-Rox-STOP-Rox-EGFP-ires-tTA vector.

<RNAi via Supernova>
pK177.CAG-loxP-STOP-loxP-mir30 (GFP RNAi) and pK178.CAG-loxP-STOP-loxP-mir30 (GFP RNAi scramble control): shRNA and its scramble control for the GFP coding region (441-461). , HM082 (SEQ ID NO: 17; CAGAAGGCTCGAGAAGGTATATTGGCTGTTGACAGTGGGCG) / HM083 (SEQ ID NO: 18; CTAAAGTAGCCCCTCTTGAATTCCGAGGCAGGCAGGCA).
The template oligonucleotide for GFP shRNA is 5'-TGCTGTTGACAGTGGAGCGAAGCCACAAACGTCATCATCATCATCATGTAGTACGAAGCCACAGATACATCATCATGATAGTACGAGCTACGATGGA-3'(HM084; SEQ ID NO: 1). The template oligonucleotide for the GFP shRNA scramble control is 5'-TGCTGTTGACAGGTGAGCGAGCTCATAGAAGCTTACAAATCTAGTTGAAGCCACAGATGGTAGATTGTTAGACTTCTACGGGA-3'(HM085; SEQ ID NO: 2). The amplification product was inserted into the XhoI / EcoRI site of the pCAG-loxP-STOP-loxP-mir30 vector.

pK225.CAG-loxP-STOP-loxP-mir30 (LacZ RNAi) and pK226.CAG-loxP-STOP-loxP-mir30 (LacZ RNAi scramble control): Coding of LacZ for knockdown of LacZ in one cell The shRNA for the region (651-671) and its scramble control were amplified by PCR using HM082 / HM083.
The template oligonucleotide for LacZshRNA is 5'-TGCTGTTGACAGTGGAGCGAAAATCGCTGATGCATTGGATACGATACACCAAATCAGCCTGGA-3'(WL090; SEQ ID NO: 3). The template oligonucleotide for the LacZ shRNA scramble control is 5'-TGCTGTTGACAGTGAGGCGAGGCAACTTCGGGTTAGTTATTATAGTGAAGCCACAGATGTATAAAATATACTAACCGAAGTTGGCCGTGCTACTCTGGA-3'(WL091; SEQ ID NO: 4). The amplification product was inserted into the XhoI / EcoRI site of the pCAG-loxP-STOP-loxP-mir30 vector.

<TALEN vector via Supernova>
pK217.CAG-FRT-STOP-FRT-α2Chn TALEN Left and pK218.CAG-FRT-STOP-FRT-α2Chn TALEN Right: α2Chn TALEN was prepared by a platinum gate TALEN kit using a platinum gate TALEN kit. The expected cleavage site is mouse α2Chn exon 6 at position 237. FRT-STOP-FRT was cut out from pK037 by Spei / SalI and inserted into the SpI / XhoI site of pK209.CMV-A2Chn TALEN Left and pK210.CMV-A2Chn TALEN Right.

<CRISPR / Cas9 vector via Supernova>
pK162 is a codon-optimized spCas9 and chimeric guide RNA expression plasmid (pX330) for humans. Donated by Zhang.
pK237. A fragment containing a portion of the pU6-Chimeric-CAG-LSL-hspCas9: CAG promoter and loxP-STOP-loxP was excised from SnaBI and AgeI of pK029 and inserted at the same site of pK162 (pX330). Since pK237 does not have a guide oligo, it is used as a control in the Supernova-CRISPR / Cas9 experiment.
pK238. pU6-gRNACrev1-CAG-LSL-hspCas9: Annealed guide oligo WL107 (SEQ ID NO: 19; CACCGTTGTCTGCTCCAGATTCCA) / WL108 (SEQ ID NO: 20; AAACTGGATTGGAGCAGACAAC), according to the target sequence cloning protocol published by the Feng Zhang group. Inserted in. The target sequence of the guide RNA is F.I. Designed using online tools provided by the Zhang group (http://crispr.mit.edu/). Genome sequence NC_0000067.6 (18054 ... 18167) from the NCBI database was used for blasting.

