JP2021106509A - Gene separation expression method, polynucleotide, and genetic transformation kit - Google Patents

Abstract

To provide a polynucleotide for separated expression of different target products in a cell population containing a plurality of cells, e.g. present in the body of a living animal.SOLUTION: A polynucleotide has a sequence encoding a first polynucleotide editing enzyme, a promoter sequence that can express the first polynucleotide editing enzyme in a host, and a recognition sequence of a second polynucleotide editing enzyme, the polynucleotide capable of expressing the first polynucleotide editing enzyme. The second polynucleotide editing enzyme recognition sequence is designed to inhibit the expression of the first polynucleotide editing enzyme in the presence of the second polynucleotide editing enzyme.SELECTED DRAWING: Figure 1

Description

The present specification discloses a polynucleotide, a kit for gene recombination, a method for separating and expressing a gene, a method for visualizing a target product, and a cell population.

Multiple fluorescence expression using a plurality of fluorescent proteins in vivo is widely used for visualizing the structure of tissues (Non-Patent Documents 1 to 3). Generally, when expressing a target product such as a fluorescent protein in the body of a living animal, gene transfer using a viral vector is performed. Further, in order to express a target product in a cell-specific manner, a gene encoding the target product is linked downstream of a promoter whose expression is induced in a specific cell, and the gene is introduced into an animal tissue.

Livet et al., 2007; Nature 450, 56-62. Weissman and Pan, 2015; GENETICS 199, 293-306. Sakaguchi et al., 2018; Elife. 7, e40350.

When a gene is introduced into an allogeneic cell population using a viral vector in vivo in a living animal, the introduced target product is expressed in all cells in contact with the viral vector. For example, adjacent cells are distinguished from each other. It was difficult to express different target products individually. According to the micromanipulation method, it is possible to express different target products in different cells, but in the tissues existing in the body of a living animal, gene transfer by the manipulation method is too inefficient and impractical. ..

One object of the present invention is to provide a polynucleotide for separating and expressing different target products in a cell population containing a plurality of cells existing in a living animal body or the like, regardless of the micromanipulation method. And.

As a result of diligent research, the present inventor has found that the problem can be solved by using a plurality of polynucleotide editing enzymes.
The present invention has been completed based on the findings and includes the following aspects.

Item 1. The first, which comprises a sequence encoding a first polynucleotide-editing enzyme, a promoter sequence capable of expressing the first polynucleotide-editing enzyme in a host, and a recognition sequence of a second polynucleotide-editing enzyme. A polynucleotide capable of expressing the polynucleotide editing enzyme of the above, and the recognition sequence of the second polynucleotide editing enzyme expresses the expression of the first polynucleotide editing enzyme in the presence of the second polynucleotide editing enzyme. The polynucleotide designed to suppress.
Item 2. The recognition sequence of the second polynucleotide editing enzyme lacks the sequence encoding the first polynucleotide editing enzyme in the presence of the second polynucleotide editing enzyme, and the first polynucleotide editing by frame shift. Item 2. The polynucleotide according to Item 1, wherein the sequence encoding the enzyme is designed so as not to be correctly translated or inverted.
Item 3. The recognition sequence for the second polynucleotide-editing enzyme is designed to degrade transcripts from the sequence encoding the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme. Item 2. The polynucleotide according to Item 1.
Item 4. A kit for gene recombination, which comprises a first polynucleotide capable of expressing a first polynucleotide editing enzyme and a second polynucleotide capable of expressing a second polynucleotide editing enzyme, wherein the first polynucleotide is capable of expressing the first polynucleotide editing enzyme. The polynucleotide 1 includes a sequence encoding the first polynucleotide editing enzyme, a promoter sequence capable of expressing the first polynucleotide editing enzyme in a host, and a recognition sequence of the second polynucleotide editing enzyme. The recognition sequence of the second polynucleotide editing enzyme, which comprises, is designed to suppress the expression of the first polynucleotide editing enzyme in the presence of the second polynucleotide editing enzyme. , The second polynucleotide is a sequence encoding a second polynucleotide editing enzyme, a promoter sequence capable of expressing the second polynucleotide editing enzyme in a host, and recognition of the first polynucleotide editing enzyme. A polynucleotide comprising a sequence, wherein the recognition sequence of the first polynucleotide editing enzyme suppresses the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme. The kit for gene recombination designed for.
Item 5. Method for separating and expressing genes, including the following first step and second step: First step: A polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme, and a second step. Second polynucleotide gene-introducing a polynucleotide for expressing a second polynucleotide-editing enzyme capable of expressing the polynucleotide editing enzyme of the above, and a second step: a polynucleotide for expressing a first target product capable of expressing a first target product. Step of gene introduction, where the polynucleotide for expressing the first polynucleotide editing enzyme can express a sequence encoding the first polynucleotide editing enzyme and the first polynucleotide editing enzyme in a host. The recognition sequence of the second polynucleotide editing enzyme, which comprises a promoter sequence and a recognition sequence of the second polynucleotide editing enzyme, is the recognition sequence of the first polynucleotide editing enzyme in the presence of the second polynucleotide editing enzyme. Designed to suppress the expression of the enzyme; the polynucleotide for expressing the second polynucleotide-editing enzyme is a sequence encoding the second polynucleotide-editing enzyme and the second polynucleotide-editing enzyme in a host. The recognition sequence of the first polynucleotide editing enzyme comprises an expressible promoter sequence and a recognition sequence of the first polynucleotide editing enzyme, and the recognition sequence of the first polynucleotide editing enzyme is a second in the presence of the first polynucleotide editing enzyme. Designed to suppress the expression of the polynucleotide editing enzyme; and the polynucleotide for expressing the first target product is capable of expressing the sequence encoding the first target product and the first target product in a host. Includes a promoter sequence and a recognition sequence for the first polynucleotide editing enzyme.
Item 6. Further, in the above two steps, the gene is introduced into a second target product expression polynucleotide capable of expressing the second target product, and the second target product expression polynucleotide is the second target product. Item 5. The method for separating and expressing a gene according to Item 5, which comprises a sequence encoding the above, a promoter sequence capable of expressing the second target product in a host, and a recognition sequence of the second polynucleotide editing enzyme.
Item 7. Visualization method of target product including the following first step, second step, and third step: First step: For expression of first polynucleotide editing enzyme capable of expressing first polynucleotide editing enzyme A step of gene-introducing a polynucleotide and a polynucleotide for expressing a second polynucleotide-editing enzyme capable of expressing a second polynucleotide-editing enzyme, a second step: a first target product capable of expressing a first target product. A step of gene-introducing an expression polynucleotide and a third step: a step of visualizing the first target product; where the first polynucleotide-editing enzyme-expressing polynucleotide is a first polynucleotide-editing enzyme. A sequence encoding the above, a promoter sequence capable of expressing the first polynucleotide editing enzyme in a host, and a recognition sequence of the second polynucleotide editing enzyme, and recognition of the second polynucleotide editing enzyme. The sequence is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme; the second polynucleotide-editing enzyme-expressing polynucleotide is the second polynucleotide. A sequence encoding a nucleotide editing enzyme, a promoter sequence capable of expressing the second polynucleotide editing enzyme in a host, and a recognition sequence of the first polynucleotide editing enzyme are included, and the first polynucleotide editing enzyme is included. The recognition sequence of the enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme; and the first polynucleotide for expressing the target product is the first. Includes a sequence encoding the target product of the above, a promoter sequence capable of expressing the first target product in a host, and a recognition sequence of the first polynucleotide editing enzyme.
Item 8. Further, in the above two steps, the gene is introduced into a second target product expression polynucleotide capable of expressing the second target product, and the second target product expression polynucleotide is the second target product. A sequence encoding the above, a promoter sequence capable of expressing the second target product in a host, and a recognition sequence of the second polynucleotide editing enzyme are included, and the second target product is obtained in the above three steps. Item 8. The method for visualizing a target product according to Item 7, which comprises visualization.
Item 9. A first group of cells into which a polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme has been introduced, and a second polynucleotide-editing capable of expressing a second polynucleotide-editing enzyme. Cell population including a second group of cells into which an enzyme-expressing polynucleotide has been introduced: Here, the first polynucleotide-editing enzyme-expressing polynucleotide includes a sequence encoding a first polynucleotide-editing enzyme. The recognition sequence of the second polynucleotide editing enzyme includes a promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and a recognition sequence of the second polynucleotide editing enzyme, and the recognition sequence of the second polynucleotide editing enzyme is a second. Designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the polynucleotide-editing enzyme; the second polynucleotide-editing enzyme-expressing polynucleotide encodes a second polynucleotide-editing enzyme. The recognition sequence of the first polynucleotide editing enzyme includes a sequence to be used, a promoter sequence capable of expressing the second polynucleotide editing enzyme in a host, and a recognition sequence of the first polynucleotide editing enzyme. , Designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
Item 10. The first cell population comprises a sequence encoding the second target product, a promoter sequence capable of expressing the second target product in a host, and a recognition sequence of the second polynucleotide editing enzyme. A polynucleotide for expressing a second target product; and / or the second cell population includes a sequence encoding the first target product and a promoter sequence capable of expressing the first target product in a host. Item 9. The cell population according to Item 9, which comprises the recognition sequence of the first polynucleotide editing enzyme and the polynucleotide for expressing the first target product.
Item 11. A sequence encoding the first polynucleotide editing enzyme, a promoter sequence capable of expressing the first polynucleotide editing enzyme in a host, a recognition sequence of the second polynucleotide editing enzyme, and a third polynucleotide editing A polynucleotide for expressing a polynucleotide editing enzyme, which comprises a recognition sequence of an enzyme, wherein the recognition sequence of the second polynucleotide editing enzyme is a first poly in the presence of the second polynucleotide editing enzyme. Designed to suppress the expression of the nucleotide editing enzyme, the recognition sequence of the third polynucleotide editing enzyme suppresses the expression of the first polynucleotide editing enzyme in the presence of the third polynucleotide editing enzyme. The polynucleotide for expressing the polynucleotide editing enzyme, which is designed as described above.
Item 12. A kit for gene recombination, which comprises a third polynucleotide-editing enzyme-expressing polynucleotide, a fourth polynucleotide-editing enzyme-expressing polynucleotide, and a fifth polynucleotide-editing enzyme-expressing polynucleotide. The third polynucleotide-editing enzyme-expressing polynucleotide includes a sequence encoding the first polynucleotide-editing enzyme, a promoter sequence capable of expressing the first polynucleotide-editing enzyme in a host, and a second polynucleotide. A polynucleotide comprising a recognition sequence of a polynucleotide editing enzyme and a recognition sequence of a third polynucleotide editing enzyme, wherein the recognition sequence of the second polynucleotide editing enzyme is a second polynucleotide editing enzyme. Designed to suppress the expression of the first polynucleotide editing enzyme in the presence of, the recognition sequence of the third polynucleotide editing enzyme is the first polynucleotide editing enzyme in the presence of the third polynucleotide editing enzyme. Designed to suppress the expression of the polynucleotide-editing enzyme, the fourth polynucleotide-editing enzyme-expressing polynucleotide comprises a sequence encoding the second polynucleotide-editing enzyme and the second polynucleotide-editing enzyme. A polynucleotide comprising a promoter sequence expressible in a host, a recognition sequence of a first polynucleotide editing enzyme, and a recognition sequence of a third polynucleotide editing enzyme, wherein the first polynucleotide editing is performed. The recognition sequence of the enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme, and the recognition sequence of the third polynucleotide editing enzyme is the third. Designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the polynucleotide-editing enzyme of the above, the fifth polynucleotide-editing enzyme-expressing polynucleotide encodes a third polynucleotide-editing enzyme. A poly containing a sequence to be used, a promoter sequence capable of expressing the third polynucleotide editing enzyme in a host, a recognition sequence of the first polynucleotide editing enzyme, and a recognition sequence of the second polynucleotide editing enzyme. The recognition sequence of the first polynucleotide editing enzyme, which is a nucleotide, is designed to suppress the expression of the third polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme, and is designed to suppress the expression of the third polynucleotide editing enzyme. The recognition sequence of the polynucleotide-editing enzyme of the third polynucleotide-editing fermentation in the presence of the second polynucleotide-editing enzyme. The kit, which is designed to suppress the expression of the element.
Item 13. Method of separating and expressing genes, including the following steps i and ii: Step i: A polynucleotide for expressing a third polynucleotide-editing enzyme capable of expressing the first polynucleotide-editing enzyme, and a second. A step of gene-introducing a fourth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the polynucleotide editing enzyme of the above and a fifth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the third polynucleotide-editing enzyme. And the second step ii: a step of gene-introducing a first target product expression polynucleotide capable of expressing the first target product and a second target product expression polynucleotide capable of expressing the second target product; Here, the third polynucleotide-editing enzyme-expressing polynucleotide includes a sequence encoding the first polynucleotide-editing enzyme, a promoter sequence capable of expressing the first polynucleotide-editing enzyme in a host, and a first. A polynucleotide comprising a recognition sequence of a second polynucleotide editing enzyme and a recognition sequence of a third polynucleotide editing enzyme, wherein the recognition sequence of the second polynucleotide editing enzyme is a second polynucleotide. Designed to suppress the expression of the first polynucleotide editing enzyme in the presence of the editing enzyme, the recognition sequence of the third polynucleotide editing enzyme is the first in the presence of the third polynucleotide editing enzyme. Designed to suppress the expression of one polynucleotide editing enzyme, the fourth polynucleotide editing enzyme expression polynucleotide includes a sequence encoding a second polynucleotide editing enzyme and the second polynucleotide editing enzyme. A polynucleotide comprising a promoter sequence capable of expressing an enzyme in a host, a recognition sequence of a first polynucleotide editing enzyme, and a recognition sequence of a third polynucleotide editing enzyme, wherein the first polynucleotide The recognition sequence of the nucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme, and the recognition sequence of the third polynucleotide editing enzyme is Designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme, the fifth polynucleotide-editing enzyme-expressing polynucleotide is the third polynucleotide-editing enzyme. A sequence encoding the above, a promoter sequence capable of expressing the third polynucleotide editing enzyme in the host, and a recognition sequence of the first polynucleotide editing enzyme. And the recognition sequence of the second polynucleotide editing enzyme, which is a polynucleotide, wherein the recognition sequence of the first polynucleotide editing enzyme is a third in the presence of the first polynucleotide editing enzyme. Designed to suppress the expression of the polynucleotide-editing enzyme of the second polynucleotide-editing enzyme, the recognition sequence of the second polynucleotide-editing enzyme exhibits the expression of the third polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme. The first target product expression polynucleotide, which is designed to be suppressed, includes a sequence encoding the first target product, a promoter sequence capable of expressing the first target product in a host, and the first target product. A polynucleotide for expressing a second target product, which comprises a recognition sequence of a polynucleotide editing enzyme, includes a sequence encoding the second target product and a promoter sequence capable of expressing the second target product in a host. , The recognition sequence of the second polynucleotide editing enzyme, and the like.
Item 14. Visualization method of target product including the following step i, step ii, and step iii: Step i: Poly for expressing a third polynucleotide editing enzyme capable of expressing the first polynucleotide editing enzyme. A nucleotide, a fourth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a second polynucleotide-editing enzyme, and a fifth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a third polynucleotide-editing enzyme. A step of introducing a gene and a second step: a polynucleotide for expressing a first target product capable of expressing a first target product, a polynucleotide for expressing a second target product capable of expressing a second target product, and a polynucleotide for expressing the second target product. Step; iii step: A step of visualizing the first target product and the second target product, wherein the third polynucleotide editing enzyme-expressing polynucleotide is the first polynucleotide. A sequence encoding an editing enzyme, a promoter sequence capable of expressing the first polynucleotide editing enzyme in a host, a recognition sequence of a second polynucleotide editing enzyme, and a recognition sequence of a third polynucleotide editing enzyme. The recognition sequence of the second polynucleotide editing enzyme, which comprises, is designed to suppress the expression of the first polynucleotide editing enzyme in the presence of the second polynucleotide editing enzyme. The recognition sequence of the third polynucleotide editing enzyme is designed to suppress the expression of the first polynucleotide editing enzyme in the presence of the third polynucleotide editing enzyme, and the fourth polynucleotide is said to be suppressed. The polynucleotide for expressing an editing enzyme includes a sequence encoding a second polynucleotide editing enzyme, a promoter sequence capable of expressing the second polynucleotide editing enzyme in a host, and a recognition sequence of one polynucleotide editing enzyme. , The recognition sequence of the third polynucleotide editing enzyme, which is a polynucleotide, wherein the recognition sequence of the first polynucleotide editing enzyme is a second in the presence of the first polynucleotide editing enzyme. Designed to suppress the expression of the polynucleotide editing enzyme, the recognition sequence of the third polynucleotide editing enzyme suppresses the expression of the second polynucleotide editing enzyme in the presence of the third polynucleotide editing enzyme. The fifth polynucleotide-editing enzyme-expressing polynucleotide is a sequence encoding a third polynucleotide-editing enzyme and the third polynucleotide-editing. A polynucleotide comprising a promoter sequence capable of expressing an enzyme in a host, a recognition sequence of a first polynucleotide editing enzyme, and a recognition sequence of a second polynucleotide editing enzyme, wherein the first polynucleotide The recognition sequence of the nucleotide editing enzyme is designed to suppress the expression of the third polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme, and the recognition sequence of the second polynucleotide editing enzyme is Designed to suppress the expression of the third polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme, the first polynucleotide for expressing the target product comprises a sequence encoding the first target product. , The promoter sequence capable of expressing the first target product in the host and the recognition sequence of the first polynucleotide editing enzyme, and the second target product expression polynucleotide is the second target product. A sequence encoding the above, a promoter sequence capable of expressing the second target product in a host, and a recognition sequence of the second polynucleotide editing enzyme.
Item 15. A third cell group into which a polynucleotide for expressing a third polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme has been introduced, and a fourth polynucleotide-editing capable of expressing a second polynucleotide-editing enzyme. A fourth cell group into which an enzyme-expressing polynucleotide has been introduced, and a fifth cell group into which a fifth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a third polynucleotide-editing enzyme has been introduced. Cell population including: Here, the polynucleotide for expressing the third polynucleotide editing enzyme is capable of expressing the sequence encoding the first polynucleotide editing enzyme and the first polynucleotide editing enzyme in the host. A polynucleotide comprising a promoter sequence, a recognition sequence of a second polynucleotide editing enzyme, and a recognition sequence of a third polynucleotide editing enzyme, wherein the recognition sequence of the second polynucleotide editing enzyme is. Designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme, the recognition sequence of the third polynucleotide-editing enzyme is that of the third polynucleotide-editing enzyme. Designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the fourth polynucleotide-editing enzyme-expressing polynucleotide, the sequence encoding the second polynucleotide-editing enzyme and the said second polynucleotide-editing enzyme. A polynucleotide comprising a promoter sequence capable of expressing 2 polynucleotide editing enzymes in a host, a recognition sequence of a first polynucleotide editing enzyme, and a recognition sequence of a third polynucleotide editing enzyme. The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme, and the third polynucleotide editing enzyme is said to be suppressed. The recognition sequence of is designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme, and the fifth polynucleotide-editing enzyme-expressing polynucleotide is the third polynucleotide. The sequence encoding the polynucleotide editing enzyme of the above, the promoter sequence capable of expressing the third polynucleotide editing enzyme in the host, the recognition sequence of the first polynucleotide editing enzyme, and the second polynucleotide editing enzyme. A polynucleotide comprising a recognition sequence, wherein the recognition sequence of the first polynucleotide editing enzyme is the presence of the first polynucleotide editing enzyme. Designed to suppress the expression of the third polynucleotide-editing enzyme in the presence, the recognition sequence of the second polynucleotide-editing enzyme is the third polynucleotide in the presence of the second polynucleotide-editing enzyme. Designed to suppress the expression of editing enzymes.

