WO2018016440A1 - Introduction agent, kit, substance introduction method, and screening method - Google Patents

Abstract

This introduction agent includes a cholesterol-dependent cytolysin, for which the optimum membrane perforation activity pH at 30-40°C is at least 0 but less than 6, and is used in the introduction of a substance into a cell. This kit comprises the introduction agent and is used in the introduction of a substance into a cell. This substance introduction method includes a step 1 for perforating a cell membrane by bringing the introduction agent into contact with the cell and for introducing a substance into the cell. This screening method for a substance uses the substance introduction method, and the substance introduced into the cell is the substance to be screened.

Description

Introduction agent, kit, substance introduction method, and screening method

The present invention relates to an introduction agent, a kit, a substance introduction method, and a screening method.
This application claims priority based on Japanese Patent Application No. 2016-141219 for which it applied to Japan on July 19, 2016, and uses the content here.

Conventionally, introduction of a substance into a cell and evaluation of the effect of the substance on the cell have been performed. For example, if a compound expected to have a therapeutic effect on a disease is found, it may be used as an active ingredient of a therapeutic agent.
So far, compounds that have activity in vitro but do not have cell membrane permeability have been overlooked because their utility could not be verified by screening using cells. However, if a membrane-impermeable compound can be introduced into a cell, an active compound that has been missed so far can be newly found. It is also possible to synthesize a novel compound having excellent activity using it as a lead compound.

There are a limited number of practically useful methods for introducing a membrane-impermeable compound into cells. Cell-penetrating-peptide (CPC) binding is one of them, but it is necessary to perform peptide binding to the compound, which is complicated, and the amount that CPC tends to be trapped in the endosome and transported to the cytoplasm is unknown However, there is a problem that there is anxiety as a function evaluation system of compounds in cells.

So far, the present inventors have developed a reversible membrane perforation method using streptococcal toxin streptolysin O (SLO). By using the reversible membrane perforation method, semi-intact cells in which the plasma membrane is partially permeable can be obtained. As shown in Patent Document 1, semi-intact cells are prepared from other cells and organs by draining the original cytoplasm while maintaining the structure and function of organelles and cytoskeleton and their relative spatial arrangement almost intact. Can be “exchanged” for the cytoplasm. For example, a pathological environment can be constructed in a cell by exchanging the cytoplasm of a semi-intact cell with the cytoplasm obtained from the pathological cell.

Japanese Unexamined Patent Publication No. 2013-213688

However, in the reversible membrane perforation method using SLO, a relatively large pore having a diameter of about 30 nm is formed in the cell, and the perforation state is such that cytoplasm exchange is possible. Therefore, there is a concern about causing damage to cells when the method is applied to the compound introduction method. In addition, cytoplasm can be added from the outside at the time of re-encapsulation of the cell membrane to promote resealing of the cell membrane and damage recovery can be promoted, but it is a problem that adjustment of the cytoplasm is a troublesome work.

The present invention has been made in view of the above circumstances, and can introduce an introduction agent, a kit, a substance introduction method, and a method that can reduce damage to cells and enable high-efficiency introduction of substances into cells. Screening methods are provided.

That is, the present invention includes the following aspects.
The introduction agent according to the first aspect of the present invention contains a cholesterol-dependent cytolytic toxin whose optimum pH for membrane perforation activity at 30 ° C. or higher and 40 ° C. or lower is in the range of 0 to less than 6, and is a substance to cells Used for introduction.
The cholesterol-dependent cytolytic toxin may be listeriolysin O.
In the introduction agent according to the first aspect, the introduction rate of the substance having a molecular weight of 1 kDa to 15 kDa may be higher than the introduction rate of the substance having a molecular weight of 30 kDa to 200 kDa.
The pH of the introducing agent may be 6 or more and 10 or less.
The cholesterol-dependent cytolytic toxin content may be 0.01 μg / mL or more and 1 μg / mL or less.

The kit according to the second aspect of the present invention includes the introduction agent according to the first aspect and is used for introducing a substance into a cell.

The substance introduction method according to the third aspect of the present invention is a method including the step 1 of bringing the introduction agent according to the first aspect into contact with a cell to perforate a cell membrane and introducing the substance into the cell.
Furthermore, after the step 1, a step 2 may be included in which a solution containing calcium ions is brought into contact with the cells in which the pores are formed, and the pores are re-encapsulated.
In the step 2, the pores may be re-encapsulated without contacting a liquid containing foreign cytoplasm with the cells in which the pores are formed.
In the step 1, the perforation may be performed by allowing the cholesterol-dependent cytolytic toxin to act on the cells under conditions of pH 6 or more and 10 or less.
The molecular weight of the substance to be introduced may be 0.1 kDa or more and 20 kDa or less.
The substance to be introduced may contain a nucleic acid.

The method for screening a substance according to the fourth aspect of the present invention is a method using the substance introduction method according to the third aspect, wherein the substance to be introduced into the cell is a test substance, and the introduction agent is brought into contact with the cell. A step A in which a cell membrane is perforated and a test substance is introduced into the cell.
In the method for screening a substance according to the fourth aspect, after step A, the cell into which the test substance is introduced or a preparation thereof is compared with a cell into which the test substance is not introduced or a preparation thereof, You may include the process B which evaluates the said to-be-tested substance.

According to the introduction agent, the kit, and the introduction method according to the above aspect, damage to cells is difficult to occur, and a substance can be introduced into cells with high efficiency.
According to the screening method according to the above aspect, since it is difficult to cause damage to cells and a test substance can be introduced into cells with high efficiency, screening of a test substance with high accuracy reflecting the original function of the cell is possible. It becomes.

It is a schematic diagram explaining the substance introduction method of this embodiment. It is a figure which shows the measurement result of the fluorescein fluorescence (10 kDa) of the cell acquired in Example 1. FIG. It is a figure which shows the measurement result of the fluorescein fluorescence (40 kDa) of the cell acquired in Example 1. FIG. It is a figure which shows the measurement result of the fluorescein fluorescence (70 kDa) of the cell acquired in Example 1. FIG. It is a figure which shows the measurement result of the FITC fluorescence (150 kDa) of the cell acquired in Example 1. FIG. It is an image which shows the comparison result of the cell form of the reseal cell perforated by LLO and the reseal cell perforated by SLO acquired in Example 2. In Example 3, it is an image which shows the result of having detected the protein phosphorylated by PKA by Western blotting. In Example 3, it is an image which shows the result of having introduced the cell impermeable cAMP analog into the cell by the substance introduction method according to the present embodiment and confirming the function of the cAMP analog in the cell. In Example 4, it is an image which shows the result of having detected the protein phosphorylated by PKA by Western blotting. 9B is a graph in which the brightness of “band-e”, which is a protein phosphorylated by PKA, with respect to the brightness of GAPDH is quantified from the detection result by Western blotting in FIG. 9A in Example 4. FIG. In Example 4, a membrane-impermeable cAMP analog or a membrane-permeable PKA activator (db-cAMP) is introduced into a cell by the substance introduction method according to this embodiment, and the cAMP analog or db-cAMP in the cell is introduced. It is an image which shows the result of having confirmed the function of this by Western blotting. In Example 4, it is the graph which quantified the brightness | luminance of the band-e with respect to the brightness | luminance of (beta) -Tubulin from the detection result by Western blotting of FIG. 10A. In Example 4, it is an image which shows the result of having introduced the membrane impermeable cAMP analog into the cell by the substance introduction method according to this embodiment and confirming the function of the cAMP analog in the cell by Western blotting. In Example 4, it is the graph which quantified the brightness | luminance of the band-e with respect to the brightness | luminance of (beta) -Tubulin from the detection result by Western blotting of FIG. 11A. FIG. 10 is a schematic diagram of each step in Example 5. In Example 5, it is an image which shows the result of having introduced the membrane-impermeable AKT inhibitor into the cell by the substance introduction method according to the present embodiment and confirming the function of the AKT inhibitor in the cell by Western blotting. In Example 5, it is the graph which quantified the brightness | luminance of phosphorylated AKT (P-AKT) with respect to the brightness | luminance of total AKT (Total-AKT) from the detection result by Western blotting of FIG. 12B.

