JPH06133784A - Method for introducing biosubstance to cell using magnetic fine particle and selective concentration and separation of cell by magnetism - Google Patents

Abstract

PURPOSE:To enable magnetic concentration and separation of a cell containing a biosubstance such as gene introduced into the cell by shooting fine magnetic particles supporting immobilized biosubstance into a cell at a high speed. CONSTITUTION:A biosubstance is immobilized on fine magnetic particles and the particles are shot into a cell at a high speed. The cell containing the introduced fine magnetic particles can be selectively and specifically concentrated or separated by a magnetic means. The method is useful for the screening of a drug resistant cell, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for introducing biological substances such as genes and enzymes into cells by magnetic fine particles and a method for selectively concentrating or separating the cells introduced by the method by magnetism. .

[0002]

2. Description of the Related Art PJ Robinson et al.
iotechnol. Bioeng., 15 , 603-606 (1973)) is a study on the use of magnetic microparticles as an enzyme immobilization carrier as an immobilization carrier for enzymes for bioreactors, focusing on its easy recovery operation. I am making a report. In this report, it was demonstrated that α-chymotrypsin or β-galactosidase was immobilized on iron oxide fine particles or cellulose-iron oxide complex and used in a complete mixed bioreactor to easily magnetically aggregate and separate the magnetic carrier. Has been done.

Further, ultrafine particles of iron having magnetism (minor axis diameter: approx.
By encapsulating 30 nm, long diameter of about 500 nm) with a synthetic polymer and immobilizing antibodies and enzymes on the outer surface of the polymer, an ultrafine particle-shaped carrier on which magnetically inducible enzymes and the like are immobilized (“ Ultrafine particle creation science and technology "Hayashi Taku et al., Published by Mita Publishing Co., pp. 231-235). Here, as a result of carrying out the reaction in a small bioreactor using a magnetic ultrafine particle carrier on which glucose oxidase was immobilized as a model enzyme, the reaction rate per unit volume was high, and the ultrafine particle carrier was easily magnetically concentrated. , Has been shown to be recoverable.

Further, DYNABEADS (registered trademark) is a fine particle of a polymer in which magnetic particles are dispersed, which is manufactured by Nippon Dynal Co., Ltd.
More imported and sold. This product is fine particles (diameter 2.8 and 4.5 μm) in which ferric oxide (ferrite, Fe ₂ O ₃ ) is dispersed in polystyrene beads for the purpose of magnetic separation of antibodies and other proteins and nucleic acids. For the beads coated with an antibody or bound to a certain protein or nucleotide, a magnet can be used in combination to rapidly separate cells and purify them by a simple operation.

However, among the above-mentioned biological substance-immobilized magnetic fine particles, those using a composite of magnetic fine particles and a polymer as a carrier have a low density as fine particles for shooting,
Improper because the impact force of the collision is weak. Also, the particle size is not sufficiently small compared to the size of the target cells. Therefore, it cannot be introduced into a plant cell having a cell wall, and in fact, there is no precedent. In addition, although it may be possible in principle with the microinjection method using a glass thin tube,
It is difficult to introduce into many cells at one time.

Also, US Pat.
No. 100,792 describes attaching a gene to a fine particle carrier,
A method of introducing it into cells at high speed and expressing its function (particle gun method) is described. Here, gold or tungsten is mainly used to increase the collision energy of the fine particles. In this U.S. Pat. No. 5,100,792, specifically used in the examples are tungsten fine particles having a diameter of 4 .mu.m, and in the detailed description of the invention, the particle size of the fine particles is about 10 nm to several .mu.m. And high density (about 10 to 20 g / cm ³ ) ferrite crystals, gold, tungsten and other metal particles, low density (1 to 2 g / cm ³ ) glass, polystyrene and latex beads.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to introduce a biological substance such as a gene into a cell and to express its function by using a magnetic fine particle having a physical property. Further, the present invention utilizes the fact that magnetic fine particles introduced into cells can be magnetically induced, and for example, only cells or cell clusters or organs in which a gene is expressed or physiological functions are expressed are selective and specific. The purpose is to physically concentrate and separate.