Different target products are isolated and expressed in a cell population containing a plurality of cells existing in a living animal body or the like.

FIG. 1 (A) shows the construct used in Example 1 (BATTLE-1). (1) shows the design of the recombinase expression vector. (2) shows the structure of the target product expression vector. FIG. 1 (B) is an example in which the mechanism of Example 1 (BATTLE-1) is represented by a scheme. FIG. 1 (C) shows the construct used in Example 2 (BATTLE-2). FIG. 2 (A) is an image when Lenti-Camk2 promoter-FRT5-Cre-FRT5-WPRE and Lenti-Camk2 promoter -loxN-FLPO-loxN-WPRE are introduced at a ratio of 1: 1. FIG. 2 (B) is a strongly enlarged view of FIG. 2 (A). FIG. 2C is a graph quantifying the ratio of mCherry + cells, YFP + cells, and mCherry + / YFP + cells in the dentate gyrus. FIG. 2 (D) is an image when Lenti-Camk2 promoter-FRT5-Cre-FRT5-WPRE and Lenti-Camk2 promoter -loxN-FLPO-loxN-WPRE are introduced at a ratio of 1: 0.01. FIG. 2 (E) is a strongly enlarged view of FIG. 2 (D). FIG. 2F is a graph quantifying the ratio of mCherry + cells, YFP + cells, and mCherry + / YFP + cells in the dentate gyrus. FIG. 3 (A) shows Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE, Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE, and Lenti- It is an image when Camk2 promoter-loxN-rcRox-FLPO-rcRox-loxN-WPRE is introduced at a ratio of 1: 1: 1. Figure 3 (B) shows Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE, Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE, and Lenti- It is an image when Camk2 promoter-loxN-rcRox-FLPO-rcRox-loxN-WPRE was introduced at a ratio of 200: 1:10. FIG. 3C shows mCherry + cells, YFP + cells, and double positive cells positive for both mCherry and YFP in the dentate gyrus subjected to Multi-sparse Transgene Expression (hereinafter referred to as “mCherry + / YFP + cells”). ) Is a quantified graph. FIG. 3D shows a fluorescence image of the hippocampus when GFP was expressed using a common conventional expression vector. FIG. 3 (E) shows a fluorescence image of the hippocampus subjected to Multi-sparse Transgene Expression. FIG. 4 (A) (left) shows a typical maximal magnified image of the CA3 region infected with the conventional virus AAV-CAG-GFP. FIG. 4A (right) shows a magnified confocal image of the box area on the left. The upper part of FIG. 4B shows a schematic diagram of the DG-CA3 synapse visualized using 4.1 times BATTLE-1-ExM. The lower part of FIG. 4B shows a typical maximum strong magnified image of the CA3 bright layer visualized using BATTLE-1-ExM. The upper left of FIG. 4 (C) shows the maximum strongly magnified photographed image of the box area of FIG. 4 (B). The upper right of FIG. 4 (C) shows an enlarged confocal image of the box area on the upper left of FIG. 4 (C). FIG. 4 (D) shows a cross-sectional line profile of the fluorescence intensity of the region surrounded by the square on the right side of FIG. 4 (C). FIG. 4D shows a typical volume-rendered three-dimensional image of the entire synaptic structure of the DG-CA3 synapse. The scale bar represents 732 nm. FIG. 4 (F) shows a three-dimensional image with the same volume rendering of FIG. 4 (D) without YFP. The left side of FIG. 4 (G) shows an enlarged image of FIG. 4 (E). The right side of FIG. 4 (G) shows the continuous continuous image reconstruction of the enclosed area on the left side of FIG. 4 (G) (left). The scale bar represents 500 nm. 5 (A) to 5 (E) are images in which the three-dimensional structure of synapses from granule neurons to hilar mossy cells is visualized on a nanoscale by volume rendering. FIG. 5A shows a weakly enlarged image of mCherry and YFP. FIG. 5 (B) shows a strongly enlarged image of FIG. 5 (A). FIG. 5C shows captured images of YFP and Homer. FIG. 5D shows captured images of YFP and Homer. FIG. 5 (D) shows captured images of YFP, Homer, and Bassoon. FIG. 5 (E) shows captured images of YFP, mCherry, Homer, and Bassoon.