≪Introducing agent≫
The introduction agent according to one embodiment of the present invention is used for substance introduction into cells, and the optimum pH of membrane perforation activity at 30 ° C. or more and 40 ° C. or less is in the range of 0 or more and less than 6. Contains cholesterol-dependent cytolytic toxin.
Hereinafter, based on the embodiment, the introduction agent of the present embodiment will be described.

In the present specification, “introducing a substance into a cell” refers to introducing a substance into the inside of the cell membrane of a cell to which the substance is to be introduced. The newly introduced substance may be the same kind of substance as the substance possessed by the cell into which the substance is to be introduced. For example, newly introducing into a cell a nucleic acid having the same sequence as the nucleic acid of a cell that is a target of substance introduction is also included in the substance introduction into the cell.

The introduction agent of this embodiment is suitably used for the substance introduction method described later. Examples of the substance to be introduced include those exemplified in the substance introduction method described later.

As used herein, “cholesterol-dependent cytolytic toxin” refers to a substance that has membrane perforation activity that binds to cholesterol and forms pores in the cell membrane (perforates the cell membrane). A toxin produced by streptococci is known as a cholesterol-dependent cytolytic toxin. The toxin is a protein that binds to cholesterol on the cell membrane and then self-assembles on the cell membrane to form pores in the cell membrane.
When the cell is contacted with cholesterol-dependent cytolytic toxin, pores are formed in the cell membrane, and the substance can be introduced through the formed pores. That is, the substance introduction means substance introduction through the pores formed in the cell membrane by cholesterol-dependent cytolytic toxin.

The existence of an optimum pH is known for the activity of cholesterol-dependent cytolytic toxin. For example, cholesterol dependent cytolytic toxin, Listeriolysin O (LLO), has an optimum pH in the range of less than 6 (Reference 1: Schuerch, DW, Wilson-Kubalek, EM and Tweten, RK (2005) Molecular basis of listeriolysin O pH dependence. PNAS, 102, 12537-12542.).
By using a cholesterol-dependent cytolytic toxin in which the optimum pH for membrane perforation activity at 30 ° C. or more and 40 ° C. or less is in the range of 0 or more and less than 6, it is possible to bring cells to cells more than when using SLO. It was found that the substance can be introduced into cells with high efficiency and low damage.
When a cell is to be treated while maintaining a good state of the cell, it is usually treated with a solution having a pH near neutral. Here, when a cholesterol-dependent cytolytic toxin having an optimum pH in the range of 0 or more and less than 6 is used, the cell state is good and the membrane perforation activity is gently exerted. Is considered to be perforated.

The membrane perforation activity is at 30 ° C. or more and 40 ° C. or less, may be 33 ° C. or more and 38 ° C. or less, and may be 35 ° C. or more and 37 ° C. or less.
The optimum pH may be from pH 1 to less than 6, may be from pH 3 to 5.5, and may be from pH 4.5 to less than 5.5.

The optimum pH of the membrane perforation activity can be measured by a known method. For example, cholesterol-dependent cytolytic toxin is brought into contact with erythrocytes cultured at the above temperature and pH conditions. Next, the membrane perforation activity can be measured based on the degree of hemolytic activity (HU) of the blood cells from which the erythrocyte membrane is destroyed and hemoglobin is eluted. Furthermore, the pH at which membrane perforation activity is maximized can be determined as the optimum pH.

The cholesterol-dependent cytolytic toxin in which the optimum pH for membrane perforation activity at 30 ° C. or more and 40 ° C. or less is in the range of 0 to less than 6, may be Listeriolysin O (LLO). The introduction agent according to one embodiment of the present invention contains listeriolysin O (LLO) and is used for substance introduction into cells.

Listeriolysin O (LLO) is a L. genus belonging to the genus Listeria. It is known to be produced by monocytogenes. LLO is L. It may be a monocytogenes LLO.
The toxin contained in the introduction agent of the present embodiment may be a protein having a function substantially equivalent to that of LLO. LLO may be a naturally occurring protein of nature. Alternatively, LLO may be a mutant or artificially modified protein having an amino acid sequence, modification, addition, or the like different from that of the natural type as long as it has a function equivalent to that of the natural type.

The reason why the LLO perforation causes less damage to the cells than the SLO perforation is probably because the diameter of the hole formed by the LLO is smaller than the diameter of the hole formed by the SLO. In addition, due to such properties, it is considered that the outflow of cytoplasmic components is suppressed as compared with the reversible membrane perforation method using SLO, the cell is hardly damaged, and the cell state is improved. In addition, since the cell state is good, a good cell state can be realized without adding cytoplasm from the outside when the cell membrane is re-encapsulated (resealed).

The introduction rate of the substance introduced by the introduction agent of this embodiment is preferably such that the introduction rate of the substance having a molecular weight of 1 kDa to 15 kDa is higher than the introduction rate of a substance having a molecular weight of 30 kDa to 200 kDa, and a molecular weight of 7 kDa to 13 kDa. It is more preferable that the introduction rate of the substance is higher than the introduction rate of the substance having a molecular weight of 35 kDa to 55 kDa.
The introduction rate of the substance can be confirmed, for example, by the method described in the examples. As shown in Examples described later, the present inventors measured the introduction rate of fluorescently labeled dextran having different molecular weights into cells, each having a molecular weight of 10 kDa, 40 kDa, 70 kDa, and 150 kDa, using LLO. In perforation by LLO, it was found that the introduction rate of dextran having a medium molecular weight (10 kDa) into cells was larger than that of dextran having other molecular weights (40 kDa, 70 kDa, 150 kDa).
Such selective permeability of medium molecules has not been confirmed by reversible membrane perforation using SLO.

According to LLO, it is considered that a pore having a diameter that can easily pass a substance having a molecular weight of 1 kDa to 15 kDa and that is difficult to pass a substance having a molecular weight of 30 kDa to 200 kDa can be formed. Therefore, it is considered that the substance that correlates with the molecular weight and can pass through the pores is sieved.
The fact that a substance can be introduced into a cell through a hole opened in the cell membrane can similarly cause components in the cell to flow out of the cell through the hole. For example, the molecular weight of enzymes contained in the cytoplasm is often about 20 kDa or less. The introduction rate of substances with a molecular weight of 1 kDa or more and 15 kDa or less is higher than the introduction ratio of substances with a molecular weight of 30 kDa or more and 200 kDa or less, so that it is difficult to cause damage to cells. Can be introduced.