<Other construction vectors>
pK046.CAG-loxP-STOP-loxP-RFP-WPRE: a loxP-STOP-loxP sequences from pBS302 vector (Gibco-BRL Co.), HM010 (SEQ ID NO: 21; GGGAATTCTAATAACTTCGTATAGCATACATTATACGAAGTTATATTAAGGGTTCCGGATCCTCGGGGACACC); a / HM007 (CCGTCGACATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCCCTCGACCTGCAGCCCAAGCTTA SEQ ID NO: 22) Amplified using. The RFP sequence was amplified from pK023.TurboRFP-N (manufactured by Evrogen) using HM026 (SEQ ID NO: 23; CGAATTTCTGCAGTCGACGGTGTACCGAGGGCC) / HM030 (SEQ ID NO: 24; GTCGCGCGCCGCATCATCTGTCGCCAGTTT). The WPRE sequence was extracted from the pK021.Lenti-Synapsin-hChR2 (H134R) -EYFP-WPRE vector using HM019 (SEQ ID NO: 25; GCGGCCGCTACTCTGgATTAAAATT) / HM020 (SEQ ID NO: 26; L.-P., Braner, M., Leewald, JF, Kay, K., Watzke, N., Wood, PG, Bamberg, E., Nagal, G., Gottschalk, A. et al. (2007). Multimodal fast optical interrogation of neural array. Nature 446, 633-639.). The three amplification products obtained were inserted into the EcoRI / SalI, SalI / NotI, and NotI sites of the pCAGplay vector, respectively.

<Supernova based on AAV>
pK168 (pAAV-EF1α-DIO-RFP-P2A-tTA-WPRE): tTA sequence pK029 (pTet-Off-Advanced Vector (Gossen M, Mammal H. (1992) Tetracycline gene expression) From promoters.Proc Natl Acad Sci USA Jun 15; 89 (12): 5547-51.)), RY78 (SEQ ID NO: 27; TGCTAGCGCCACCATGTACTAGACTGGA) / RY79 (SEQ ID NO: 28;
P2A-tRFP sequence from pK025 (TurboRFP (Evrogen, Moscow, Russia)) to RY69 (SEQ ID NO: 29; AGGATCCGGAAGCGGAGCTACTAACTTCAGCCCGCTGAAGCAGGCTGGTACGGTACGGACTGAGCTGACTGGACTCGTACGGACTGAGCTGACTGGACTCGACTGGACT
pK168 (pAAV-EF1α-DIO-tTA-P2A-RFP-WPRE) provides tTA and P2A-tRFP with pK167 (pAAV-EF1α-DIO-EYFP) vector (Addgene: 27056, Cambridge, MA, US). It was prepared by inserting it into the / BamHI site and the BamHI / AscI site.
pK170 (pAAV-TRE-Cre-WPRE): The Cre sequence was amplified from pK031 (pCAG-nlsCre-WPRE). The pK170 was produced by inserting Cre into the EcoRI / BglII site of the pK169 (pAAV-TRE-MCS-WPRE) vector (provided by Dr. Yu Hayashi).

<Other AAV vectors>
pK165 (pAAV-EF1α-RFP-WPRE) is available from Iwata, R.M. et. al. (2015). Developmental RacGAP α2-Chimerin Signaling Is A Signaling of the Morphological Features of Dendritic Spines in Adults in Adult.
pK229 (pAAV-EF1α-EGFP-WPRE): The EGFP sequences were from pK024 (pCAsalEGFP) to WL103 (SEQ ID NO: 31; CTGAATTCGCCACCACTGGGTGAGCAAGGGGCGAGGGAGC) / WL104 (SEQ ID NO: 32; To make pK229, the RFP in pK165 (pAAV-EF1α-RFP-WPRE) was replaced with EGFP by subcloning into the EcoRI / BamHI site.