The invention disclosed herein relates to a method for expressing a target product in which only a part of the cells express the target product in a cell population containing a plurality of cells using two or more kinds of polynucleotide editing enzymes. In the first aspect, as shown in FIGS. 1 (A) and 1 (1), first, a polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme, and a second polynucleotide. A polynucleotide having a recognition sequence for an editing enzyme and a polynucleotide for expressing a second polynucleotide editing enzyme capable of expressing a second polynucleotide editing enzyme, and a poly having a recognition sequence for the first polynucleotide editing enzyme. A nucleotide is introduced into a cell population containing a plurality of cells such as a tissue. The polynucleotide editing enzymes expressed from the two types of polynucleotides recognize each other's recognition sequences existing in each polynucleotide and suppress the activity of the other polynucleotide editing enzyme. As a result, as shown in FIG. 1 (B), cells in which the first polynucleotide editing enzyme is predominantly expressed and cells in which the second polynucleotide editing enzyme is predominantly expressed appear in the cell population. In addition, cells in which the expression level of the first polynucleotide editing enzyme and the expression level of the second polynucleotide editing enzyme antagonize each other will appear. In the example of FIGS. 1 (A) and 1 (1), Cre recombinant is used as the first polynucleotide editing enzyme, and FLPO recombinant is used as the second polynucleotide editing enzyme. When the polynucleotide editing enzyme is fully expressed, a polynucleotide for expressing the target product is subsequently introduced as shown in FIGS. 1 (A) and 1 (2). In the examples of FIGS. 1 (A) and 1 (2), the Cre recombinase recognition sequence loxp / lox2722 is designed to sandwich the inverted mCherry gene in the first polynucleotide for expressing the target product. By the function of Cre recombinase, the mCherry gene is ranked with respect to the promoter EF1α, and mCherry is expressed. Further, in the second polynucleotide for expressing the target product, FRT / FRT3, which is a recognition sequence of FLPO recombinant, is designed to sandwich the inverted YFP gene, and the function of FLPO recombinant allows the YFP gene to be generated. It ranks with respect to the promoter EF1α, and YFP is expressed. Therefore, as shown in FIG. 1 (B), the cells in which the first polynucleotide editing enzyme is predominantly expressed expresses mCherry and therefore emits red fluorescence. In addition, cells in which the second polynucleotide editing enzyme is predominantly expressed expresses YFP, and therefore emits green fluorescence. In addition, neither fluorescence is expressed in cells in which the expression level of the first polynucleotide editing enzyme and the expression level of the second polynucleotide editing enzyme antagonize each other. 2 (A) and 2 (B) show an example of actual gene transfer into the dentate gyrus. Since cells expressing red fluorescence, cells expressing green fluorescence, and cells not expressing fluorescence coexist, the shape of each cell, particularly the running of nerve fibers and dendrites, can be clearly discriminated. As described above, according to the first aspect, the number of cells expressing the target product can be controlled, and different target products can be expressed in a plurality of adjacent cells.

The second aspect is, as shown in FIGS. 1 (C) and 1 (1), first, a polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme, and a second polynucleotide. A recognition sequence of an editing enzyme and a polynucleotide having a recognition sequence of a third polynucleotide editing enzyme; a second polynucleotide capable of expressing a second polynucleotide editing enzyme A polynucleotide for expressing an editing enzyme, the first A polynucleotide having a recognition sequence of 1 polynucleotide editing enzyme and a recognition sequence of a 3rd polynucleotide editing enzyme; a polynucleotide for expressing a third polynucleotide editing enzyme capable of expressing a third polynucleotide editing enzyme. Then, the recognition sequence of the first polynucleotide editing enzyme and the polynucleotide having the recognition sequence of the second polynucleotide editing enzyme are gene-introduced into a cell population including a plurality of cells such as a tissue. The polynucleotide editing enzymes expressed from the three types of polynucleotides recognize each other's recognition sequences existing in each polynucleotide and suppress the activity of the other polynucleotide editing enzyme. As a result, in the cell population, cells in which the first polynucleotide-editing enzyme is predominantly expressed, cells in which the second polynucleotide-editing enzyme is predominantly expressed, and cells in which the third polynucleotide-editing enzyme is predominantly expressed. Appears. In addition, cells in which the expression levels of two or three polynucleotide editing enzymes are antagonistic will also appear. In the examples of FIGS. 1 (C) and 1 (1), Cre recombinant is used as the first polynucleotide editing enzyme, FLPO recombinant is used as the second polynucleotide editing enzyme, and Dre is used as the third polynucleotide editing enzyme. I am using a recombinant. When the polynucleotide editing enzyme is fully expressed, a polynucleotide for expressing the target product is subsequently introduced in the same manner as in FIGS. 1 (A) and 1 (2). FIGS. 3 (A) and 3 (B) show examples of actual gene transfer into the dentate gyrus. Since cells expressing red fluorescence, cells expressing green fluorescence, and cells not expressing fluorescence coexist, the shape of each cell, particularly the running of nerve fibers and dendrites, can be clearly discriminated. Moreover, the number of cells expressing fluorescence can be controlled as compared with the first aspect. Here, in the second aspect, for example, one of a plurality of polynucleotide editing enzymes to be used is not directly edited into the polynucleotide for expressing the target product, but only to suppress the expression of other polynucleotide editing enzyme-expressing polynucleotides. Can be used. This is called shadowing. In the example of FIGS. 1 (C) and 1 (1), Dr recombinase is used for shadowing. By doing so, the number of cells expressing the target product can be further controlled.

1. 1. First aspect 1-1. Polynucleotides This section describes the structure of the polynucleotides used in the first aspect. The polynucleotide may include a polynucleotide for expressing an editing enzyme and a polynucleotide for expressing a target product.

1-1-1. Polynucleotides for Expressing Editing Enzymes This section relates to polynucleotides capable of expressing the polynucleotide editing enzymes used in the first aspect. In the present specification, a polynucleotide capable of expressing a polynucleotide editing enzyme is referred to as a polynucleotide for expressing an editing enzyme or a polynucleotide for expressing a polynucleotide editing enzyme.

A polynucleotide editing enzyme is a general term for an enzyme that can edit a target polynucleotide (hereinafter, also referred to as a target polynucleotide) in the cell. Editing a polynucleotide involves (i) recombining the target polynucleotide with another polynucleotide, (ii) deleting the entire sequence of the target polynucleotide, and (iii) making the target polynucleotide part of the nucleotide. It may include the deletion or addition of nucleotides, (iv) inversion of the target polynucleotide, (v) degradation of transcripts from the target polynucleotide, and the like.

Editing of a polynucleotide is preferably carried out by relying on the polynucleotide editing enzyme to recognize a particular nucleotide sequence present within the target polynucleotide. Therefore, the polynucleotide editing enzyme is preferably a sequence-specific polynucleotide editing enzyme. A particular nucleotide sequence recognized by a polynucleotide editing enzyme is called a recognition sequence.

The promoter sequence for expressing the polynucleotide editing enzyme is the mRNA of the polynucleotide editing enzyme encoded by the polynucleotide for expressing the editing enzyme before editing in the cell of the host into which the polynucleotide for expressing the editing enzyme is introduced. It is not restricted as long as it can be transferred. The host is not limited as long as it is a multicellular organism. Preferably, the host is mammals such as mice, rats, humans, guinea pigs, monkeys, dogs, cats, gerbils; birds such as chickens; insects such as silk moths and silk moths; fish such as zebrafish: sea squirts, sea urchins. Such as marine organisms; gerbils; pranaria and the like. For example, in mammalian cells, various types of cytomegalovirus promoter, SV40 promoter, Cumate promoter, translation extender 1α (EF1α) promoter, CAGGS promoter, MSCV promoter, chicken β-actin (CAG) promoter, etc. are used regardless of cell type. It is a promoter that can be expressed in mammalian cells. The phosphoglycerate kinase (PGK) promoter is a promoter that can be expressed in undifferentiated stem cells of mammals and the like. As the promoter, a promoter that expresses tissue-specifically can be used. For example, a 1.3 kb calcium / calmodulin-dependent protein kinase (CamKII (1.3 kb)) promoter can be used in nervous system cells. The α-myosin heavy chain (αMHC) promoter can be used in cardiomyocytes.

The polynucleotide for expressing the editing enzyme before being edited may have a sequence other than the promoter required for expressing the polynucleotide editing enzyme from the sequence encoding the polynucleotide editing enzyme in the host cell. For example, poly A signal, WPRE sequence, multicloning site, etc.

The polynucleotide for expressing an editing enzyme may be prepared by using a vector such as a plasmid vector or a viral vector as a skeleton. Examples of the plasmid vector include a pCAGGS vector and a pCDNA vector. Examples of the viral vector include adeno-associated virus vector, lentiviral vector, retroviral vector, adenoviral vector and the like. Adeno-associated virus vectors can contain from 1 to 10 cellotypes. The type of vector can be selected depending on the host.

The polynucleotide may be DNA or RNA. Further, it may be single-stranded or double-stranded.

In this embodiment, the editing enzyme expression polynucleotide may include a first editing enzyme expression polynucleotide and a second editing enzyme expression polynucleotide.

(1) Polynucleotide for expressing the first editing enzyme The polynucleotide for expressing the first editing enzyme is a sequence encoding the first polynucleotide editing enzyme and a promoter capable of expressing the first polynucleotide editing enzyme in a host. Contains sequences. The promoter sequence is located upstream of the sequence encoding the first polynucleotide editing enzyme so that the first polynucleotide editing enzyme is expressed by the promoter sequence in the host.

In addition, the polynucleotide for expressing the editing enzyme includes the recognition sequence of the second polynucleotide editing enzyme. The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme. Suppression of expression means that the second polynucleotide-editing enzyme exerts its function, and (i) the sequence encoding the first polynucleotide-editing enzyme contained in the polynucleotide for expressing the editing enzyme is another polynucleotide. (Ii) The entire sequence encoding the first polynucleotide editing enzyme is deleted, (iii) A nucleotide deletion or addition occurs in the sequence encoding the first polynucleotide editing enzyme, and a frame shift occurs. The first polynucleotide-editing enzyme is not translated correctly, (iv) the sequence encoding the first polynucleotide-editing enzyme is inverted, and (v) from the sequence encoding the first polynucleotide-editing enzyme. It may include aspects that degrade the transcribed transcript.