The introduction agent of this embodiment should just contain a cholesterol dependent cytolytic toxin. The introduction agent of this embodiment may consist essentially of a cholesterol-dependent cytolytic toxin, or may contain an optional component other than the toxin. As used herein, “substantially composed only of a cholesterol-dependent cytolytic toxin” means that the introduction agent of the present embodiment comprises only a cholesterol-dependent cytolytic toxin, or detects any component other than the toxin. It means that only trace amount below the limit is included.
The content of cholesterol-dependent cytolytic toxin contained in 100% by mass of the introduction agent may be, for example, from 1% by mass to 100% by mass, or from 10% by mass to 98% by mass, 50 mass% or more and 90 mass% or less may be sufficient, and 60 mass% or more and 80 mass% or less may be sufficient.

The size of the substance that can pass through the pores varies depending on the concentration of cholesterol-dependent cytolytic toxin that acts on the cells. Therefore, the concentration of cholesterol-dependent cytolytic toxin that acts on cells may be adjusted according to the molecular weight of the substance to be introduced.

The form of the introduction agent is not particularly limited, and may be various forms such as a solid (powder, granule, etc.) and a liquid.
Examples of the powder include a dried product of cholesterol-dependent cytolytic toxin. As an example, the moisture content of the powder may be 0% by mass or more and 20% by mass or less, and may be 5% by mass or more and 10% by mass or less.
Examples of the granular material include a compact of the powder.
Examples of the liquid include a dispersion or solution of cholesterol-dependent cytolytic toxin in which cholesterol-dependent cytolytic toxin is dispersed or dissolved in a medium. Examples of the medium include aqueous solvents, and specific examples include various media used for cell culture such as water, buffer, and serum-free medium.

The introduction agent of this embodiment is preferably used for cultured cells, and may be added to a medium for culturing cultured cells. The introduction agent of this embodiment may be provided and used as a medium in which cultured cells can be cultured.
In the present specification, “culture” refers to breeding or growing cells outside a living body (individual). The period of breeding or growth may be, for example, from 1 minute to 7 days, from 5 minutes to 16 hours, or from 10 minutes to 1 hour.
In addition, “medium” is a concept that generally refers to a cell culture medium. When the introduction agent is a medium, the remaining components obtained by removing the cholesterol-dependent cytolytic toxin from the introduction agent are not limited to the medium, as long as the cells can be cultured even for a very short period of time. It may be a buffer or water. Examples of components that can be contained in the medium include components contained in ordinary cell culture media. Specific examples of the component include nutritional components such as glucose, sodium chloride, vitamins and minerals, amino acids, growth factors, cell growth factors, differentiation-inducing factors, antibacterial agents, and antifungal agents. .

When the introduction agent is provided as a medium capable of culturing cultured cells, the content of cholesterol-dependent cytolytic toxin contained in the introduction agent is 0.01 μg / mL or more and 1 μg / mL or less per mL of the introduction agent. It is preferably 0.025 μg / mL or more and 0.6 μg / mL or less, more preferably 0.03 μg / mL or more and 0.5 μg / mL or less, and 0.05 μg / mL or more and 0.0. It is particularly preferably 3 μg / mL or less. The introduction agent containing the cholesterol-dependent cytolytic toxin in the above-mentioned concentration range can cause less damage to the cells and make the substance introduction effect into the cells more uniform.

In LLO, the introduction efficiency of a substance of medium molecule (about 10 kDa or less) is high due to the influence of the diameter of pores formed in the cell membrane, but larger molecules can be introduced depending on the concentration.
In the case where it is desired to obtain a tendency that the introduction rate of the substance having a molecular weight of 1 kDa or more and 15 kDa or less is higher than the introduction rate of the substance having a molecular weight of 30 kDa or more and 200 kDa or less, cholesterol contained in the introduction agent in the above concentration range. The content of the dependent cytolytic toxin can be in the range of 0.05 μg / mL to 0.3 μg / mL.

When the introduction agent is provided as a medium capable of culturing cultured cells, the pH of the introduction agent may be pH 6 or more, 10 or less, 6.5 or more, 8 or less, or 7.0 or more, 7 .5 or less. The pH is a measured value in a temperature range of 30 ° C. or higher and 40 ° C. or lower.
With the introduction agent within the above range, the cells can be cultured better. Furthermore, it is preferable from the viewpoint that the membrane perforation activity of cholesterol-dependent cytolytic toxin having an optimum pH in the range of 0 to less than 6 is exhibited more gently.

Conventionally, in the reversible membrane perforation method using SLO, cells are damaged by perforation of the cell membrane, and depending on the technique, cell death tends to occur.
LLO is considered to have an optimum pH in the range of 0 to less than 6, and has not been tried to be used for substance introduction.
However, the present inventors have surprisingly found that by using LLO, it is difficult to cause damage to cells, and substances can be introduced into cells with high efficiency.
According to the introduction agent of the present embodiment, since it contains a cholesterol-dependent cytolytic toxin having an optimum pH in the range of 0 or more and less than 6, it is difficult to cause damage to the cell, and the substance is highly efficient in the cell. Can be introduced inside.

≪Kit≫
The kit which concerns on one Embodiment of this invention is equipped with the above-mentioned introduction agent, and is used for the substance introduction | transduction of a cell.
Hereinafter, the kit of this embodiment will be described based on the embodiment.

In addition to the introduction agent, the kit of this embodiment may further include reagents such as a medium and a buffer, and reagents that promote reseal of the cell membrane such as calcium salt, ATP, and ATP regeneration system. .

The kit of this embodiment can be suitably used for the substance introduction method of this embodiment described later. The kit of this embodiment can be provided with instructions describing the substance introduction method of this embodiment.
In this way, by introducing reagents that can be used in the substance introduction method of the present embodiment, which will be described later, into a kit, the substance introduction method can be performed more easily and in a short time.

≪ Substance introduction method ≫
A substance introduction method according to an embodiment of the present invention is a method including the step 1 of bringing a substance into contact with a cell by bringing the aforementioned introduction agent into contact with the cell to perforate a cell membrane.

The introduction agent contains the cholesterol-dependent cytolytic toxin. That is, the substance introduction method of this embodiment may be a method including the step 1a of bringing the cholesterol-dependent cytolytic toxin into contact with a cell, perforating the cell membrane of the cell, and introducing the substance into the cell. .

The introduction agent that may be used in the substance introduction method may be a liquid containing cholesterol-dependent cytolytic toxin. Alternatively, it may be a medium containing cholesterol-dependent cytolytic toxin. Alternatively, it may be a liquid medium containing cholesterol-dependent cytolytic toxin.
Hereinafter, the substance introduction method of the present embodiment will be described by taking as an example the case where the introduction agent is a liquid medium containing a cholesterol-dependent cytolytic toxin. By culturing the cells in the medium (introducing agent), the introducing agent is brought into contact with the cells.
Hereinafter, the substance introduction method of the present embodiment will be described based on the embodiment with reference to FIG.

The substance introduction method of this embodiment is
A perforating step of perforating a cell membrane by contacting the introduction agent with cells;
An introducing step of introducing a substance into the cells through the holes formed in the cell membrane in the perforating step;
After the introduction step, a re-encapsulation step of bringing a solution containing calcium ions into contact with the cells in which the pores are formed and re-encapsulating (resealing) the pores.

(Punching process)
The perforation step is a step of perforating the cell membrane by bringing the introduction agent into contact with the cells.
(A) to (c) of FIG. 1 are schematic diagrams for explaining a perforation process.
As shown in FIG. 1A, the well W contains a medium M1 (introducing agent) containing cholesterol-dependent cytolytic toxin 1 (hereinafter sometimes referred to as “toxin 1”) and cells C. Contained. Examples of the cholesterol-dependent cytolytic toxin include those exemplified for the introduction agent.

