JPS63283569A - Device for introducing gene utilizing laser beam - Google Patents

Abstract

PURPOSE:To enable introduction of genes into a suspended cell, by providing a vibration part for dropping cells in a sheath flow at a given interval, laser device for irradiating the cells during the dropping with laser beam pulses and perforating the cells, etc. CONSTITUTION:Cells 6 are dropped from a sheath flow state thereof in one row through a vibration part 10 and irradiated with laser beam pulses to perfo rate cell membranes. The perforated cells 6 are then dropped into a suspension 14 containing genes mixed therein. The genes in the suspension 14 are then taken from the perforated openings into the cells 6, which close the perforated openings by self reparative action to complete the introduction of the genes. When the cells 6 are dropped into the genetic suspension 14, a rotating plate can be rotated to change positions of dropping within a given range without dropping the cells 6 onto a constant place of a vessel 12.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a device for introducing genes into various types of cells.

(Prior art) Methods for introducing genes into cells include (1) chemical introduction methods such as the calcium protonate coprecipitation method and dextran method; (2) applying electric pulses to cells to create holes in the cell membrane. Known methods include the electric pulse method for introducing genes, and (3) the artificial laser beam method for introducing genes by making holes in fixed cells with a laser beam.

(Problems to be solved by the invention) Among the disadvantages of conventional methods, such as being time-consuming and troublesome, the introduction efficiency is not very high, the introduction efficiency is expensive, and the introduction efficiency varies depending on the size of the gene. It has some of the following.

The object of the present invention is to provide a gene transfer device that solves these problems.

(Means for Solving the Problems) To explain with reference to FIG. 1 showing an embodiment, the gene transfer device of the present invention includes a sheath flow section (8) for aligning cells (6) and flowing them; A vibrating part (10) that drips flowed cells (6) at regular intervals, and a vibrating part (10) that drips flowed cells (6) at regular intervals;
6), a laser device (16) that irradiates a laser beam pulse to perforate the cell, and a container (12) that contains a suspension containing genes (14) and receives the dropped cells (6). There is.

(Function) When the laser pulse beam is irradiated onto the cells (6) dropped through the vibrating part (10) from the sheath flow state in which the cells (6) flow in a line, the cell membrane is perforated. The perforated cells (6) are dropped into a suspension containing genes (14). Then, the genes in the suspension (14) are taken into the cells (6) through the perforated opening. The cell (6) closes the perforation by its self-repairing action, completing the gene transfer.

(Example) FIG. 1 represents one embodiment of the invention.

Reference numeral 2 denotes a cell container containing a cell suspension 4, and an outlet for the cell suspension 4 is provided at the bottom thereof. 6 is a cell.

A tube 8 is attached to the outlet at the bottom of the cell container 2 to allow the cells 6 to flow in an aligned manner. The inner diameter of the tube 8 is set to a size suitable for flowing the cells 6 together with the solvent in a line. The tube 8 constitutes a sheath flow section.

At the tip of the tube 8 is an ultrasonic vibrator 10 as a vibrating part.
is attached. The cell suspension 4 that flows out from the tip of the tube 8 in the form of a sheath flow passes through the ultrasonic transducer 1
Cells 6 are separated individually by 0 and sequentially dropped at regular intervals.

A container 12 for receiving cells 6 to be dropped is provided below the tip of the tube 8. The container 12 contains a suspension 14 containing DNA to be introduced into cells.

Reference numeral 16 denotes a laser oscillator, which pulses and oscillates a laser beam, and irradiates the cells 6 which are individually separated and dropped by the ultrasonic transducer 10 with laser beam pulses. This creates a hole in the cell membrane of the cell 6. The pulse width and interval of the laser beam pulses from the laser oscillator 16 can be adjusted depending on the size of the cells 6 and the dropping period.

Since the laser oscillator 16 only needs to have enough output to make a hole in the cell 6, various laser devices such as a gas laser device and a semiconductor laser device can be used.

The cells 6 perforated by the laser beam pulse are placed in the container 1
The DNA in the suspension 14 is taken into the cell 6 until the perforation is self-repaired by the self-repair function of the cell 6 .

FIG. 2 shows an embodiment in which the container 12 is rotated within a plane in the embodiment shown in FIG.