Here, the recognition sequence recognized by the first polynucleotide editing enzyme (also referred to as the first recognition sequence) and the recognition sequence recognized by the second polynucleotide editing enzyme (also referred to as the second recognition sequence) are referred to as a recognition sequence. Nucleotide sequences must be different. The recognition of a sequence different from the recognition sequence that the polynucleotide editing enzyme should originally recognize is called a cross-reactivity. For example, the activity value of the first polynucleotide editing enzyme when the first polynucleotide editing enzyme recognizes the first recognition sequence to be originally recognized and exerts the enzyme activity is set to 100%. At this time, the activity value of the cross reaction in which the first polynucleotide editing enzyme recognizes the recognition sequence of another polynucleotide editing enzyme and exerts the enzyme activity is 5% or less, preferably 2% or less, more preferably 1%. Hereinafter, it is more preferably 0%.
The activity value of the cross-reaction of the second polynucleotide editing enzyme is also 5% or less, preferably 2% or less, more preferably 1% or less, still more preferably 0%.
Polynucleotide editing enzymes can include, for example, sequence-specific recombinases, Cas proteins, and the like. Preferably, it is a sequence-specific recombination.

The sequence-specific recombination recognizes the two recognition sequences present on the target polynucleotide and can delete or reversely edit the target region sandwiched between the two recognition sequences. The sequence-specific recombinase may include Cre recombinase, FLPO recombinase, Dre recombinase, Vika recombinase and the like.

Cre recombinant is a type I topoisomerase derived from bacteriophage P1. The recognition sequence for Cre-recombinase is loxP and its variants (eg lox2722, loxN, etc.). Cre recombinant is an enzyme identified by a sequence registered in, for example, GenBank ID: AY056050.1. The sequences of loxP and its variants are GenBank: MN389527.1, MH282425.1, KP686439.1.

The FLPO recombinant is derived from Saccharomyces cerevisiae. The recognition sequence of the FLPO recombinant is FRT and its variants (eg, FRT3, FRT5, etc.). The FLPO recombinant is an enzyme identified by the sequence registered in, for example, GenBank ID: AAB59340.1. The sequences of FRT and its variants are MN3895277.1, LT726874.1, BD013315.1.

Dre recombinant is a bacteriophage D6 derived recombinant. The recognition sequence of the Dre recombinant is rox. Dr recombinase is an enzyme identified by a sequence registered in, for example, GenBank ID: AY735669.1. The sequence of rox is JN195816.1.

Vika recombination is a recombination derived from bacteriophage Vibrio corallilyticu. The recognition sequence of the Vika recombinant is vox. Vika recombinant is an enzyme identified by a sequence registered in, for example, GenBank ID: ZP_05884863.1. The sequence of vox is CP020455 REGION: 73449. .. 73482.

Cas protein may include Cas9, Cas3, Cas13 and the like. Cas9 and Cas3 are genome editing enzymes, and Cas13 is an RNA editing enzyme. The Cas protein exerts its function by being expressed intracellularly together with a polynucleotide encoding an sgRNA sequence. The sequence in which the sgRNA sequence hybridizes becomes the recognition sequence for the Cas protein.

In the polynucleotide for expressing the editing enzyme, the positional relationship between the sequence encoding the first polynucleotide editing enzyme and the second recognition sequence is not limited as long as the sequence encoding the first polynucleotide editing enzyme is edited. For example, when the second polynucleotide editing enzyme is a recombinant, two second recognition sequences can be arranged so as to sandwich the sequence encoding the first polynucleotide editing enzyme. In this case, when the two second recognition sequences are both arranged in the same direction (order) from the promoter side, the sequence encoding the first polynucleotide editing enzyme is due to the function of the second polynucleotide editing enzyme. It is cut out between the two second recognition sequences. Therefore, the sequence encoding the first polynucleotide editing enzyme is deleted. Of the two second recognition sequences placed so as to sandwich the sequence encoding the first polynucleotide editing enzyme, the second recognition sequence located upstream of the sequence encoding the first polynucleotide editing enzyme is ranked. And when the second recognition sequence located downstream of the sequence encoding the first polynucleotide editing enzyme is placed in the opposite direction (inversion), there is an inversion between the two second recognition sequences. It is done and becomes the reverse. When the promoter is upstream of the sequence encoding the inverted first polynucleotide editing enzyme, the original first polynucleotide editing enzyme is derived from the sequence encoding the inverted first polynucleotide editing enzyme. An mRNA having a sequence different from the sequence encoding the above will be transcribed. As a result, the first polynucleotide editing enzyme fails.

The two second recognition sequences may be arranged so as to sandwich a promoter sequence for transcribing the sequence encoding the first polynucleotide editing enzyme instead of the sequence encoding the first polynucleotide editing enzyme. .. Alternatively, the two second recognition sequences may be designed to sandwich both the promoter sequence and the sequence encoding the first polynucleotide editing enzyme.

When the polynucleotide editing enzyme is a Cas protein, a sequence encoding sgRNA, which is a recognition sequence of the second polynucleotide editing enzyme, should be inserted in the vicinity of the sequence encoding the first polynucleotide editing enzyme. Therefore, the sequence encoding the first polynucleotide editing enzyme is deleted by the function of the second polynucleotide editing enzyme. At this time, the sgRNA may be in the sequence encoding the first polynucleotide editing enzyme, or may be an artificial sequence.

(2) Polynucleotide for expressing the second editing enzyme The polynucleotide for expressing the second editing enzyme can express the sequence encoding the second polynucleotide editing enzyme and the second polynucleotide editing enzyme in the host. Includes promoter sequence. The promoter sequence is located upstream of the sequence encoding the second polynucleotide editing enzyme so that the second polynucleotide editing enzyme is expressed by the promoter sequence in the host.

The polynucleotide for expressing the editing enzyme includes the recognition sequence of the first polynucleotide editing enzyme. The recognition sequence of the first polynucleotide-editing enzyme is designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the first polynucleotide-editing enzyme.

The above 1-1-1. Of "polynucleotide", "editing", "editing enzyme", "recombinase", "Cas protein", "recognition sequence", "promoter" and the like. For terms common to (1), see 1-1-1 above. The explanation described in (1) is incorporated here.

Here, when a recombinant is used as the first polynucleotide editing enzyme, it is preferable that the second polynucleotide editing enzyme is also a recombinant. When a Cas protein is used as the first polynucleotide editing enzyme, it is preferable that the second polynucleotide editing enzyme is also a Cas protein.

1-1-2. Target Product Expression Polynucleotides In the present specification, a polynucleotide for expressing a target product is referred to as a target product expression polynucleotide. The polynucleotide for expressing the target product includes a sequence encoding the target product, a promoter sequence capable of expressing the target product in the host, and a recognition sequence of the polynucleotide editing enzyme. The target product is a product intended to be artificially expressed in a host. The product is not limited as long as it is a product that is biosynthesized intracellularly from the sequence encoded by the polynucleotide. The product may be RNA or polypeptide. For example, for morphological observation of cells, the target product is preferably a fluorescent protein. Examples of the fluorescent protein include mCherry, Green Fluorescent Protein (GFP, eGFP), Green Fluorescent Protein-Yellovariant (YFP), Blue Fluorescent Protein (BFP), Cyan Fluorescent Protein (BFP), Cyan Fluorescent Protein Can be done. The target product may have additional sequences such as a nuclear localization signal (NLS) tag, HA tag, His tag, FLAG tag, and c-myc tag.

The polynucleotide for expressing the target substance is described in 1-1-1 above. Similar to the polynucleotide for expressing the editing enzyme described in the above, the host cell may have a sequence other than the promoter required for expressing the target product from the sequence encoding the target product. For example, poly A signal, WPRE sequence, multicloning site, etc.

The polynucleotide for expressing an editing enzyme may be prepared by using a vector such as a plasmid vector or a viral vector as a skeleton.

(1) First Target Product Expression Polynucleotide The first target product expression polynucleotide includes a sequence encoding the first target product and a promoter sequence capable of expressing the first target product in a host. Includes the recognition sequence of the first polynucleotide editing enzyme.
The definition of "first" is intended to have a recognition sequence (first recognition sequence) of the first polynucleotide editing enzyme described in 1-1-1 (1) above.

Here, when a recombinant is used as the first polynucleotide editing enzyme, the sequence encoding the first target product is sandwiched between the two first recognition sequences. At this time, for example, in the polynucleotide for expressing the target product before editing, the sequence encoding the first target product is inverted with respect to the promoter sequence in advance, and the first polynucleotide editing enzyme is the first. When the function is exerted on the polynucleotide for expressing the target product, the sequence encoding the first target product in the inverted position becomes the order and the first target product can be designed to be correctly expressed. Alternatively, in the polynucleotide for expressing the target product before editing, the sequence encoding the first target product of the rank with respect to the promoter sequence is sandwiched between the first recognition sequences of the two ranks, and the first polynucleotide is sandwiched. It can be designed so that the sequence encoding the first target product is deleted when the editing enzyme exerts its function on the polynucleotide for expressing the first target product.

The first recognition sequence may be designed so that the sequence encoding the first target product is edited, but it may be a promoter sequence for expressing the sequence encoding the first target product, or a promoter sequence. And may be designed to act on the sequence encoding the first target product.

The above 1-1-1. Of "polynucleotide", "editing", "editing enzyme", "recombinase", "Cas protein", "recognition sequence", "promoter" and the like. For terms common to (1), see 1-1-1 above. The explanation described in (1) is incorporated here.

(2) Polynucleotide for expressing the second target product The polynucleotide for expressing the second target product includes a sequence encoding the second target product and a promoter sequence capable of expressing the second target product in the host. Includes a recognition sequence for a second polynucleotide editing enzyme.

The definition of "second" is intended to have the recognition sequence of the second polynucleotide editing enzyme described in 1-1-1 (2) above.

The above 1-1-1. Of "polynucleotide", "editing", "editing enzyme", "recombinase", "Cas protein", "recognition sequence", "promoter" and the like. For terms common to (1), see 1-1-1 above. The explanation described in (1) is incorporated here. In addition, the explanation of terms common to the polynucleotide for expressing the first target product is incorporated herein by reference.

1-2. Kit This section describes a gene recombination kit containing a first editing enzyme expression polynucleotide and a second editing enzyme expression polynucleotide used in the first aspect.

The polynucleotide included in the kit may be in a dry state or may be dissolved in a buffer or the like. The buffer for dissolving the polynucleotide may contain a chelating agent such as ethylenediaminetetraacetic acid, β-mercaptoethanol and the like, in addition to Tris-HCl and the like for adjusting the pH.

Further, the gene recombination kit may include a first target product expression polypeptide, a first target product expression polypeptide, and a second target product expression polypeptide.

1-3. Gene Separation and Expression Method and Cell Population In which Genes are Separated and Expressed In this section, the above 1-1. A method for separating and expressing a gene using the polynucleotide described in the above will be described. Separation expression is intended to express a different target product in a cell population containing a plurality of cells such as a tissue, or to express the target product only in a part of cells.

The method for separating and introducing the gene is as follows: a first polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the first polynucleotide-editing enzyme and a second polynucleotide-editing enzyme-expressing polypeptide capable of expressing the second polynucleotide-editing enzyme. It includes a first step of gene-introducing a nucleotide and a second step of gene-introducing a first target product-expressing polynucleotide capable of expressing the first target product. Further, in the second step, a second target product expression polynucleotide may be introduced. Gene transfer is performed on a host cell population containing a plurality of cells such as tissues and cell clusters. The introduction may be performed in vivo or in vitro. The induction may be systemic or topical. For topical administration, the polynucleotide may be delivered to the target site by injection, or the target site may be surgically exposed and the polynucleotide may be delivered.

The description of the first polynucleotide-editing enzyme-expressing polynucleotide and the second polynucleotide-editing enzyme-expressing polynucleotide is described in 1-1-1. The explanation of is used here. The description of the first target product expression polynucleotide and the second target product expression polypeptide is described in 1-1-2. The explanation of is used here.