In the case of conventional application examples of magnetic particles or plastic beads containing magnetic particles (composite magnetic microspheres), there are all magnetic particles outside the cell, and especially in cells having a hard cell wall such as plants. It has not been possible to put magnetic particles inside, and the present invention has not been demonstrated worldwide.

[0009]

The first invention of the present application is a method for introducing a biological substance into a cell, wherein the magnetic fine particles having the biological substance immobilized thereon are shot into the cell at a high speed. The second invention is a selective concentration / separation method of cells, which is characterized by selectively concentrating or separating magnetically the cells into which magnetic particles have been introduced by the introduction method.

Any magnetic substance may be used as the magnetic property of the magnetic fine particles used in the present invention. For example, a ferromagnetic substance, a paramagnetic substance or a superparamagnetic substance is used. Examples of the material of the magnetic fine particles include metals, metal oxides, non-metal-metal composites, ceramic composites, organic magnetic materials containing natural and synthetic organic compounds with the above substances, and composites thereof, fluorescent substances, Photo-responsive substance, magneto-optical functional substance,
Examples include electron mediators, electrochemically functional substances, quantum mechanically functional substances, and semiconductor substances, but when magnetically inducing separation or concentration, the magnetic transfer speed of cells containing fine particles is high. Therefore, a ferromagnetic compound, for example, magnetite, other compound fine particles of a ferromagnetic element such as iron, cobalt and nickel, and magnetic fine particles containing chromium, manganese, aluminum, yttrium, tellurium, tungsten, titanium and the like are preferable. Among such ferromagnetic compounds, magnetite does not show toxicity to living organisms,
It is particularly preferable in that it is stable.

With respect to the particle size of the magnetic particles, the maximum particle size can be from 1/5 of the target cell to the size of the ultrafine particles, and it is preferable to use ultrafine particles having an average particle size of 5 nm to 100 nm or aggregated particles thereof. Yes, preferably with an average particle size of 10 nm to several μm
Magnetic fine particles are used. The density of the magnetic fine particles is usually 1 to 21 g / cm ³ , preferably ³ to 8 g / cm ³ .
Therefore, magnetite (density: about 5 g / cm ³ ), hematite (density: about 5 g / cm ³ ), cobalt iron oxide (density: about 3 g / cm ³ ), barium ferrite (density: about 5
g / cm ³ ), carbon steel, tungsten steel, KS steel having a density of about 8 g / cm ³ , and iron compounds of iron oxide and zinc, magnesium, manganese, or nickel having a density of about 4 to 5 g / cm ³ ( It is preferable to use fine particles of a Ferrox cube material or fine particles of a rare earth cobalt magnet (density: about 8 g / cm ³ ).

The shape of the magnetic fine particles is preferably non-spherical and has an angle in order to easily penetrate and break through the cell wall or cell membrane, and when compared with the same cross-sectional area, the moment of inertia is larger than that of spherical fine particles, and the cell wall or Needle-shaped and rod-shaped cross-sections are preferred because they easily penetrate and break through the cell membrane. In the present invention, the biological substance to be immobilized on the magnetic fine particles refers to animals, plants, protozoa, physiologically active substances such as microorganisms, intracellular organs, biological fine particles, etc., for example, genes,
Enzymes, antibodies, proteins, pheromones, allomones, mitochondria, viruses and the like can be mentioned.

In the present invention, the term "immobilization" means to hold a biological substance on magnetic fine particles, mainly by physical adsorption or other biochemical affinity. Immobilization of the biological substance on the magnetic fine particles can be performed, for example, by mixing a solution of the magnetic fine particles and a buffer solution of the biological substance, placing the mixture on the tip of a bullet, and air-drying.

The target cells in the present invention are:
Examples include animal cells (including human cells), plant cells, other biological cells, organs, tissues and the like. In the present invention,
"Shoot at high speed" means that the initial velocity of particles is 50 to 400 m / s.
Is about, for example, U.S. Patent No. 5,100,792
It can be carried out by the particle gun method described in Japanese Patent No. 4-25626 and Japanese Patent Application No. 4-25626.