<Animal>
Emx1 Cre KI-ΔNeo Mice (Iwasato, T., Datwani, A., Wolf, AM, Nishiyama, H., Taguchi, Y., Tonegawa, S., Knopfer, S. and Itohara, S. (2000). Cortex-restricted disorder of NMDR1 impairs neuronal patterns in the barrel cortex. Nature 406, 726-731 .; Iwa S., Itohara, S., and Crair, M.C. (2008) Cortical adenylyl cyclase 1 is required for thalamocortical synapse maturation and aspects of layer IV barrel development The Journal of Neuroscience 28, 5931-5943;... Iwasato, T., Inan, M., Kanki, H., Erzurumlu, R.S., Itohara, S., and Crair, MC (2008). IV barrel development. The Journal of Neuroscience 28, 5931-5943.), CAG-loxP-CAT-loxP-GFP reporter mice (Kawamoto, S., Niwa, H. G., Takeda, J., Tabayashi, K., and Miyazaki, J.-i. (2000). A novel reporter mouse strat express synapse enhanced green
upon Cre-mediated recombination. FEBS Letters 470, 263-268.), RNZ Reporter Mouse (Kobayashi, Y., Sano, Y., Vannoni, E., Goto, H., Ikeda, Tsu Oba, A., Kawasaki, H., Kamba, S., Lipp, H.-P., et al. (2013). , Α2-Chn flox mice (Iwata, R., Ohi, K., Kobayashi, Y., Masada, A., Iwama, M., Yasuda, Y., Yamamori, H., Taka ., Itohara, S., et al. (2014). RacGAP α2-Chimaerin Function in Development in Devosts Cognitive Ability in Adult. Cell Report.
Foets for IUE and viral infections were obtained by mating males and females overnight. Noon on the day when the vaginal plug was observed was defined as the embryonic period of 0.5. The day of birth was counted as 0 days after birth.

<In utero electroporation (IUE)>
IUE was performed based on a previous report (Mizuno, H., Hirano, T., and Tagawa, Y. (2007). Evidence for activity-dependent cerebral cortex wiring: formal cerebral engineering) projection neuron activity. The Journal of Neuroscience 27, 6760-6770.). Briefly, pregnant mice were anesthetized with pentobarbital sodium (50 mg / kg body weight) dissolved in saline.
After washing the abdomen with 70% ethanol, a midline laparotomy of about 3 cm was performed and the uterus was removed. For microinjection of DNA, a glass capillary tube (GC150TF-10; Harvard Apparatus) was pulled using a micropipette puller (PC-10; Narisige). A total 0.5 μL of DNA solution was injected into the right lateral ventricle of the foetation using a mouse-controlled pipette system (Drummond Scientific). By holding with tweezers-type electrodes (CY650P10, Unique Medical Imada, Japan), Square electric pulses (40 V; 50 ms) to the foetation via the electroporator (CUY21SC; NepaGene) via the uterus. It was transmitted 5 times at a rate. The positions of the electrodes for gene transfer into cortical neurons were based on previous reports (Saito, T., and Nakatsuji, N. (2001). Effective gene transfer in vivo. Development 240, 237-246.). After electroporation, the uterus was returned to the abdominal cavity and the abdominal wall and skin were sutured by surgical suture.
The plasmid for IUE was purified using the NucleoBond Xtra MIDI kit (manufactured by Macherey-Nagel). The DNA lysate compositions used in each experiment are summarized below.

In Cre-based Supernova labeling, pK031.TRE-Cre (5 ng / μL) and pK029.CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE (1 μg / μL) or pK038.CAG-loxP-STOP-loxP -EGFP-ires-tTA-WPRE (1 μg / μL) or pK039.CAG-loxP-STOP-loxP-AmCyan-ires-tTA-WPRE (1 μg / μL) was used.

In the Flpe-based Supernova label of cortical neurons, pK036.TRE-Flpe-WPRE (5 ng / μL) and pK037.CAG-FRT-STOP-FRT-RFP-ires-tTA-WPRE (1 μg / μL) or pK068.CAG- FRT-STOP-FRT-EGFP-ires-tTA-WPRE (1 μg / μL) was used.

In the Flpe-based Supernova labeling of hippocampal CA1 neurons, pK036.TRE-Flpe-WPRE (50 ng / μL) and pK037.CAG-FRT-STOP-FRT-RFP-ires-tTA-WPRE (1 μg / μL) were used.

In Dre-based Supernova labeling, pK073.TRE-Dre-WPRE (5 ng / μL) and pK129.CAG-Rox-STOP-Rox-RFP-ires-tTA-WPRE (1 μg / μL) or pK038.CAG-loxP-STOP. -LoxP-EGFP-ires-tTA-WPRE (1 μg / μL) or pK224.CAG-Rox-STOP-Rox-EGFP-ires-tTA-WPRE (1 μg / μL) was used.

In Flpe-SnGFP and Dre-SnGFP labeling of cortical neurons in Emx1-Cre KI mice, pK046.CAG-loxP-STOP-loxP-RFP-WPRE (1 μg / μL) was co-introduced as a control.

As co-expression experiments, pK031.TRE-Cre (10 ng / μL), pK038.CAG-loxP-STOP-loxP-EGFP-ires-tTA-WPRE (1 μg / μL) and pK098.CAG-loxP-STOP-loxP- nlstagRFP-ires-tTA-WPRE (1 μg / μL) or pK029.CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE (1 μg / μL) and pK102.CAG-loxP-STOP-loxP-PSD95eGFP-re Used with tTA-WPRE (1 μg / μL).