As shown in FIG. 1 (b), when cell C is cultured in medium M1 (introducing agent) containing toxin 1, toxin 1 binds to cholesterol in the cell membrane of cell C, thereby binding to the cell membrane of cell C. To do.

The medium M1 containing the toxin 1 can be obtained, for example, by adding the toxin 1 to a known medium used for cell culture.
The medium component obtained by removing toxin 1 from medium M1 is not particularly limited, and a medium used for cell culture may be used, and a conventionally known medium may be used. Specific examples of the known medium include, for example, DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimum Essential Media), GMEM (Glasgow's MEM), and D-PBS (Dulbecco's Phosphate-Suffefer-Suffe medium Etc.

The concentration of toxin 1 contained in medium M1 (introducing agent) to be brought into contact with cells is preferably 0.01 μg / mL or more and 1 μg / mL or less, and 0.025 μg / mL or more and 0.6 μg / mL or less. Is more preferably 0.05 μg / mL or more and 0.3 μg / mL or less, and particularly preferably 0.08 μg / mL or more and 0.1 μg / mL or less. The medium M1 containing the toxin 1 in the above-described concentration range has less damage to the cells and can make the substance introduction effect into the cells more uniform.

The amount ratio of the toxin 1 contained in the medium M1 and the cell C can be appropriately determined according to the cell type of the cell. For example, it is considered that the higher the cholesterol content in the cell membrane of the cell, the less the amount of toxin required for membrane perforation. When the cholesterol content in the cell membrane is high, the amount (concentration) of the toxin contained in the medium M1 may be adjusted to decrease, and when the cholesterol content in the cell membrane is low, the amount of the toxin contained in the medium M1 ( The density may be adjusted in the increasing direction.

When cultured in medium M1 containing toxin 1, cells C are preferably cultured at a temperature of 0 ° C. or higher and 10 ° C. or lower, and preferably cultured at a temperature of 2 ° C. or higher and 5 ° C. or lower. That is, the temperature of the medium M1 is preferably 0 ° C. or higher and 10 ° C. or lower, and more preferably 2 ° C. or higher and 5 ° C. or lower. For example, the temperature of the culture medium M1 may be controlled by placing the culture container in which the well W is formed on ice. In the temperature range, the membrane perforation activity of toxin 1 is suppressed. Therefore, when the culture medium M1 containing the toxin 1 is brought into contact with the cell C, the toxin 1 binds to the cell membrane of the cell C, but the action of perforation is hardly exerted, and the degree of perforation of the cell membrane can be controlled more easily.

The culture time for culturing the cells C in the medium M1 (introducing agent) containing the toxin 1 may be appropriately determined according to the type of toxin and the cell type. An example of the culture time is about 1 to 30 minutes.

The cell into which the substance is introduced is not particularly limited, and examples thereof include cells such as animal cells, plant cells, and insect cells, and microorganisms such as Escherichia coli, Bacillus subtilis, and yeast. The cells may constitute cell aggregates such as cell clusters, spheroids, tissues, embryoid bodies, organs and the like.

After culturing the cells in the medium M1 (introducing agent), the medium M1 is replaced with the medium M2 not containing the toxin 1, the cells are cultured in the medium M2, and the cell membrane of the cell C is perforated.
By exchanging the medium M1 containing the toxin 1 for the medium M2 not containing the toxin 1, the toxins 1 other than those bound to the cells C are removed from the reaction system in the well W.

When cultured in the medium M2, the cells C are preferably cultured at 30 ° C. or higher and 40 ° C. or lower, more preferably cultured at 33 ° C. or higher and 38 ° C. or lower, and cultured at 35 ° C. or higher and 37 ° C. or lower. More preferably. That is, the temperature of the medium M2 is preferably 30 ° C. or higher and 40 ° C. or lower, more preferably 33 ° C. or higher and 38 ° C. or lower, and further preferably 35 ° C. or higher and 37 ° C. or lower. By culturing in the temperature range, the membrane perforation activity of toxin 1 is more exerted, the cell membrane of cell C is perforated, and a cell Cp having a pore formed in the cell membrane is obtained. Since the toxin 1 other than the toxin 1 bound to the cell C is removed from the medium M2, it is possible to prevent the toxin 1 from entering the cell further from the hole and perforating the organelle in the cytoplasm.

The culture time for culturing the cells C in the medium M2 may be appropriately determined according to the type of toxin and the cell type. An example of the culture time is about 1 to 30 minutes.

As the medium M2, the medium exemplified in the medium M1 may be used, but the TB (Transport Buffer) medium used in the examples can be exemplified as a particularly preferable medium. The TB medium is prepared in consideration of the composition of cytoplasmic ions, and the influence of perforation of the cell membrane can be reduced.

The perforation is preferably performed by allowing the toxin 1 to act on the cell C under the condition of pH 6 to 10, more preferably 6.5 to 8 and more preferably 7.0 to 7 More preferably, it is carried out under the condition of 5 or less. That is, the pH of the medium M2 is preferably 6 or more and 10 or less, more preferably 6.5 or more and 8 or less, and even more preferably 7.0 or more and 7.5 or less. The pH is a measured value in a temperature range of 30 ° C. or higher and 40 ° C. or lower.
Within the above range, the cells can be cultured more favorably, and the membrane perforation activity of toxin 1 is also exhibited more gently.

The perforation is preferably performed by allowing toxin 1 to act on cell C under conditions of 30 ° C. or higher and 40 ° C. or lower and pH 6 or higher and 10 or lower, and under conditions of 33 ° C. or higher and 38 ° C. or lower and pH 6.5 or higher and 8 or lower. More preferably, it is carried out under the conditions of 35 ° C. to 37 ° C. and pH 7.0 to 7.5. That is, the medium M2 is preferably 30 ° C. or higher and 40 ° C. or lower and pH 6 or higher and 10 or lower, more preferably 33 ° C. or higher and 38 ° C. or lower, and pH 6.5 or higher and 8 or lower, more preferably 35 ° C. or higher and 37 ° C. or lower, and pH 7.0 or higher. 5 or less is more preferable.

(Introduction process)
The introducing step is a step of introducing a substance into the cell through the hole formed in the cell membrane in the perforating step.
FIG. 1D is a schematic diagram for explaining the introduction process. As shown in FIG. 1 (d), the well W contains a medium M3 containing the substance 3 to be introduced into the cells Cp, and cells Cp in which pores have been formed in the cell membrane in the perforation step.
As shown in FIG. 1 (d), by culturing the cell Cp in the medium M3, the substance 3 passes through the hole formed in the cell Cp and is introduced into the cell membrane of the cell Cp.

The medium M3 containing the substance 3 can be obtained, for example, by adding the substance 3 to the medium M2 in the drilling step. The amount of the substance 3 contained in the medium M3 can be appropriately set according to the type of the substance 3.

In the substance introduction method of the present embodiment, the substance introduced into the cell is not particularly limited as long as it can be introduced into the cell by the method. The molecular weight of the substance may be, for example, a molecular weight of 0.1 kDa to 20 kDa, 1 kDa to 15 kDa, or 5 kDa to 10 kDa. When introducing a substance in the above range, the outflow of components contained in the cytoplasm is less likely to occur, so that it is more difficult to cause damage to cells, and the advantages of the substance introduction method of the present embodiment are more easily exhibited and better. Substances can be introduced into cells with high efficiency in the cellular state.