The cells introduced with the magnetic fine particles as described above can be selectively concentrated or separated by magnetism. This concentration or separation can be performed as follows, for example. For example, the solution in which the cells are dispersed is transferred to a transparent container such as a test tube, and a magnet is brought into close contact with the side wall of the transparent container to concentrate the cells. After that, the other solution portion is removed with a pipette or the like to separate only the cells containing the magnetic fine particles. If necessary, repeat this operation several times.

The present invention is to improve the efficiency of gene introduction, recombination, and breeding of recombinants; introduction and magnetic induction of substances having physiological effects (plants, animals), selective separation; drugs such as anticancer agents; genetically modified cells. Bioreactor (for analysis-industrial production); Magnetic recombination sensing; Intracellular information combined with magnetic means and all other chemical, biochemical, physical properties, methods It can be applied to a method of recognizing from the outside depending on the method.

[0017]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. (Example 1) Gene introduction and expression by magnetite Magnetite (average particle size 0.3 μm, density 5.2 g / cm ³ ) (see FIG. 1) was used as the magnetic particles, and a particle gun method was used to discharge particles (Japanese Patent Application No. -25626 was used) to introduce and express the gene.

For the gene, plasmid DNA, pBI22
1 (manufactured by Clonetech, USA) was used. This plasmid carries the β-glucuronidase (GUS) gene,
This is the Cauliflower Mosaic Virus (CaMV) 35
It is connected between the S promoter (pCaMV35S) and the nopaline synthase gene (NOS) terminator (pAnos).

The operation of coating the plasmid DNA on the magnetite fine particles and attaching it to the plastic bullet was carried out as follows. Prepare a solution of 50 mg of magnetite particles suspended in 1 ml of 100% ethanol, and use this solution and a TE buffer solution of plasmid DNA (DNA concentration of 200-50
(Between 0 ng / μl) and an appropriate amount of ultrasonic waves, the mixture was lightly irradiated with ultrasonic waves and left to stand for a while, and then the mixed solution was placed on the tip of a 1-2 μl plastic bullet and air dried.

The gene introduction treatment was carried out by aseptic collection of the tobacco suspension culture cells BY-2 (obtained from the Faculty of Agriculture, Hokkaido University) on the 4th day on a filter paper by means of a fine particle ejector. It was carried out using a bullet placed on a sample table and air-dried under the following firing conditions. The firing condition is the distance to the sample
The pressure was adjusted to 10 cm, the degree of vacuum in the container for storing the sample was adjusted to 100 mmHg, and the supply pressure of nitrogen gas used to accelerate the polyacetal bullet was set to about 28 kg / cm ² (initial speed is about 200 to 250 m / sec). At this time, as a control experiment, those in which the gene was not attached to the magnetite microparticles were also shot under the same firing conditions as the tobacco cultured cells.

In order to evaluate that the gene is introduced into cultured tobacco cells and its function is expressed, the artificial substrate 5-
Bromo-4-chloro-3-indolyl-glucuronide (X
-Gluc) was performed. After bombarding magnetite microparticles, the cultured tobacco cells incubated at 25 ° C for 24 hours were immersed in X-Gluc's phosphate buffer solution together with the filter paper.
Incubated at 24 ° C for 24 hours. As a result, about 10 blue spots were observed on the filter paper.

A stereoscopic micrograph of this spot is shown in FIG. About 100 or more blue tobacco cells (cell mass) were confirmed. This is because, as a result of the introduction of the gene, the action thereof forms the enzyme β-glucuronidase in tobacco cells, enzymatically decomposes the artificial substrate X-Gluc, and produces the blue pigment indigotin. An example of a small amount of this tobacco cell observed with a biological microscope is shown in FIG. It was confirmed that the color of the cells in which the gene was expressed was clearly changed to blue as compared to the cells in which the gene was not expressed. Further, upon detailed observation, it was observed that the intracellular nucleolus, nuclear region and cytoplasm were blue. In this experiment, about 10
The introduction and expression of the gene was confirmed in more than 00 cells, and the introduction and expression of the gene of the biological material was succeeded for the first time by magnetite.