In a supernova-mediated sparse gene knockdown experiment using CAG-loxP-CAT-loxP-GFP reporter Tg mice, pTRE-Cre (10 ng / μl), pK098.CAG-loxP-STOP-loxP-nlstagRFP- ires-tTA-WPRE (1 μg / μl) and pK177.CAG-loxP-STOP-loxP-mir30 (GFP RNAi) (1 μg / μl) or pK178.CAG-loxP-STOP-loxP-mir30 (GFP RNAi scramble control) ) (1 μg / μl)-containing DNA lysate was used.

In a supernova-mediated sparse gene knockdown experiment using RNZ mice, pTRE-Cre (10 ng / μl), pK039: pCAG-loxP-STOP-loxP-AmCyan-ires-tTA-WPRE (1 μg / μL) and pK225 : A DNA lysate containing pCAG-loxP-STOP-loxP-mir30 (LacZ RNAi) (1 μg / μl) or pK226: pCAG-loxP-STOP-loxP-mir30 (LacZ RNAi scramble control) (1 μg / μL). Using.

In an experiment of α2-Chn gene manipulation via TALEN by the Supernova system, pK036.TRE-Flpe-WPRE (50 ng / μl), pK037.CAG-FRT-STOP-FRT-RFP-ires-tTA-WPRE (1 μg / μl) ), PK217.CAG-FRT-STOP-FRT-α2Chn TALEN Left (1 μg / μl), and pK218.CAG-FRT-STOP-FRT-α2Chn TALEN Right (1 μg / μl).

<Preparation of virus>
AAV was prepared using an AAV-DJ / 8 Helper Free Packaging System (Cell Biolabs, San Diego, CA, USA). The pAAV expression vector is pAAV-DJ (Agilent Technologies: 240071) and pHelper (Agilent Technologies: 240071), and the complete medium (DMEM product) containing pencillin and streptomycin. HEK293T or HEK293FT cells in sodium bicarbonate solution) were transfected. After 72 hours of transfection, cells were gently scraped from the flask and centrifuged at 2,500 rpm for 30 minutes. The supernatant was filtered through a 0.45 μm membrane filter (Millipore, Bedford, MA, USA) and transferred to an ultracentrifugation tube. The virus precipitate was suspended in 100 μL of cold PBS and stored at -80 ° C. The titer of each virus was determined by quantitative PCR using primer set WL123 (SEQ ID NO: 33; GTATGGAGCAAGGGCAAG) / WL124 (SEQ ID NO: 34; AGGCGGAGATTGCAGTGAG). Virus samples were treated with DNase I at 37 ° C. for 30 minutes. DNase I was inactivated by incubation at 65 ° C. for 10 minutes. Standard curves were prepared using a 10-fold dilution series (10-10 ⁸ copies / μL) of pre-purified pK165 plasmid digested with EcoRI.

<Injection of virus>
P10-P13 mice were anesthetized with 1.7-2.5% isoflurane. The injection site of the skin was opened and a hole was made in the skull using a hand drill (MINIMO, Tokyo, Japan). The virus was injected into the left hippocampus or skin layer. During the virus infusion, the dose of isoflurane was reduced to 1.5%. In the hippocampal CA1 region and the deep skin layer, the injection site of labeled neurons was located approximately 1 mm outside the sagittal suture and -1.5 mm to -0.8 mm from the dural surface. In the virus injection of P2 mice, the injection site was located 1.5 mm to the left of the human character suture and -1.5 mm to -1.0 mm from the dural surface. Virus delivery was performed at a rate of 220 nL / min using a programmed 34 gauge oblique angle NanoFil seedle (World Precision Instruments, Sarasota, FL, USA) syringe pump (KD Scientific). The viral vector used for injection is shown below.