In the substance introduction method of this embodiment, the substance introduced into the cell may have a property of impermeable to the cell membrane.
In the present specification, the “substance having cell membrane impermeability” means a substance that does not dissolve in the lipid bilayer of the cell membrane and cannot permeate the lipid bilayer. According to the substance introduction method of the present embodiment, even a cell membrane impermeable substance can be introduced into cells through the pores with high efficiency.

In this embodiment, the substance to be introduced into the cells is contained in the medium M3. However, the substance introduction technique is limited to the above technique as long as the substance can be introduced through the pores formed in the cell membrane. It is not something. Examples of the method of introducing a substance include a method of bringing a substance into contact with the cell Cp, a method of spraying a substance directly on the cell Cp, a method of dropping a substance directly on the cell Cp, and the like.

In the substance introduction method of the present embodiment, the substance introduced into the cell may be a compound or an organic compound. The substance may contain a nucleic acid. Examples of the nucleic acid include antisense nucleic acid, miRNA, siRNA, shRNA, ribozyme, aptamer and the like. These compounds can be active ingredients of nucleic acid drugs. Compounds containing these nucleic acids are normally impermeable to cell membranes, but according to the substance introduction method of this embodiment, they can be efficiently introduced into cells.

(Re-encapsulation process)
The re-encapsulation step is a step of bringing the solution containing calcium ions into contact with the cells in which the pores are formed and re-encapsulating the pores after the introduction step.
FIG. 1E is a schematic diagram for explaining the re-encapsulation process. As shown in FIG. 1 (e), the medium M4 contains calcium ions (Ca ²⁺ ).

In the present specification, “re-encapsulation (reseal)” means that the opening of the hole formed in the cell membrane in the perforation step is completely or partially closed. Re-encapsulation is caused by removal of toxin 1 from the cell membrane by at least one of endocytosis and exocytosis, and is promoted by the presence of calcium ions. The cell Cp cultured in the medium M4 containing Ca ²⁺ has the cell membrane resealed to become a cell C. The substance 3 introduced into the cell in the introduction step is efficiently held in the cell C by re-encapsulation.

As an example, the concentration of the calcium ions brought into contact with the cells may be 0.1 mmol / L or more and 10 mmol / L or less, and may be 0.5 mmol / L or more and 5 mmol / L or less. That is, the concentration of calcium ions contained in the medium M4 may be 0.1 mmol / L or more and 10 mmol / L or less, and may be 0.5 mmol / L or more and 5 mmol / L or less.

The liquid containing calcium ions can be obtained, for example, by adding a calcium salt to the medium M3 in the introduction step. Examples of the calcium salt to be added include CaCl ₂ .

In the re-encapsulation step, it is preferable to re-encapsulate the pores without bringing a solution containing foreign cytoplasm into contact with the cells in which the pores are formed. The cytoplasm contains a factor that promotes at least one of endocytosis and exocytosis. In the conventional re-encapsulation, re-encapsulation was performed by bringing a solution containing foreign cytoplasm into contact with the cells in which the pores were formed. In the conventional method, since the cytoplasm component is released to the outside by perforation, it is considered that it was effective to replenish the released cytoplasm. On the other hand, according to the substance introduction method of the present embodiment, since it is difficult for damage to cells, re-encapsulation is considered to be performed well without adding cytoplasm from the outside when re-encapsulating the cell membrane.

In the present specification, “foreign cytoplasm” means the cytoplasm of cells other than the substance introduction target cell that has been treated with an introduction agent and perforated. For example, the cytoplasm of the cell Cp in which pores are formed in the cell membrane does not correspond to the foreign cytoplasm. In the example shown in FIG. 1, the foreign cytoplasm is a cytoplasm obtained from cells other than the cells C and Cp accommodated in the well W. The type of foreign cytoplasm may be a cytoplasm of a different type of cell from the cell into which the substance is introduced, a cytoplasm of the same type of cell, or a cytoplasm prepared from a tissue.

In the re-encapsulation step, a liquid containing a foreign cytoplasm may be brought into contact with the cells in which the pores are formed. In that case, for example, the foreign cytoplasm is at least one of the medium M3 and the medium M4. The cells may be cultured by adding them.

In the substance introduction method of the present embodiment, cells may be cultured in a medium containing an ATP regeneration system, or cells may be cultured in a medium containing an ATP regeneration system in at least one of the introduction step and the re-encapsulation step. Good. For example, the cells may be cultured by adding an ATP regeneration system to at least one of the medium M3 and the medium M4. Examples of the ATP regeneration system include a combination of ATP, creatine kinase, and creatine phosphate.

In the substance introduction method of the present embodiment, the drilling process and the introduction process are performed as independent processes, but the drilling and the substance introduction may occur almost simultaneously. In this case, in the substance introduction method of the present embodiment, it is exemplified that the substance 3 is added to the M3 medium after the perforation process. However, the substance 3 may be added to the medium M2 of the perforation process.
Moreover, although the re-encapsulation process was implemented in the substance introduction method of this embodiment, the re-encapsulation process is not an essential process in the substance introduction method of this embodiment.
“Step 1” indicates the above-mentioned “drilling step” and the above-mentioned “introduction step”. “Step 1a” indicates the above-mentioned “perforation step” and the above-mentioned “introduction step” in the case where cholesterol-dependent cytolytic toxin is used as a specific example of the introduction agent. “Step 2” indicates a re-encapsulation step.

As described above, according to the substance introduction method of the present embodiment, by using the above-described introduction agent, it is difficult to cause damage to cells, and a substance can be introduced into cells with high efficiency. In addition, when the cell membrane is re-encapsulated, re-encapsulation is satisfactorily performed without adding cytoplasm from the outside, and the substance is retained in the cell.

≪Screening method≫
The screening method according to an embodiment of the present invention is a method using the above-described substance introduction method, wherein the substance to be introduced into the cell is a test substance, and the introduction agent is contacted with the cell to perforate a cell membrane. A method comprising the step A of introducing a test substance into the cell.
Hereinafter, based on the embodiments, the screening method of the present embodiment will be described.

The screening method of this embodiment is:
A perforation step of perforating a cell membrane by contacting the introduction agent with a cell;
An introducing step of introducing a test substance into the cells through the holes formed in the cell membrane in the perforating step;
An evaluation step of comparing the cell into which the test substance is introduced and the cell into which the test substance is not introduced, and evaluating the test substance.

Examples of test substances include expression products of gene libraries, synthetic low molecular weight compound libraries, peptide libraries, nucleic acid libraries, antibodies, bacterial release substances, cells (microorganisms, plant cells, animal cells) extracts and cultures. Examples include supernatants, purified or partially purified polypeptides, marine organisms, plant or animal extracts, soils, random phage peptide display libraries, and the like.
Moreover, the substance introduce | transduced into the cell illustrated in the above-mentioned substance introduction | transduction method can be illustrated as a suitable thing.

The perforating step and the introducing step are as described in the substance introducing method. Moreover, as a method for introducing a test substance, the method exemplified in the above-described substance introduction method can be exemplified.
Further, the perforation process and the introduction process may be independent processes, and the perforation and the substance introduction may occur almost simultaneously.
Moreover, you may implement the above-mentioned re-encapsulation process after an introduction process and before an evaluation process.
“Step A” indicates the above-mentioned “perforation step” and the above-mentioned “introduction step” when the substance to be introduced into the cell is a test substance. “Process B” refers to an evaluation process described later.