Example 2 Selective Concentration and Separation of Cells Introduced with Gene-Adhered Magnetite Microparticles and Separated and Incubated with Introduced Genes by Injecting Magnetite Microparticles Coated with Plasmid pBI221 Was taken in an appropriate amount, suspended in phosphate buffer, transferred to a small glass sample container, and an experiment of magnetic induction of the cells was performed. The container was shaken to evenly disperse the tobacco cells (Fig. 4). Then, a magnet (paper clip with a magnet for stationery) was brought into close contact with the upper right part of the container, and a photograph after 2 minutes is shown in FIG. As is clear from FIG. 5, most of the tobacco cells in the container were concentrated on the upper right side of the container, and only cells containing magnetite fine particles having magnetism were selectively separated and concentrated by magnetism. As a control experiment, the experiment was conducted under the same conditions when magnetite fine particles and tobacco cultured cells were simply mixed. In this case, first, only the magnetite particles quickly gather near the magnet,
Coarse particles were formed. At first, it was observed that some tobacco cells moved by being affected by the movement of particles, but when the concentration operation with a magnet was carried out about 3 times, only magnetic magnetite fine particles moved and aggregated, and the tobacco cells were completely magnetic. I stopped responding to.

Thus, it was confirmed that only tobacco cells having magnetite in their cells could be selectively concentrated and separated by magnetism. (Example 3) Growth of Tobacco Cultured Cell Transformant Under the same conditions as in the above Example, only plasmid DNA was coated as pBI121 on magnetite fine particles and similarly introduced into tobacco cultured cells. The plasmid DNA used in this experiment causes recombination into the plant chromosome.

The binary vector pBI121 is shown in FIG. The plasmid DNA, pBI121, has a β-glucuronidase (GUS) gene as a reporter gene whose expression site can be observed by staining. This gene is a promoter that works in plants to allow expression in plants
It is surrounded by (promoter, P) and terminator (poly-adenylation site, pA). NPTII
Is surrounded by the nopaline synthase gene promoter (Pnos) and the same gene terminator (pAnos), and GUS is surrounded by the cauliflower mosaic virus (CaMV) 35S promoter (pCaMV 35S) and pAnos. It also has a kanamycin resistance gene as a marker gene.

The cells bombarded with magnetite were screened by magnetically selectively separating and collecting the cells as in the above-mentioned examples, and then the drug-resistant cells were screened in a medium containing an antibiotic such as geneticin. On this occasion,
Both screening with and without magnetic screening were performed to compare screening efficiency. As a result, it was estimated that, for example, the selection efficiency of resistant callus at least about 10 to 20 times higher was higher than that of the experimental example in which magnetic screening was not performed. When the transgene was confirmed by the PCR method, the presence of the transgene was confirmed. In addition, the results of Southern hybridization analysis also confirmed bands specific to the transgene, and these cells were considered to be transformants.

As described above, it was shown that the new magnetic screening method can significantly efficiently perform the process of screening drug-resistant cells, which requires time and labor when growing transformants.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to introduce a biological substance such as a gene into a cell and to express its function by using magnetic fine particles, and to express a gene or a cell having a physiological function. Only cell clusters and organs can be selectively and specifically concentrated and separated.

[Brief description of drawings]

FIG. 1 is a photograph showing the particle structure of magnetite fine particles.

FIG. 2 is a photograph showing the morphology of organisms for tobacco cells (cell mass).

FIG. 3 is a photograph showing the morphology of an organism when a small amount of the tobacco cells shown in FIG. 2 is taken and observed under a biological microscope.

FIG. 4 is a photograph showing the morphology of an organism showing a state in which tobacco cells are uniformly dispersed.

FIG. 5 is a photograph showing the morphology of organisms 2 minutes after the tobacco cells were uniformly dispersed and a magnet was brought into close contact with the upper right part of the container.

FIG. 6 shows the binary vector pBI121.

Claims (2)

[Claims] 1. A method for introducing a biological substance into a cell, wherein magnetic fine particles having a biological substance immobilized thereon are shot into the cell at a high speed. 2. A method for selectively concentrating / separating cells by magnetically selectively concentrating or separating cells into which magnetic microparticles have been introduced by the method according to claim 1.