The gene transfer method can be selected depending on the vector serving as the skeleton of the polynucleotide for expressing the editing enzyme and the polypeptide for expressing the target product. For example, when the vector is a plasmid, it can be introduced by a known method such as lipofection or electroporation. In the case of a viral vector, it can be introduced by utilizing the infectivity of the virus. In addition, the amount of polynucleotide to be introduced follows a known amount. ^{10 13} copies to 10 ⁸ in the case the vector is a plasmid, in the case of viral vectors is about ^{10 13} copies of 10 ^8.

The introduction ratio of the first editing enzyme expression polynucleotide and the second editing enzyme expression polynucleotide is based on 1: 1 and the first target product expression polynucleotide and the second target product expression polynucleotide. Can be appropriately set according to how to express.

The first step and the second step may be performed continuously, but there may be a time difference between the first step and the second step. The time difference is about 2 to 4 weeks. Further, the second step may be repeated. When the second step is repeated, the first second step can be performed, and then the second second step can be performed in accordance with the cell turnover.

When each polynucleotide is introduced into a cell population of a host, a cell population expressing the target product is obtained.

The cell population includes a first group of cells into which a first polynucleotide-editing enzyme-expressing polynucleotide has been introduced and a second cell group into which a second polynucleotide-editing enzyme-expressing polynucleotide has been introduced. How the cell population expresses the target product depends on the design of the recognition sequence of each polynucleotide editing enzyme present in the polynucleotide for expressing the target product. 1-1-1 above. As described in the above, when the polynucleotide editing enzyme exerts its function, it is possible to express the target product or not to express the target product.

2. Second aspect 2-1. Polynucleotide 2-1-1. Polynucleotides for Expressing Editing Enzymes This section relates to polynucleotides capable of expressing the polynucleotide editing enzymes used in the second aspect. Descriptions of terms common to the first aspect, such as "polynucleotide", "editing", "editing enzyme", "recombinase", "Cas protein", "recognition sequence", "promoter", "vector", etc. Use here.

Further, the activity value of the cross reaction used in this embodiment in which each polynucleotide editing enzyme recognizes the recognition sequence of another polynucleotide editing enzyme and exerts the enzyme activity is 5% or less, preferably 2% or less, more preferably. Is 1% or less, more preferably 0%.

(1) Polynucleotide for expressing a third editing enzyme The first polynucleotide for expressing an editing enzyme can express a sequence encoding the first polynucleotide editing enzyme and the first polynucleotide editing enzyme in a host. Includes promoter sequence. The promoter sequence is located upstream of the sequence encoding the third polynucleotide editing enzyme so that the first polynucleotide editing enzyme is expressed by the promoter sequence in the host.
In addition, the first polynucleotide for expressing an editing enzyme includes a recognition sequence for a second polynucleotide editing enzyme and a recognition sequence for a third polynucleotide editing enzyme.

Here, the recognition sequence recognized by the first polynucleotide editing enzyme (also referred to as the first recognition sequence), the recognition sequence recognized by the second polynucleotide editing enzyme (also referred to as the second recognition sequence), and the second The recognition sequence recognized by the polynucleotide editing enzyme of No. 3 (also referred to as the third recognition sequence) needs to have a different nucleotide sequence.

(2) Polynucleotide for expressing the fourth editing enzyme The polynucleotide for expressing the fourth editing enzyme is a sequence encoding the second polynucleotide editing enzyme and a promoter capable of expressing the second polynucleotide editing enzyme in the host. Contains sequences. The promoter sequence is located upstream of the sequence encoding the second polynucleotide editing enzyme so that the second polynucleotide editing enzyme is expressed by the promoter sequence in the host.
In addition, the polynucleotide for expressing an editing enzyme includes a recognition sequence of a first polynucleotide editing enzyme and a recognition sequence of a third polynucleotide editing enzyme.

(3) Polynucleotide for expressing the fifth editing enzyme The polynucleotide for expressing the fifth editing enzyme is a sequence encoding the third polynucleotide editing enzyme and a promoter capable of expressing the third polynucleotide editing enzyme in the host. Contains sequences. The promoter sequence is located upstream of the sequence encoding the third polynucleotide editing enzyme so that the third polynucleotide editing enzyme is expressed by the promoter sequence in the host.

The third polynucleotide editing enzyme is described in 1-1-1. Among the polynucleotide editing enzymes described in the above, the polynucleotide editing enzymes are different from the first polynucleotide editing enzyme and the second polynucleotide editing enzyme.

Here, when a recombinant is used as the first polynucleotide editing enzyme, it is preferable that the other polynucleotide editing enzyme is also a recombinant. When a Cas protein is used as the first polynucleotide editing enzyme, it is preferable that the other polynucleotide editing enzyme is also a Cas protein.
In addition, the polynucleotide for expressing an editing enzyme includes a recognition sequence of a first polynucleotide editing enzyme and a recognition sequence of a second polynucleotide editing enzyme.

2-1-2. Target product expression polynucleotide The target product expression polynucleotide used in this embodiment is the same as in the first aspect.

2-2. Kit In this section, a gene recombination containing a third editing enzyme expression polynucleotide, a fourth editing enzyme expression polynucleotide, and a fifth editing enzyme expression polynucleotide used in the first aspect. The kit for use will be described.

The polynucleotide included in the kit may be in a dry state or may be dissolved in a buffer or the like. The buffer for dissolving the polynucleotide may contain a chelating agent such as ethylenediaminetetraacetic acid, β-mercaptoethanol and the like, in addition to Tris-HCl and the like for adjusting the pH.

Further, the gene recombination kit may include a first target product expression polypeptide, a first target product expression polypeptide, and a second target product expression polypeptide.

2-3. Gene Separation and Expression Method and Cell Population In this section, the above 1-1. A method for separating and expressing a gene using the polynucleotide described in the above will be described. Separation expression is intended to express a different target product in a cell population containing a plurality of cells such as a tissue, or to express the target product only in a part of cells.

The method for separating and introducing the gene is to express a third polynucleotide-editing enzyme capable of expressing a third polynucleotide-editing enzyme and a fourth polynucleotide-editing enzyme capable of expressing a fourth polynucleotide-editing enzyme. Step i for gene transfer of the polynucleotide for expression and the polynucleotide for expressing the fifth polynucleotide editing enzyme capable of expressing the fifth polynucleotide editing enzyme, and the first purpose capable of expressing the first target product. The ii step of gene transfer of the product expression polynucleotide is included. Further, in the second step, a polynucleotide for expressing a second target product may be introduced. Gene transfer is performed on a host cell population containing a plurality of cells such as tissues and cell clusters. The introduction may be performed in vivo or in vitro. The induction may be systemic or topical. For topical administration, the polynucleotide may be delivered to the target site by injection, or the target site may be surgically exposed and the polynucleotide may be delivered.

A description of the third polynucleotide-editing enzyme-expressing polynucleotide, the fourth polynucleotide-editing enzyme-expressing polynucleotide, and the fifth polynucleotide-editing enzyme-expressing polynucleotide is described in 2-1-1. The explanation of is used here. The description of the first target product expression polynucleotide and the second target product expression polypeptide is described in 1-1-2. The explanation of is used here.

Further, the gene transfer method, the amount of nucleotides at the time of transfer, the time difference between the steps, etc. are the same as those in the first aspect. Here, the polynucleotide editing enzyme used for shadowing can be 10 to 1000 times the amount of nucleotides of the polynucleotide for expressing another polynucleotide editing enzyme. In such a case, the ii step may be performed immediately after the i-th step.

When each polynucleotide is introduced into a cell population of a host, a cell population expressing the target product is obtained.

The cell population includes a third group of cells into which a third polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a first polynucleotide-editing enzyme has been introduced, and a fourth group capable of expressing a second polynucleotide-editing enzyme. A fourth cell group into which a polynucleotide for expressing a polynucleotide-editing enzyme was introduced, and a fifth cell into which a polynucleotide for expressing a fifth polynucleotide-editing enzyme capable of expressing a third polynucleotide-editing enzyme was introduced. Including groups. How the cell population expresses the target product depends on the design of the recognition sequence of each polynucleotide editing enzyme present in the polynucleotide for expressing the target product. 1-1-1 above. As described in the above, when the polynucleotide editing enzyme exerts its function, it is possible to express the target product or not to express the target product.

3. 3. Method for Visualizing Target Product This specification discloses a method for visualizing a target product. The visualization method is described in 1-3. Or the above 2-3. A step of visualizing the first target product and the second target product after performing the separation and expression of the gene in the above is included.

The visualization method is not limited as long as the target product can be visualized, and the target product can be visualized by preparing a frozen tissue section, a paraffin-embedded section, or the like, and performing direct observation or observation by immunohistochemical staining. Visualization also includes extended microscopy.

The visualization method will be described by taking as an example the case where the target product is a fluorescent protein and the target product in the tissue of a host other than human is visualized. After anesthetizing a non-human host, transcardiat the host with ice-cooled 4% (w / v) paraformaldehyde (PFA) -phosphate buffered saline (PBS) using a perista pump. Perfusion fixation.

Next, the tissue is removed and fixed with the same fixing solution for 24 hours. After transferring the fixed tissue to 30% (w / v) sucrose / PBS and draining the fixative, the fixed brain was implanted in an OCT compound (Sakura) and frozen in isopentane and liquid nitrogen to prepare a frozen block. do. The embedding block can be sliced into sections 40-100 μm thick using a cryostat. The sliced sections are placed on a slide glass, sealed with a cover glass using an encapsulant such as glycerin, and microscopically examined.

In addition, the sliced sections may be immunostained with an antibody against the expressed fluorescent protein.

Visualization of the fluorescent protein can be performed with a fluorescence microscope, a confocal laser scanning microscope, or the like.

Further, the sliced sections may be subjected to expansion microscopy (Expansion Microscope) to expand the sections, and then immunostained and visualized. Extended microscopy is known as an online method of Asano et al, 2018 (Curr Protocol Cell Biol. 80: e56.). For example, sliced Free-floating sections are subjected to antigen recovery with citric acid buffer (10 mM citric acid + 0.05% Tween 20, pH 8.0) at about 80 ° C. for 30 minutes, and then washed with PBS. Sections are treated with blocking buffer containing 1% bovine serum albumin (BSA) at 23-27 ° C. and PBS containing 0.1% Triton X-100 for 1 hour for permeation and blocking. Next, the section is reacted with an antibody solution containing an antibody against the target product. Wash with blocking buffer and high salinity PBS (PBS + 350 mM NaCl), followed by washing with PBS for 5 minutes each for at least 6 hours at room temperature, followed by fixation with acryloyl-X / PBS (0.1 mg / mL). Wash with PBS. For gelation of the resin, the sections were divided into gelling solutions (monomer solution (8.6% [w / v] sodium acrylate, 2.5% [w / v] acrylamide, 0.15% [w / v]. ] N, N'-methylenebisacrylamide, 11.7% [w / v] NaCl in PBS), ammonium persulfate (APS), tetramethylethylenediamine (TEMED) and 4-hydroxy-2,2,6,6 -Tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO) is placed in a mixed solution at a ratio of, for example, 47: 1: 1: 1, and then the sections are incubated on ice for about 30 minutes. Then, the section is transferred to a gelation chamber and gelled at 37 ° C. for about 2 hours.

Tissue is excised from the gel section and digestion buffer (50 mM Tris-HCl [pH 8.0], 23 mM ethylenediaminetetraacetic acid [EDTA], 0.5% Triton X-100, 0.8 M guanidine HCl, and 8 U / Incubate in mL Proteinase K) overnight. The sections are washed with PBS and then stored in the dark at 4 ° C. until expansion. For expansion of the section, the section is washed with water and then transferred to an imaging chamber, where silicon sheets are sandwiched between the sections on both sides, the section and the silicon sheet are sandwiched between coverslips, and the specimen for microscopic examination is placed. To make.