(Evaluation process)
The test substance introduced into the cell can exert its function inside the cell.
The evaluation step is a step of evaluating the test substance by comparing a cell into which the test substance is introduced and a cell into which the test substance is not introduced.
What is necessary is just to set a comparison item suitably according to the objective of screening. For example, if the active ingredient of a therapeutic or preventive agent for disease A is screened, the degree of phenotype associated with disease A may be used as a comparison item. More specifically, for example, when the degree of phenotype associated with disease A in cells into which the test substance is introduced is lower than the degree of phenotype associated with disease A in cells into which the test substance has not been introduced, It can be evaluated that the test substance can be an active ingredient of a therapeutic or preventive agent for disease A.
The cell comparison may be performed on a cell preparation such as a cell extract, a slice, or an amplification product of a nucleic acid obtained from the cell.

According to the screening method of the present embodiment, since the substance introduction method of the present embodiment is used, it is difficult to cause damage to cells, the test substance can be introduced into cells with high efficiency, and the original function of the cell is reflected. High-accuracy screening of test substances becomes possible.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

(Reagents)
The reagents used in the examples are shown below.
Listeriolysin O (LLO): (CEDARLANE, model number: CLPRO320)
・ Streptricin O (SLO): (manufactured by Bio Academia, model number: 01-531)
・ FITC-dextran 150: (TdB, model number: FD150)
Dextran, Fluorescein, 40,000 MW, Anionic, Lysine Fixable: (Invitrogen, model number: D1845)
Dextran, Fluorescein, 10,000 MW, Anionic, Lysine Fixable (Fluoro-Emerald): (Invitrogen, model number: D1820)
Dextran, Fluorescein, 70,000 MW, Anionic, Lysine Fixable: (Invitrogen, model number: D1822)

[Example 1] Introduction of molecules into cells using LLO In order to confirm whether Listeriolysin O (LLO) can reversibly open holes in cells and introduce molecules into cells, fluorescently labeled dextran having various molecular weights is used. Was introduced into the cells.

(1) Perforation step First, after washing HeLa cells with PBS, LLO was diluted with DMEM (-FCS), and LLO was diluted 1100 times (0.45 μg / mL), 2200 times diluted (0.23 μg / mL), Cells were cultured on ice in DMEM (−FCS) containing 3300-fold dilution (0.15 μg / mL), 4400-fold dilution (0.11 μg / mL), and 5500-fold dilution (0.091 μg / mL). After culturing, LLO was allowed to adhere on the cells. After washing the above cells three times with PBS cooled on ice, TB (transport buffer: 25 mM Hepes, 1.15 mM KOAC, 250 μM MgCl ₂ , 2 mM EGTA, pH 7.2) previously added was added, and 37 Incubation was performed at 0 ° C. for 10 minutes to form perforations in the cell membrane.

(2) Introduction step Next, the cells were washed once with TB, and then ATP regeneration system (1 mM ATP, 50 μg / mL creatine kinase, 2.62 mg / mL creatine phosphate), GTP (1 mM), Glucose (1 mg / mL) And 10 kDa, 40 kDa or 70 kDa fluorescein-labeled dextran (Fluorescein-dextran, concentration 100 μg / mL) or 150 kDa FITC-labeled dextran (FITC-dextran, concentration 100 μg / mL), It was incubated at 37 ° C. for 30 minutes and introduced into the cells.

(3) Re-encapsulation step Next, 1 mM CaCl ₂ was further added and incubated at 37 ° C. for 5 minutes to induce re-encapsulation (reseal) of the cell membrane.

(4) Quantification of fluorescence The cells were washed with PBS, exchanged with DMEM, and cultured for 1 hour at 37 ° C. in a 5% CO ₂ environment. Next, the cells were detached with trypsin, and the FITC fluorescence of the cells was quantified by Flowcytometry.
The results are shown in FIGS. % In the graph indicates the percentage of cells plotted in the gate out of the cells detected by Flowcytometry, with the gate set so as not to include the region of fluorescence intensity detected for intact cells. ing.

As shown in FIGS. 2 to 5, when the cell membrane was perforated with a medium containing 1/5500 (0.091 μg / mL) LLO, 10 kDa dextran was retained in 96% or less of cells, At 40 kDa, 60% or less, at 70 kDa, 5% or less, and at 150 kDa, 7% or less, large molecules tended not to be introduced into cells. In addition, the size of the molecule to be introduced depends on the LLO concentration. For example, in the case of 1/1100 (0.45 μg / mL) LLO operated at the highest concentration, dextran at 10 kDa is 97% or less, and 90 at 40 kDa. Dextran was introduced into cells of less than or equal to 70%, 53% or less at 70 kDa, and 42% or less at 150 kDa.
Under the conditions of the concentration used in this example, dextran up to about 10 kDa can freely pass through the pores formed by LLO, and in the case of dextran having a higher molecular weight, it can easily pass depending on the concentration of LLO. Was found to change.

[Example 2] Comparison of introduction of molecules into cells using LLO and introduction of cells into cells using SLO Cell morphology was compared between reseal cells using LLO and reseal cells using SLO (Fig. 6).

(1) Drilling step HeLa cells were cultured for 5 minutes on ice in DMEM (-FCS) containing LLO (0.15 μg / mL) or SLO (0.125 μg / mL) to act on LLO or SLO. The cells were washed with PBS, and then incubated at 37 ° C. for 10 minutes with TB containing warm propidium iodide (PI). PI is a cell membrane-impermeable nucleic acid stain, and only cells with holes in the cell membrane are stained.

(2) Introduction step After that, the above ATP regeneration system, GTP, glucose and 10 kDa Fluorescein-dextran are added in the presence (cyrosol +) or absence (cyrosol−) of cytoplasm (1.5 mg / mL) prepared from L5178Y cells. Incubation was carried out at 37 ° C. for 30 minutes and introduced into the cells.

(3) Re-encapsulation step 1 mM CaCl ₂ was added and incubated at 37 ° C. for 5 minutes, and the cell membrane was closed again.

(4) Observation of cells The cells were replaced with DMEM and cultured in a 37 ° C., 5% CO ₂ environment for 30 minutes. After culture, the cells were observed using a confocal laser microscope. The results are shown in FIG.

As shown in FIG. 6, resealed cells that had been punctured with LLO remained clean and intact, with or without the addition of cytoplasm. On the other hand, in resealed cells that had been punctured with SLO (particularly when there was no cytoplasm), a protruding membrane (bleb) from the cell membrane was observed. Although the bleb seen in resealed cells by SLO disappears over time, it has become clear that LLO can introduce molecules into cells without destroying the integrity of the cell membrane.

[Example 3] Functional analysis of cells after introduction of molecules by LLO 1
Next, a membrane-impermeable molecule was introduced into the cell using LLO without the addition of exogenous cytoplasm and the function of the molecule in the cell was assayed. It was a membrane impermeable analog of cAMP used for introduction. Since cAMP activates PKA, the phosphorylation of the substrate protein by PKA after introduction of cAMP analog was verified.

(1) Detection of PKA substrate protein in intact HeLa cells (control)
First, as a control, PKA substrate protein was detected in intact HeLa cells (FIG. 7). Membrane-permeable PKA inhibitor (H89) or okadaic acid (OA), which is a serine / threonine phosphatase inhibitor and increases the phosphorylation level of PKA substrate, is added to intact HeLa cells, 0 minutes, 30 minutes, 60 minutes, Incubated for 90 or 120 minutes. A lysate was prepared from the cultured cells, and a protein band phosphorylated by PKA was detected by Western blotting using an anti-phospho PKA substrate antibody. The results are shown in FIG.