Normal labeling with AAV, AAV-EF1α-RFP- WPRE the (5.5 × 10 ¹¹ genome copies / mL) was diluted 10 to 10 ³ times with PBS. Diluted or undiluted AAV-EF1α-RFP-WPRE, AAV-EF1α-GFP-WPRE (3.5x10 ¹¹ genome copy / mL) and PBS were mixed in equal amounts. One mouse was injected with 1000 nL of virus mixture.
Labeling with AAV-SnRFP, AAV-TRE-Cre-WPRE (2.3x10 ¹³ genome copy / mL) was diluted ^{10 2} to 10 ^{6 times with PBS.} Diluted or undiluted AAV-TRE-Cre-WPRE, AAV-EF1α-DIO-tTA-RFP (1.7x10 ¹³ genome copy / mL), and AAV-EF1α-GFP-WPRE (3.5x10 ¹¹ genome) Copy / mL) PBS was mixed in equal amounts and used for infusion. In FIG. 17 (B) ~ (G) , was used to AAV-TRE-Cre-WPRE was diluted 10 ⁵ fold. One mouse was injected with 1000 nL of virus mixture.
In the injection of AAV-SnRFP to RNZ mouse, AAV-TRE-Cre-WPRE the (2.3 × 10 ¹³ genome copies / mL) was diluted 10 ⁵ times in PBS, AAV-EF1α-DIO- tTA-RFP (1.7x10 ^{Equal amounts were mixed with 13} genome copies / mL). One mouse was injected with 1000 nL of virus mixture. In the injection of AAV-SnRFP to [alpha] 2-Chn floxed mouse, AAV-TRE-Cre-WPRE the (2.3 × 10 ¹³ genome copies / mL) was diluted 10 ⁴ times with PBS, AAV-EF1α-DIO- tTA-RFP ( It was mixed in equal amounts with ^{1.7x10 13 genome copy / mL).} One mouse was injected with 300 nL of virus mixture.

The configurations and combinations thereof in each of the embodiments described above are examples, and the configurations can be added, omitted, replaced, and other changes are possible without departing from the spirit of the present invention. Moreover, the present invention is not limited to each embodiment, but is limited only to the scope of claims.

Claims (4)

A method for controlling the expression of a target gene in a dense non-human cell group by expressing and labeling a gene encoding a fluorescent protein specifically for each cell.
(1) A first vector containing a first regulator containing a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE.
A cassette containing the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side, and a cassette located downstream of the cassette, said exogenous. A second vector containing a second regulator, including a gene encoding the fluorescent protein and a gene encoding a tetracycline-regulated transactivator (tTA), under the control of an expression promoter.
A fifth control in which one or more genes, including a guide RNA, are operably linked to the cassette, a gene encoding the Cas9 protein located downstream of the cassette and under the control of the exogenous expression promoter. A third vector containing the factor, or
The cassette, the vector of the third A containing the regulator of the third A including TALEN left, located downstream of the cassette and under the control of the exogenous expression promoter, and the cassette and the cassette. A fourth vector containing a fourth regulator, including TALEN light, located downstream and under the control of the exogenous expression promoter, was introduced into cells and labeled with the fluorescent protein in a cell-specific manner. Edit the target gene,
(2) A first vector containing a first regulator containing a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE.
A cassette containing the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side, and a cassette located downstream of the cassette, said exogenous. A second vector containing a second regulator, including a gene encoding the fluorescent protein and a gene encoding a tetracycline-regulated transactivator (tTA), under the control of an expression promoter.
The site-specific recombinant enzyme recognition sequence, the target gene, and the site-specific recombinant enzyme recognition sequence are introduced into cells having chromosomes containing the site-specific recombinant enzyme recognition sequence in this order from the 5'side, and the target is cell-specifically labeled with the fluorescent protein. Knock out a gene or
(3) A first vector containing a first regulator containing a tetracycline response factor (TRE) and a gene encoding a site-specific recombinant enzyme under the control of the TRE.
A cassette containing the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side, and a cassette located downstream of the cassette, said exogenous. A second vector containing a second regulator, including a gene encoding the fluorescent protein and a gene encoding a tetracycline-regulated transactivator (tTA), under the control of an expression promoter.
A cassette containing the exogenous expression promoter, the site-specific recombinant enzyme recognition sequence, the transcription termination sequence, and the site-specific recombinant enzyme recognition sequence in this order from the 5'side, and a cassette located downstream of the cassette, said exogenous. A third vector containing an RNAi-inducible nucleic acid and a third regulator, which is under the control of an expression promoter, is introduced into cells, and the target gene is knocked down in a cell-specific manner labeled with the fluorescent protein. the, way. The method according to claim 1, wherein the cell is a nerve cell . The method according to claim 1 or 2, wherein each vector is introduced region-specifically into a mouse having pre-cells . The method of claim 3, wherein the region is the hippocampus .

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