Hereinafter, the contents of the present invention will be described in detail with reference to Examples, but the present invention is not construed as being limited to the Examples.
In addition, the animal experiments shown below were conducted in accordance with the guidelines of Kansai Medical University, and were approved by the Animal Experiment Committee of Kansai Medical University.

I. Example 1
In Example 1 (BATTLE-1), genes were separated and introduced using two types of recombinases.

1. 1. Introduction vector (1) First recombinase expression vector The design of the first recombinase expression vector is shown in the upper part of FIGS. 1 (A) and 1 (1). For this vector, Cre recombinase (GenBank: AY056050.1 or less, sometimes simply referred to as "Cre") was used as the first recombinase. A 1.3 kb calcium / calmodulin-dependent protein kinase (CamKII (1.3 kb)) promoter was used as the promoter for expressing the Cre recombinase gene. In addition, the recognition sequence FRT5 of FLPO recombinase, which is a second recombinase, was inserted so as to sandwich the Cre recombinase gene. This vector is a lentiviral vector having a CamkIIa promoter, FRT5 sequence, Cre, FRT5 sequence, and WPRE sequence from the 5'side. Specifically, the synthesized FRT5-Cre-FRT5 sequence was inserted into the BamHI-EcoRI site of the Lentiviral backbone vector (Addgene 20943). Hereinafter, the first recombinase expression vector is also referred to as Lenti-Camk2 promoter -FRT5- Cre-FRT5-WPRE.

(2) Second Recombinase Expression Vector The design of the second recombinase expression vector is shown in the lower part of FIGS. 1 (A) and 1 (1). For this vector, FLPO recombinase (hereinafter, may be simply referred to as "FLPO") was used as the second recombinase. A 1.3 kb calcium / calmodulin-dependent protein kinase (CamKII (1.3 kb)) promoter was used as the promoter for expressing the FLPO recombinase gene. In addition, the recognition sequence LoxN of Cre recombinase, which is the first recombinase, was inserted so as to sandwich the FLPO recombinase gene. This vector is a lentiviral vector having a CamkIIa promoter, LoxN sequence, FLPO, LoxN sequence, and WPRE sequence from the 5'side. Specifically, the synthesized loxN-FLPO-loxN sequence was inserted into the BamHI-EcoRI site of the Lentiviral backbone vector (Addgene 20943). Hereinafter, the second recombinase expression vector is also referred to as Lenti-Camk2 promoter-loxN-FLPO-loxN-WPRE.

(3) First Target Product Expression Vector The structure of the first target product expression vector is shown in the upper part of FIGS. 1 (A) and 1 (2). The first target product expression vector AAV-EF1α promoter-DIO-mCherry (Addgene plasmid 47636) was used. The first target product vector is an AAV vector having an EF1α promoter, a loxP sequence, a lox2722 sequence, an inverted mCherry sequence, an inverted loxP sequence, an inverted lox2722 sequence, and a WPRE sequence from the 5'side. When Cre recombinase acts on this vector, the mCherry sequence is recombined into a ranking sequence and mCherry is expressed.

(4) Second Target Product Expression Vector The structure of the first target product expression vector is shown in the lower part of FIGS. 1 (A) and 1 (2). The second target product vector is an AAV vector having an EF1α promoter, an FRT sequence, an FRT3 sequence, an inverted EYFP sequence, an inverted FRT sequence, an inverted FRT3 sequence, and a WPRE sequence from the 5'side. This vector was prepared by inserting the synthesized NheI-EYFP-BSrGI into the NheI-BSrGI site of pAAV-EF1α-F-FLEX-Kir2.1-T2A-tdTomato (Addgene 60661). Hereinafter, the second target product expression vector is also referred to as AAV-EF1α promoter-Fflex-EYFP. When FLPO recombinase acts on this vector, the EYFP sequence is recombined into the standing sequence and EYFP is expressed.

2. Virus packaging All the viral vectors produced were packaged into viruses at Gunma University.

3. 3. Gene transfer and observation (1) Stereotaxic Injection
Mice were anesthetized and fixed in a world precision instruments.

Tapered glass capillaries were made with a micropipette puller (Sutter). A tapered glass capillary and a Hamilton syringe with a 26G needle were filled with mineral oil and used to inject the virus. In the first injection, a viral solution packaged with 1 μL of recombinase was placed at the specified coordinates of the hippocampal DG-CA2-3 region of the right hemisphere from bregma (-1.94 mm AP, + 2.14 mm ML, -2.32 mm DV). Was injected into. When injecting, mix 1: 1 (each viral load is 5 x 10 ⁸ copies) or 1: 0.01 (5 x 10 ⁸ copies: 5 x 10 ⁶ copies) and mix each into different mice. Infused.

Three weeks later, the virus that packaged the target product in the second injection was injected at the same site as in the first injection. AAV-EF1α promoter-DIO-mCherry (1 μL, viral load 1.43 × 10 ¹³ copies) and AAV-EF1α promoter-Fflex-EYFP (1 μL, viral load 1.43 × 10 ¹³ copies) were used as viruses that packaged the target product. Used as a mixture.
Mice were perfused and fixed one week after the second infusion.

(2) Preparation of tissue sections Adult mice were anesthetized with a mixture of medetomidine, midazolam, and butorphanol, and then ice-cooled using a perista pump to 4% (w / v) paraformaldehyde (PFA) -phosphate buffered saline. Water (PBS) was perfused transcardiacically.

Next, the brain was removed and fixed in the same fix for 24 hours, the fixed tissue was transferred to 30% (w / v) sucrose / PBS, placed in OCT compound (Sakura), and the fixed brain was isopentane and It was frozen in liquid nitrogen and embedded. The embedding block was sliced into anterior-posterior sections with a thickness of 60 μm using a cryostat (using Leica).

(3) Fluorescence Imaging A fluorescence image was acquired using an inverted confocal fluorescence microscope (Zeiss, LSM700, Olympus FV3000).

Fluorescence images were acquired using an inverted confocal fluorescence microscope with a 20x objective to analyze the segregation and localization of YFP and mCherry. Using ZenBlue 2012 software (Zeiss), from the square region on the cell body of DG granule neurons (400 μm × 400 μm × 2 μm) and the square region on the cell body of CA2-3 neurons (3.9 μm x 3.9 μm) in the confocal image. The fluorescence intensity was measured. Whether or not YFP and mCherry were co-expressed was determined to be strongly expressed when both fluorescence intensities were higher than the background fluorescence (Arbitrary unit 10, fluorescence intensity 10 in the range of fluorescence intensity 0-255). that's all).

4. Results Figure 2 shows the fluorescent images of the fluorescent proteins mCherry and YFP introduced by BATTLE-1. FIG. 2 (A) shows a fluorescence image (microscope magnification) when Lenti-Camk2 promoter -FRT5-Cre-FRT5-WPRE and Lenti-Camk2 promoter-loxN-FLPO-loxN-WPRE were introduced at a ratio of 1: 1. × 50 times). FIG. 2 (B) is a strongly magnified view (microscope magnification × 250 times) of FIG. 2 (A). mCherry-positive (hereinafter, “mCherry +”) cells are cells in which Cre predominantly survives and is expressed, and are shown in red in the fluorescence image. YFP-positive (hereinafter, “YFP +”) cells are cells in which FLPO predominantly survives and is expressed, and are shown in green in the fluorescence image. YFP and mCherry were localized in different cells. FIG. 2C is a graph quantifying the proportion of mCherry + cells, YFP + cells, and double positive cells (hereinafter referred to as “mCherry + / YFP + cells”) in which both mCherry and YFP are positive in the dentate gyrus. Is. Counting for a total of 827 granule neurons in the dentate gyrus of 5 mice, mCherry + cells: YFP + cells: mCherry + / YFP + cells were 61.3 ± 5.8%: 38 ± 5.8%: 0.7 ± 0.2% (mean ± SEM). )Met. Counting for a total of 292 pyramidal neurons in 5 mice in the CA2-3 region of the hippocampus, mCherry + cells: YFP + cells: mCherry + / YFP + cells were 78 ± 2.7%: 21.4 ± 2.7%: 0.6 ± 0.4. It was% (mean ± SEM). Only a few cells showed co-expression of mCherry and YFP.

FIG. 2 (D) shows a fluorescence image (microscope magnification) when Lenti-Camk2 promoter -FRT5-Cre-FRT5-WPRE and Lenti-Camk2 promoter-loxN-FLPO-loxN-WPRE were introduced at a ratio of 1: 0.01. × 50 times). FIG. 2 (E) is a strongly magnified view (microscope magnification × 250 times) of FIG. 2 (D). FIG. 2 (F) is a graph quantifying the ratio of mCherry + cells, YFP + cells, and double positive cells (hereinafter referred to as “mCherry + / YFP + cells”) in which both mCherry and YFP are positive in the dentate gyrus. Is. Counting for a total of 1153 granule neurons in the dentate gyrus of 3 mice, mCherry + cells: YFP + cells: mCherry + / YFP + cells were 98.7 ± 0.2%: 1.1 ± 0.2%: 0.2 ± 0.1% (mean ± SEM). )Met. Counting for a total of 442 pyramidal neurons in 4 mice in the CA2-3 region of the hippocampus, mCherry + cells: YFP + cells: mCherry + / YFP + cells were 96.2 ± 0.8%: 3.6 ± 0.9%: 0.1 ± 0.1. It was% (mean ± SEM).
From the above results, it was shown that the BATTLE-1 gene separation and introduction method can express two different genes in a tissue in different cells.

Figures 2 (G) and 2 (H) show the scheme of the BATTLE-1 mechanism. As shown in FIGS. 2 (A) to 2 (C), by making the ratio of the two recombinase expression vectors the same, it is possible to express the two target products separately in almost the same number of cells. did it. It was also shown that by changing the ratio of the two recombinase expression vectors, the number of cells expressing the target product can be adjusted according to the ratio.

II. Example 2
In Example 2 (BATTLE-2), genes were separated and introduced using three types of recombinases.

1. 1. Introduction vector (1) Third recombinase expression vector The design of the third recombinase expression vector is shown in the upper part of FIGS. 1 (C) and 1 (1). For this vector, Cre recombinase (hereinafter, may be simply referred to as "Cre") was used as the first recombinase. A 1.3 kb calcium / calmodulin-dependent protein kinase (CamKII (1.3 kb)) promoter was used as the promoter for expressing the Cre recombinase gene. In addition, the recognition sequence FRT5 of FLPO recombinase, which is the second recombinase, and the recognition sequence Rox of Dre recombinase, which is the third recombinase, were inserted so as to sandwich the Cre recombinase gene. This vector is a lentiviral vector having a CamkIIa promoter, FRT5 sequence, inverted Rox sequence, Cre, inverted Rox sequence, FRT5 sequence, and WPRE sequence from the 5'side. Specifically, the above I.1. The synthesized NheI-rox-FRT5-EcoRI sequence was inserted into the NheI-EcoRI site of the Lenti-Camk2 promoter -FRT5-Cre-FRT5-WPRE prepared in (1). Subsequently, the synthesized BamHI-lox N-rox-AgeI was inserted into the BamHI-AgeI site of the Lentivirus-CamK2-FRT5-Cre-FRT5-WPRE vector into which NheI-rox-FRT5-EcoRI was inserted. Hereinafter, the third recombinase expression vector is also referred to as Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE.