As shown in FIG. 7, when H89 was allowed to act for 30 to 120 minutes, it disappeared, and when okadaic acid was allowed to act, two bands were found at 150 kDa and 50 kDa. These bands are thought to be substrate proteins that react sensitively to the activation or deactivation of PKA. Among these, the band of 150 kDa was used as an index of phosphorylation of substrate protein when cAMP membrane impermeable analog was added to resealed cells by LLO.

(2) Intracellular introduction of cAMP analog using LLO Intracellular introduction of cAMP analog using LLO was performed by the following method.

(2-1) Perforation step First, HeLa cells were allowed to act on ice for 5 minutes in the presence or absence of LLO (0.15 μg / mL). The cells were washed with PBS and then incubated with PBS at 37 ° C. for 10 minutes.

(2-2) Introduction step After that, 1 mM 8-OH-cAMP (membrane impermeable activator), 1 mM Rp-8-OH-cAMP (membrane impermeable inhibitor) is present together with the ATP regeneration system, GTP, and glucose. Cells were incubated at 37 ° C. for 30 minutes in the absence or absence.

(2-3) Re-encapsulation process After that, a reseal operation with CaCl ₂ was performed.

(2-4) Detection of PKA substrate protein The cells were further cultured at 37 ° C. in a 5% CO ₂ environment for 1 hour. Thereafter, the cells were solubilized to prepare a lysate, and a protein band phosphorylated by PKA was detected by Western blotting using an anti-phospho PKA substrate antibody. The results are shown in FIG.

As shown in FIG. 8, the 150 kDa band was detected slightly stronger when cAMP active analog was introduced and significantly weaker when cAMP inactive analog was introduced, as in the intact control experiment.
This means that the membrane-impermeable cAMP analog introduced by the LLO treatment certainly functions in the cell.

[Example 4] Functional analysis of cells after molecule introduction by LLO 2
Next, again, a membrane-impermeable molecule was introduced into the cell using LLO without the addition of exogenous cytoplasm, and the intracellular function of the molecule was assayed. It was a membrane impermeable analog of cAMP used for introduction. Since cAMP activates PKA, the phosphorylation of the substrate protein by PKA after introduction of cAMP analog was verified.

(1) Detection of PKA substrate protein in intact HeLa cells (control)
First, as a control, PKA substrate protein was detected in intact HeLa cells (FIGS. 9A and 9B). To intact HeLa cells, membrane permeable 10 μM, 100 μM, 1000 μM or 2000 μM PKA activator (db-cAMP) or 0.3 μM, 3 μM, 30 μM or 60 μM PKA inhibitor (H89) is added for 1 hour. Cultured. Intact HeLa cells with no drug added as control 1 and DMSO added as control 2 were also cultured for 1 hour in the same manner. A lysate was prepared from the cultured cells, and a protein band phosphorylated by PKA was detected by Western blotting using an anti-phospho PKA substrate antibody. The results are shown in FIG. 9A. In FIG. 9A, “band-a” to “band-i” indicate a plurality of bands that change in a concentration-dependent manner with respect to db-cAMP. GAPDH is a loading control.

As shown in FIG. 9A, nine bands were found that varied in a concentration-dependent manner with respect to db-cAMP. These bands are thought to be substrate proteins that react sensitively to PKA activation. Among these, “band-e” was used as an index of phosphorylation of the substrate protein when cAMP membrane impermeable analog was added to resealed cells by LLO.

FIG. 9B is a graph obtained by quantifying the band-e luminance with respect to the GAPDH luminance.
Also from FIG. 9B, it was confirmed that the band-e luminance with respect to the GAPDH luminance increased in a concentration-dependent manner with respect to db-cAMP.

(2) Intracellular introduction of cAMP analog using LLO 1
Intracellular introduction of cAMP analog using LLO was carried out by the following method.

(2-1) Perforation Step First, HeLa cells were allowed to act on ice for 5 minutes in the presence of LLO (0.15 μg / mL). The cells were washed with TB and then incubated with TB for 10 minutes at 37 ° C.

(2-2) Introduction step Thereafter, the cells are treated in the presence of 1 mM 8-OH-cAMP (membrane-impermeable activator) or 1 mM db-cAMP (membrane-permeable activator) together with the ATP regeneration system, GTP, and glucose. Incubated for 30 minutes at 37 ° C.

(2-3) Re-encapsulation process After that, a reseal operation with CaCl ₂ was performed.

(2-4) Evaluation process (detection of PKA substrate protein)
Further, the cells were cultured for 1 hour in an environment of 37 ° C. and 5% CO ₂ . Thereafter, the cells were solubilized to prepare a lysate, and a protein band phosphorylated by PKA was detected by Western blotting using an anti-phospho PKA substrate antibody. The results are shown in FIG. 10A. β-Tubulin is a loading control.

As shown in FIG. 10A, the band-e was remarkably detected when the cAMP active analog, which is a membrane-impermeable activator, was introduced, as in the intact control experiment.
On the other hand, the band-e was detected slightly weaker when db-cAMP, which is a membrane permeability activator, was introduced, compared to the intact control experiment.

FIG. 10B is a graph obtained by quantifying the band-e luminance with respect to the β-tubulin luminance from the detection result of Western blotting in FIG. 10A.
FIG. 10B also confirmed that the band-e luminance relative to the β-tubulin luminance was higher when the membrane-impermeable cAMP analog was introduced than when the membrane-permeable activator db-cAMP was introduced. .

From the above, it was suggested that more membrane impermeable cAMP analog was introduced into the cell and functioned by LLO treatment than db-cAMP, which is a membrane permeability activator.

(3) Intracellular introduction of cAMP analog using LLO 2
Intracellular introduction of cAMP analog using LLO was carried out by the following method.

(3-1) Perforation Step First, HeLa cells were allowed to act on ice for 5 minutes in the presence or absence (control) of LLO (0.15 μg / mL). The cells were washed with TB and then incubated with TB for 10 minutes at 37 ° C.

(3-2) Introduction Step After that, the cells were incubated at 37 ° C. for 30 minutes in the presence or absence of 1 mM 8-OH-cAMP (membrane impermeable activator) together with ATP regeneration system, GTP and glucose.

(3-3) Re-encapsulation process After that, a reseal operation with CaCl ₂ was performed.

(3-4) Evaluation process (detection of PKA substrate protein)
Further, the cells were cultured for 1 hour in an environment of 37 ° C. and 5% CO ₂ . Thereafter, the cells were solubilized to prepare a lysate, and a protein band phosphorylated by PKA was detected by Western blotting using an anti-phospho PKA substrate antibody. The results are shown in FIG. 11A. β-Tubulin is a loading control.

As shown in FIG. 11A, in the HeLa cells using the substance introduction method of the present embodiment, the band-e was remarkably detected as in the intact control experiment.
On the other hand, when the LLO treatment was not performed, the band-e was detected slightly weaker than the intact control experiment.

FIG. 11B is a graph obtained by quantifying the band-e luminance with respect to the β-tubulin luminance from the detection result of Western blotting in FIG. 11A.
Also from FIG. 11B, it was confirmed that the band-e luminance with respect to the β-tubulin luminance increases when the substance introduction method of this embodiment is used.