(2) Fourth Recombinase Expression Vector The design of the fourth recombinase expression vector is shown in the middle of FIGS. 1 (C) and 1 (1). For this vector, FLPO recombinase (hereinafter, may be simply referred to as "FLPO") was used as the second recombinase. A 1.3 kb calcium / calmodulin-dependent protein kinase (CamKII (1.3 kb)) promoter was used as the promoter for expressing the FLPO recombinase gene. In addition, the recognition sequence LoxN of Cre recombinase, which is the first recombinase, and the recognition sequence Rox of Dre recombinase, which is the third recombinase, were inserted so as to sandwich the FLPO recombinase gene. This vector is a lentiviral vector having a CamkIIa promoter, LoxN sequence, inverted Rox sequence, FLPO, inverted Rox sequence, LoxN sequence, and WPRE sequence from the 5'side. Specifically, the above-mentioned I. 1. 1. The synthesized BamHI-loxN-rox-AgeI sequence was inserted into BamHI-AgeI of the Lenti-Camk2 promoter-loxN-FLPO-loxN-WPRE prepared in (2). Subsequently, the synthesized NheI-rox-loxN-EcoRI was inserted into the NheI-EcoRI site of the Lenti-Camk2 promoter-loxN-FLPO-loxN-WPRE in which BamHI-loxN-rox-AgeI was inserted. Hereinafter, the fourth recombinase expression vector is also referred to as Lenti-Camk2 promoter-loxN-rcRox-FLPO-rcRox-loxN-WPRE.

(3) Fifth Recombinase Expression Vector The design of the fifth recombinase expression vector is shown in the lower part of FIGS. (C) and (1). For this vector, Dre recombinase (NLS-HA-Dre, addgene51272, hereinafter, sometimes simply referred to as "Dre") was used as the second recombinase. NLS is a nuclear localization signal and HA is an HA tag. A 1.3 kb calcium / calmodulin-dependent protein kinase (CamKII (1.3 kb)) promoter was used as the promoter for expressing the Dre recombinase gene. In addition, the recognition sequence LoxN of Cre recombinase, which is the first recombinase, and the recognition sequence FRT5 of FLPO recombinase, which is the second recombinase, were inserted so as to sandwich the Dre recombinase gene. This vector is a lentiviral vector having a CamkIIa promoter, FRT5 sequence, LoxN sequence, Dre, LoxN sequence, FRT5 sequence, and WPRE sequence from the 5'side. Specifically, the synthesized BamHI-FRT5-LoxN-Dre-LoxN-FRT5-EcoRI sequence was inserted into the BamHI-EcoRI site of the Lentiviral backbone vector (Addgene 20943). Hereinafter, the fifth recombinase expression vector is also referred to as Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE. Since the target product expression vector does not have a Dre recombinase recognition sequence, the Dre recombinase will attack the third recombinase expression vector and / or the fourth recombinase expression vector infected with the same cells.

(4) Target product expression vector I. 1. 1. The vectors described in (3) and (4) were used. 1 (C) and 1 (2) are the same as those in FIGS. 1 (A) and 1 (2).

2. Virus packaging All the viral vectors produced were packaged into viruses at Gunma University.

3. 3. Gene transfer and observation (1) Stereotaxic Injection
The virus was injected in the same manner as in Example 1. However, the virus is the above II. 1. 1. The one prepared in the above was used.

In the first injection, Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE, Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE and Lenti- Camk2 promoter-loxN-rcRox-FLPO-rcRox-loxN-WPRE was mixed at a ratio of 1: 1: 1 (each virus amount was 5 × 10 ⁸ copies), and 1 μL was injected into different mice.

Three weeks after the first injection, the second injection was performed in the same manner as in Example 1.
Mice were perfused and fixed one week after the second infusion.

(2) Multisparse Transgene Expression
The above II. 1. 1. In (1), Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE, Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE and Lenti-Camk2 promoter After injecting -loxN-rcRox-FLPO-rcRox-loxN-WPRE at 200: 1:10, the target product expression vector was further added to the above II. 1. 1. It was injected with the same injection amount as in (1). Mice were perfused and fixed 2 weeks after injection.

(3) Conventional vector injection As a control, conventional AAV-CAG-GFP virus (Addgene 37825-AAVrg, 1 μL, viral load 1 × 10 ¹³ copies) was infected at the same site in different mice, and one week after the injection, the mice were infected. Was perfused and fixed.

4. Results Figure 3 shows the fluorescent images of the fluorescent proteins mCherry and YFP introduced by BATTLE-2. FIG. 3 (A) shows Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE, Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE, and Lenti- It is a fluorescence image (microscope magnification × 250 times) when Camk2 promoter-loxN-rcRox-FLPO-rcRox-loxN-WPRE was introduced at a ratio of 1: 1: 1. Figure 3 (B) shows Lenti-Camk2 promoter-FRT5-loxN- (NLS-HA-Dre) -loxN-FRT5-WPRE, Lenti-Camk2 promoter-FRT5-rcRox-Cre-rcRox-FRT5-WPRE, and Lenti- It is a fluorescence image (microscope magnification × 250 times) when Camk2 promoter-loxN-Rox-FLPO-Rox-loxN-WPRE was introduced at a ratio of 200: 1:10. mCherry-positive (hereinafter, “mCherry +”) cells are cells in which Cre predominantly survives and is expressed, and are shown in red in the fluorescence image. YFP (hereinafter, “YFP +”) cells are cells in which FLPO predominantly survives and is expressed, and are shown in green in the fluorescence image. Double-negative cells in which both mCherry and YFP are negative (hereinafter referred to as "mCherry- / YFP-cells") are cells in which Dre is significantly expressed or infected with a virus packaged with a target product expression vector. It is a cell that did not.

Counting for a total of 618 granule neurons in the dentate gyrus of 3 mice, the number of mCherry + cells: YFP + cells: mCherry + / YFP + cells per ^{400 μm 2 was 63.1 ± 10.2: 13.9 ± 1.8: 0.3 ± 0.2 (} mean ± SEM). Counting for a total of 364 pyramidal neurons in 4 mice in the CA2-3 region of the hippocampus, mCherry + cells: YFP + cells: mCherry + / YFP + cells per ^{400 μm 2: 39.8 ± 8.5: 5.1 ± 0.4:} The number was 0.6 ± 0.4 (mean ± SEM).

FIG. 3C shows a double positive cell (hereinafter referred to as “mCherry + / YFP + cell”) in which both mCherry + cells and YFP + cells in the dentate gyrus and both mCherry and YFP are positive, which have undergone Multisparse Transgene Expression. It is a graph which quantified the ratio of. Counting for a total of 66 granule neurons in the dentate gyrus of 4 mice, the number of mCherry + cells: YFP + cells: mCherry + / YFP + cells per ^{400 μm 2 was 3.4 ± 0.9: 4.9 ± 0.8: 0 (mean ±).} It was SEM). Counting for a total of 364 pyramidal neurons in 4 mice in the CA2-3 region of the hippocampus, mCherry + cells: YFP + cells: mCherry + / YFP + cells per ^{400 μm 2: 1.1 ± 0.4: 1.9 ± 0.3:} The number was 0 (mean ± SEM). FIG. 3 (E) shows a fluorescence image of the hippocampus subjected to Multisparse Transgene Expression. In contrast, FIG. 3D shows a fluorescence image of the hippocampus when GFP was expressed using a common conventional expression vector. Comparing FIG. 3 (D) with FIG. 3 (E), it is clear that the Multisparse Transgene Expression labeled the dendrites and axons morphology strongly, sparsely and clearly.

III. Experimental Examples As shown in FIG. 4 (A), it is difficult to clearly visualize the entire synaptic structure by the conventional method using AAV expressing GFP. In order to clearly visualize the entire structure of synaptic connections, we have developed a hybrid technology BATTLE-1EX that combines BATTLE-1 and ExM. In this section, the entire synaptic structure was visualized on a nanoscale by multicolor immunohistochemical staining using the BATTLE-1 system.

1. 1. Gene transfer The BATTLE-1 system shown in Example I was used.

2. CamKIIa immunostaining
Free-floating sections were incubated for 1 hour in blocking buffer containing 3% (v / v) goat serum and 0.3% Triton-X PBS, followed by the addition of a mouse monoclonal antibody against CamKIIa (1: 300, Abcam ab22609) 3 Incubated in% goat serum and 0.3% Triton-X-added PBS solution at 4 ° C. for 16 hours. One section was then washed 3 times for 5 minutes with 0.3% Triton-X-added PBS. Subsequently, the secondary antibody, Alexa Fluor 647 Plus-labeled anti-mouse goat antibody (1: 1000, A32728, Thermo Fisher Scientific) was incubated for 2 hours, and the sections were washed 3 times for 15 minutes with 0.3% Triton-X-added PBS. All sections were mounted on glass slides using Vector Shield and visualized using 4', 6-diamidino-2-phenylindole (DAPI, Vector Lab).

3. 3. Expansion Microscopy of BATTLE-1
ExM (Expansion Microscopy) was implemented with minor modifications to the online method of Asano et al, 2018. Free-floating sections were antigen-recovered with citric acid buffer (10 mM citric acid + 0.05% Tween 20, pH 8.0) at 80 ° C. for 30 minutes, and then washed with PBS four times for 5 minutes. Sections were treated with blocking buffer containing 1% bovine serum albumin (BSA) at 26 ° C. and PBS containing 0.1% Triton X-100 for 1 hour for permeization and blocking. Next, in the blocking buffer, chicken primary polyclonal antibody against GFP (1: 500, ab13970, Abcam), rat monoclonal antibody against RFP (1: 100, 5f8-100, Chromotek), mouse monoclonal antibody against Bassoon (1: 100, ab82958). , Abcam), Homer1 (1: 100, 160003, SYSY) was added with a rabbit polyclonal antibody to prepare an antibody solution, and sections were placed in the antibody solution and reacted at 4 ° C. for 2 days. It was then washed 4 times with blocking buffer for 5 minutes. As secondary antibodies, Alexa488 dye-labeled anti-chicken donkey antibody (1: 200, 703-545-155, Jackson), Alexa568 dye-labeled anti-rat donkey antibody (1: 100, ab175710, Abcam), Atto647N dye-labeled anti-mouse goat antibody (1: 100, ab175710, Abcam) 1: 100, 50185, Rockland), CF405M dye-labeled anti-rabbit goat antibody (1: 100, 20373, Biotium) was used. The secondary antibody was added to a blocking buffer at a predetermined dilution ratio, and the washed sections were placed therein and incubated at 4 ° C. for 1 day. It was washed with blocking buffer and high salt concentration PBS (PBS + 350 mM NaCl) 3 times in a row, followed by PBS for 5 minutes each. The cells were fixed with acryloyl-X / PBS (0.1 mg / mL) for 6 hours or more at room temperature, and then washed 5 times with PBS for 5 minutes each time.

For gelation, the sections were subjected to a gelling solution (monomeric solution (8.6% [w / v] sodium acrylate, 2.5% [w / v] acrylamide, 0.15% [w / v] N, N'-methylenebis). Acrylamide, 11.7% [w / v] NaCl in PBS), ammonium persulfate (APS), tetramethylethylenediamine (TEMED) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4) -Hydroxy-TEMPO) was placed in a mixed solution in a ratio of 47: 1: 1: 1 and then the sections were incubated on ice for 30 minutes, after which the sections were placed in a gelling chamber (slide glass). , The section was transferred to a solution in which coverslips were sandwiched on both sides of the sample as a spacer, and another slide glass was stacked), and the mixture was gelled at 37 ° C. for 2 hours.