From the above, it was suggested that db-cAMP, a membrane permeability activator, was introduced into cells by LLO treatment and functioned.

[Example 5] Functional analysis of cells after introduction of molecules by LLO 3
Next, different membrane impermeable molecules were introduced into the cells using LLO without the addition of exogenous cytoplasm and the function of the molecules in the cells was assayed. An Akt inhibitor was used for introduction. Akt is a serine / threonine kinase having a PH (Plekstrin Homology) domain at the N-terminus. Akt is an important intracellular signaling factor that controls cell death (apoptosis). Akt is activated by phosphorylation of Thr308 and Ser473. Therefore, the phosphorylation of Akt after introduction of the Akt inhibitor was verified.

(1) Intracellular introduction of an Akt inhibitor using LLO Intracellular introduction of an Akt inhibitor using LLO was performed by the following method. In addition, the schematic diagram of each process is shown to FIG. 12A. In FIG. 12A, the upper arrow shows the outline of the operation in each step. The lower square indicates the type and timing of the reagent added for each sample.

(1-1) Drilling step First, HeLa cells were allowed to act on ice for 5 minutes in the presence of LLO (0.15 μg / mL). The cells were washed with TB and then incubated with TB for 10 minutes at 37 ° C.

(1-2) Introduction Step After that, the cells were incubated with ATP regeneration system, GTP and glucose in the presence or absence of an Akt inhibitor at 37 ° C. for 30 minutes. As an Akt inhibitor, a peptide (Akt-in) composed of an amino acid sequence represented by 1 mM of SEQ ID NO: 1 (AVDTHPDRLWAWEKF), a TAT-labeled peptide (TAT) composed of an amino acid sequence represented by SEQ ID NO: 2 (YGRKKRRQRRRAVTHPDRLWAWEKF) of 50 μM. -Akt-in) or 31.2 μM triciribine.

(1-3) Re-encapsulation step Thereafter, a reseal operation with CaCl ₂ was performed.

(1-4) Evaluation process (detection of PKA substrate protein)
Further, the cells were cultured for 2 hours in an environment of 37 ° C. and 5% CO ₂ . As shown in FIG. 12A, TAT-Akt-in and trisiribine were added to the medium and cultured after resealing. Thereafter, EGT was added and incubated for 5 minutes. Thereafter, cells were solubilized to prepare lysates, and phosphorylated Akt and total Akt were detected by Western blotting using anti-phosphorylated Akt (P-Akt) antibody and anti-total Akt (Total-Akt) antibody. The result is shown in FIG. 12B.

As shown in FIG. 12B, phosphorylated Akt was significantly and strongly detected by EGT stimulation in cells cultured in the absence of an Akt inhibitor.
On the other hand, in cells cultured in the presence of an Akt inhibitor, phosphorylated Akt was detected significantly weaker than cells cultured in the absence of an Akt inhibitor.

FIG. 12C is a graph in which the luminance of phosphorylated Akt (P-Akt) with respect to the luminance of total Akt (Total-Akt) is quantified based on the detection result of Western blotting in FIG. 12B.
FIG. 12C also confirms that in the cells cultured in the absence of the Akt inhibitor, the brightness of phosphorylated Akt (P-Akt) relative to the brightness of total Akt (Total-Akt) is significantly increased by EGT stimulation. It was. In addition, in the cells cultured in the presence of the Akt inhibitor, the brightness of phosphorylated Akt (P-Akt) relative to the brightness of total Akt (Total-Akt) is higher than in the cells cultured in the absence of the Akt inhibitor. It was confirmed that it decreased significantly.
In addition, phosphorylated Akt (total-Akt) with respect to the luminance of the cells cultured in the presence of membrane-impermeable Akt-in and cells cultured in the presence of membrane-permeable TAT-Akt-in (Total-Akt) There was no significant difference in the brightness of (P-Akt). Furthermore, in cells cultured in the presence of membrane-permeable triciribine, the brightness of phosphorylated Akt (P-Akt) relative to the brightness of total Akt (Total-Akt) was particularly significantly reduced.

From this, the membrane-impermeable Akt inhibitor (Akt-in) was introduced into the cell to the same extent as the membrane-permeable Akt inhibitor (TAT-Akt-in) by LLO treatment and functioned. Was suggested.

Each configuration in each embodiment, a combination thereof, and the like are examples, and the addition, omission, replacement, and other changes of the configuration can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

According to the introduction agent, kit, and introduction method of the present embodiment, damage to cells is difficult to occur, and a substance can be introduced into cells with high efficiency.
According to the screening method of the present embodiment, it is difficult to cause damage to cells, and the test substance can be introduced into the cell with high efficiency, so that it is possible to screen the test substance with high accuracy reflecting the original function of the cell. It becomes.

1 ... Toxin, 3 ... Substance, C ... Cell, Cp ... Cell, M1 ... Medium, M2 ... Medium, M3 ... Medium, M4 ... Medium, W ... Well

Claims (14)

1. An introduction agent used for introducing a substance into cells, containing a cholesterol-dependent cytolytic toxin having an optimum pH for membrane perforation activity at 30 ° C. or higher and 40 ° C. or lower in a range of 0 to less than 6.
2. The introduction agent according to claim 1, wherein the cholesterol-dependent cytolytic toxin is Listeriolysin O.
3. The introduction agent according to claim 1 or 2, wherein an introduction rate of the substance having a molecular weight of 1 kDa to 15 kDa is higher than an introduction rate of the substance having a molecular weight of 30 kDa to 200 kDa.
4. The introduction agent according to any one of claims 1 to 3, wherein the pH of the introduction agent is 6 or more and 10 or less.
5. The introduction agent according to any one of claims 1 to 4, wherein a content of the cholesterol-dependent cytolytic toxin is 0.01 µg / mL or more and 1 µg / mL or less.
6. A kit comprising the introduction agent according to any one of claims 1 to 5 and used for introducing a substance into a cell.
7. A substance introduction method comprising the step 1 of bringing the introduction agent according to any one of claims 1 to 5 into contact with a cell to perforate a cell membrane and introducing the substance into the cell.
8. Furthermore, the said substance introduction | transduction method of Claim 7 including the process 2 which makes the cell which the said pore formed contact after the said process 1, and re-encloses the said hole.
9. 9. The substance introduction method according to claim 8, wherein in the step 2, the pores are re-encapsulated without contacting a liquid containing foreign cytoplasm with the cells in which the pores are formed.
10. The substance introduction method according to any one of claims 7 to 9, wherein in the step 1, the perforation is performed by allowing the cholesterol-dependent cytolytic toxin to act on the cells under a condition of pH 6 or more and 10 or less.
11. 11. The substance introduction method according to claim 7, wherein the substance to be introduced has a molecular weight of 0.1 kDa to 20 kDa.
12. The substance introduction method according to any one of claims 7 to 11, wherein the substance to be introduced contains a nucleic acid.
13. Using the substance introduction method according to any one of claims 7 to 12,
    The substance to be introduced into the cell is a test substance;
    A screening method for a substance comprising the step A of bringing the introduction agent into contact with a cell, perforating a cell membrane, and introducing a test substance into the cell.
14. The method further comprises a step B of comparing the cell into which the test substance is introduced or a preparation thereof with a cell into which the test substance is not introduced or a preparation thereof after the step A and evaluating the test substance. 14. The method for screening a substance according to 13.

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