The hippocampal region is excised from the gel section and digested buffer (50 mM Tris-HCl [pH 8.0], 23 mM ethylenediaminetetraacetic acid [EDTA], 0.5% Triton X-100, 0.8 M guanidine HCl, and 8 U / mL Proteinase K). Incubated overnight in. Sections were washed 3 times with PBS for 5 minutes and then stored in the dark at 4 ° C until dilated. For expansion of the section, the sample was washed 5 times with water for 10 minutes and then transferred to an imaging chamber where silicon sheets were sandwiched on both sides of the section and the section and silicon sheet were sandwiched between coverslips.

4. The same observation confocal microscope as in Example 1 was used.
Co-expression of CamKIIa and fluorescent protein was determined based on the expression in the cytoplasm of the cell body in confocal images obtained using an Olympus confocal microscope (FV3000).

The magnification was calculated using the physical distance between the two landmark positions in the pre- and post-magnification images measured by Imaris 9.1 (Bitplane).

The maximum intensity projected image was generated using software Zenblack (Zeiss), software FV31S-SW (Olympus), and Imaris 9.1 (Bitplane). 3D volume rendering (FIGS. 4 (E) and 4 (F) and FIG. 5) was generated after subtracting the background using Imaris 9.1 (Bitplane).

5. Line profiling analysis of fluorescence intensity of all synapses In order to quantify the nanoscale structure of the entire synapse and the distribution of Homer and Bassoon, the region surrounded by a square (478 nm × 1094 nm) of the entire synapse region was measured. The Image J program (National Institutes for Health [NIH]) was used for line profiling analysis of fluorescence intensities of YFP, mCherry, Homer, and Bassoon.
All experiments were performed 3 times each.

6. Results Figure 4 (A) shows an example of GFP expression by a conventional expression system. In addition, FIG. 4 (B) shows an example of gene expression by the BATTLE-1 system shown in Example 1. It is clear that nerve cells can be observed at high resolution by the method of Example 1 and extended microscopy.

The hippocampal slices transgeniced with BATTLE-1 were magnified 4.1 times by extended microscopy (bottom of FIG. 4 (B)). In this region (44.3 μm × 44.3 μm × 6.8 μm), 14 YFP-positive presynaptic terminals could be stochastically observed to form the mCherry-positive postsynaptic spines of CA3 neurons and the entire synapse. Other cases consisting of mCherry-positive presynaptic terminals with YFP-positive postsynaptic spines were also observed (FIG. 5 (B)). From this result, it is possible to clearly visualize the entire synaptic structure consisting of YFP-positive presynaptic terminals from the dentate gyrus and mCherry-positive postsynaptic spines of CA3 pyramidal neurons on a nanoscale by combining BATTLE-1 and extended microscopy. Shown.

Furthermore, the localization of the presynaptic protein Bassoon and the postsynaptic protein Homer could be identified simultaneously throughout the synaptic structure (Fig. 4 (C)). Volume rendering was performed to observe the three-dimensional structure of the entire synapse and the three-dimensional localization of the DG-CA3 synapse Bassoon and Homer in the hippocampus (FIGS. 4 (E) and 4 (F)). Further, FIG. 4 (G) shows the nanoscale structure of the entire synapse after continuous image reconstruction. Further, images of the three-dimensional structure of synapses from granule neurons to hilar mossy cells visualized on a nanoscale by volume rendering are shown in FIGS. 5 (A) to 5 (E).

It is shown that by introducing different genes into individual cells by the gene separation and introduction method of the present invention, it is possible to observe the localization of individual cells and the connection with other cells, which could not be observed by the conventional high-resolution microscopic observation method. Was done.

Claims (15)

The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
A polynucleotide capable of expressing the first polynucleotide editing enzyme, including
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The polynucleotide. The recognition sequence of the second polynucleotide-editing enzyme lacks the sequence encoding the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme, and the first polynucleotide-edited by frameshift. Designed so that the sequence encoding the enzyme is not translated correctly or is inverted,
The polynucleotide according to claim 1. The recognition sequence for the second polynucleotide-editing enzyme is designed to degrade transcripts from the sequence encoding the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The polynucleotide according to claim 1. A first polynucleotide capable of expressing a first polynucleotide editing enzyme, and
A second polynucleotide capable of expressing a second polynucleotide editing enzyme, and
Is a genetic recombination kit containing
The first polynucleotide is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The second polynucleotide is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The above-mentioned genetic recombination kit. Method for separating and expressing a gene, which comprises the following first step and second step:
First step: A polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme, and a polynucleotide for expressing a second polynucleotide-editing enzyme capable of expressing a second polynucleotide-editing enzyme. And the second step: the step of gene-introducing the polynucleotide for expressing the first target product capable of expressing the first target product,
here,
The polynucleotide for expressing the first polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
Including
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme;
The polynucleotide for expressing the second polynucleotide editing enzyme is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
Including
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme; and the expression of the first target product. Polynucleotide for
The sequence encoding the first target product and
A promoter sequence capable of expressing the first target product in a host and
The recognition sequence of the first polynucleotide editing enzyme and
including. Further, in the above two steps, a gene transfer of a second target product expression polynucleotide capable of expressing the second target product is included.
The polynucleotide for expressing the second target product is
The sequence encoding the second target product and
A promoter sequence capable of expressing the second target product in a host and
The recognition sequence of the second polynucleotide editing enzyme and
including,
The method for separating and expressing a gene according to claim 5. Visualization method of target product including the following first step, second step, and third step:
First step: A polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme, and a polynucleotide for expressing a second polynucleotide-editing enzyme capable of expressing a second polynucleotide-editing enzyme. Process of gene transfer,
Second step: a step of gene transfer of a first target product expression polynucleotide capable of expressing the first target product, and a third step: a step of visualizing the first target product;
here,
The polynucleotide for expressing the first polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
Including
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme;
The polynucleotide for expressing the second polynucleotide editing enzyme is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
Including
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme; and the expression of the first target product. Polynucleotide for
The sequence encoding the first target product and
A promoter sequence capable of expressing the first target product in a host and
The recognition sequence of the first polynucleotide editing enzyme and
including. Moreover,
The two steps include gene transfer of a second target product expression polynucleotide capable of expressing the second target product.
The polynucleotide for expressing the second target product is
The sequence encoding the second target product and
A promoter sequence capable of expressing the second target product in a host and
The recognition sequence of the second polynucleotide editing enzyme and
Including
The three steps include visualizing the second target product.
The method for visualizing a target product according to claim 7. A first cell group into which a polynucleotide for expressing a first polynucleotide-editing enzyme capable of expressing a first polynucleotide-editing enzyme has been introduced, and
Includes a second group of cells into which a second polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a second polynucleotide-editing enzyme has been introduced.
Cell population:
here,
The polynucleotide for expressing the first polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
Including
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme;
The polynucleotide for expressing the second polynucleotide editing enzyme is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
Including
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme. The first cell population is
Expression of a second target product, which comprises a sequence encoding the second target product, a promoter sequence capable of expressing the second target product in a host, and a recognition sequence of the second polynucleotide editing enzyme. Polynucleotide; and / or said second cell population
A sequence encoding the first target product, a promoter sequence capable of expressing the first target product in a host, and a recognition sequence of the first polynucleotide editing enzyme are designated as a poly for expressing the first target product. With nucleotides
including,
The cell population according to claim 9. The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
A polynucleotide for expressing a polynucleotide editing enzyme, including
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The polynucleotide for expressing the polynucleotide editing enzyme. A polynucleotide for expressing a third polynucleotide-editing enzyme,
A polynucleotide for expressing a fourth polynucleotide-editing enzyme,
A polynucleotide for expressing a fifth polynucleotide editing enzyme,
Is a genetic recombination kit containing
The polynucleotide for expressing the third polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fourth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fifth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the third polynucleotide editing enzyme and
A promoter sequence capable of expressing the third polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the second polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the third polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the third polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The kit. Method of separating and expressing a gene, which comprises the following step i and step i:
Step i: A third polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the first polynucleotide-editing enzyme and a fourth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the second polynucleotide-editing enzyme. And a step of gene-introducing a fifth polynucleotide editing enzyme-expressing polynucleotide capable of expressing a third polynucleotide editing enzyme, and a third step: expression of a first target product capable of expressing a first target product. A step of gene-introducing a polynucleotide for expressing a second target product and a polynucleotide for expressing a second target product capable of expressing the second target product;
here,
The polynucleotide for expressing the third polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fourth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fifth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the third polynucleotide editing enzyme and
A promoter sequence capable of expressing the third polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the second polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the third polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the third polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The polynucleotide for expressing the first target product is
The sequence encoding the first target product and
A promoter sequence capable of expressing the first target product in a host and
The recognition sequence of the first polynucleotide editing enzyme and
Including
The polynucleotide for expressing the second target product is
The sequence encoding the second target product and
A promoter sequence capable of expressing the second target product in a host and
The recognition sequence of the second polynucleotide editing enzyme and
including. A method for visualizing a target product, which includes the following i-step, ii-step, and iii-step:
Step i: A third polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the first polynucleotide-editing enzyme and a fourth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the second polynucleotide-editing enzyme. And a step of gene-introducing a fifth polynucleotide editing enzyme-expressing polynucleotide capable of expressing a third polynucleotide editing enzyme, and a third step: expression of a first target product capable of expressing a first target product. A step of gene-introducing a polynucleotide for expressing a second target product and a polynucleotide for expressing a second target product capable of expressing the second target product;
Third iii step: A step of visualizing the first target product and the second target product,
here,
The polynucleotide for expressing the third polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fourth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fifth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the third polynucleotide editing enzyme and
A promoter sequence capable of expressing the third polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the second polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the third polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the third polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The polynucleotide for expressing the first target product is
The sequence encoding the first target product and
A promoter sequence capable of expressing the first target product in a host and
The recognition sequence of the first polynucleotide editing enzyme and
Including
The polynucleotide for expressing the second target product is
The sequence encoding the second target product and
A promoter sequence capable of expressing the second target product in a host and
The recognition sequence of the second polynucleotide editing enzyme and
including. A third cell group into which a third polynucleotide-editing enzyme-expressing polynucleotide capable of expressing the first polynucleotide-editing enzyme has been introduced, and
A fourth cell group into which a fourth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a second polynucleotide-editing enzyme has been introduced, and
A fifth cell group into which a fifth polynucleotide-editing enzyme-expressing polynucleotide capable of expressing a third polynucleotide-editing enzyme has been introduced, and
Including, cell population:
here,
The polynucleotide for expressing the third polynucleotide editing enzyme is
The sequence encoding the first polynucleotide editing enzyme and
A promoter sequence capable of expressing the first polynucleotide editing enzyme in a host and
The recognition sequence of the second polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the first polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fourth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the second polynucleotide editing enzyme and
A promoter sequence capable of expressing the second polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the third polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the second polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the third polynucleotide-editing enzyme is designed to suppress the expression of the second polynucleotide-editing enzyme in the presence of the third polynucleotide-editing enzyme.
The fifth polynucleotide-editing enzyme-expressing polynucleotide is
The sequence encoding the third polynucleotide editing enzyme and
A promoter sequence capable of expressing the third polynucleotide editing enzyme in the host, and
The recognition sequence of the first polynucleotide editing enzyme and
The recognition sequence of the second polynucleotide editing enzyme and
Is a polynucleotide containing
The recognition sequence of the first polynucleotide editing enzyme is designed to suppress the expression of the third polynucleotide editing enzyme in the presence of the first polynucleotide editing enzyme.
The recognition sequence of the second polynucleotide-editing enzyme is designed to suppress the expression of the third polynucleotide-editing enzyme in the presence of the second polynucleotide-editing enzyme.

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