The container 12 is placed on a rotating plate 18, and the rotating plate 18
is rotated in a plane by a rotary drive device 20.

The ultrasonic transducer 10 is inserted from the tip of the tube 8 to the container 12.
The falling position of the cells 6 that are individually separated and dropped is
The relative positional relationship between the tip of the tube 8 and the container 12 is determined so that the tip of the tube 8 and the container 12 are located eccentrically from the center of rotation of the container 12.

In this embodiment, when the cells 6 being dropped are irradiated with a laser beam pulse from the laser oscillator 16 to perforate the cells 6 and dropped into the DNA suspension in the container 12, the plate 1 rotates once.
By rotating the cell 8, the dropping location can be changed within a predetermined range without causing the cells 6 to fall to a fixed location in the container 12. This allows a wide range of genes to contribute to the introduction, improving the efficiency of gene introduction.

FIG. 3 shows the DNA in the container 12 in the embodiment of FIG.
This embodiment is equipped with a mechanism for applying vibration to the suspension 14.

The container 12 is placed on a vibrating plate 22, and the vibrating plate 22 is driven by a vibrating table 24 to vibrate.

In this embodiment, when the cells 6 are punctured and fall into the container 12, the DNA in the container 12 is vibrated by the vibrating plate 22! ! I The turbid liquid vibrates and the DN
A is stirred and the efficiency of DNA introduction into the cells 6 is improved.

In the embodiments shown in FIGS. 2 and 3, if the rotating plate 18 also serves as the diaphragm 22 and the container 12 is rotated and vibrated, the DNA introduction efficiency can be further improved.

FIG. 4 shows the DNA in the container 12 in the embodiment of FIG.
@Represents an embodiment equipped with a mechanism for cooling the suspension.

The container 12 is placed in contact with a Peltier element 28 as a heat exchange element via an aluminum plate 26.

The Peltier element 28 is activated by being energized by the temperature control section (path shown), and can absorb heat or generate heat. In this embodiment, the Peltier element 28 absorbs heat and cools the DNA suspension 1.
4 is used for cooling.

The Peltier element 28 has fins 30 for heat exchange.
is provided, air is supplied by a fan 32, and heat from the Peltier element 28 is released. 34 is a filter.

In this example, the temperature of the DNA suspension 14 is controlled by the Peltier device 2.
The perforated cells 6 with laser beam pulses from a laser oscillator 16 are placed in a DNA suspension 14 while being cooled by a
By dropping the DNA into the cells 6, the time for the cell membrane lipids of the cells 6 to repair can be extended, and the probability that DNA will be introduced into the cells 6 can be increased during this time.

(Effects of the Invention) In the present invention, genes are introduced into cells by making holes with a laser beam while dropping the cells at regular intervals and dropping them into a suspension containing genes. It is now possible to introduce genes into floating cells, which was not possible using the method, and the range of application is widened.

Furthermore, since cells are perforated with a laser beam, genes can be introduced into cells with their cell walls still attached. That is, in the chemical introduction method and the electric pulse method, gene introduction cannot be performed until the cell wall is removed and the cells are converted into a protoplast state, so the step of removing the cell wall is not necessary.

[Brief explanation of drawings]

Fig. 1 is a schematic front sectional view showing one embodiment, Fig. 2 is a schematic perspective view of main parts showing another embodiment, Fig. 3 is a schematic front view showing still another embodiment, and Fig. 4 is a further FIG. 7 is a schematic front sectional view showing another embodiment. 2...Cell container, 4...Cell suspension, 6...Cells, 8...Tube, IO...Ultrasonic vibration child, 12... Container, 14... DNA suspension, 16... Laser device, 18... Rotating plate, 22... Vibration plate, 28... Peltier element.

Claims (3)

[Claims] (1) A sheath flow section that aligns and flows cells, and a vibration section that drops cells that have been made into a sheath flow at regular intervals;
A gene transfer device that includes a laser device that irradiates cells being dropped with laser beam pulses to perforate them, and a container that contains a suspension containing a mixture of genes and receives the cells that have been dropped. (2) The gene transfer device according to claim 1, wherein the container is mounted on a mechanism that vibrates and/or rotates within a plane. (3) The gene according to claim 1, wherein the container is provided with a heat exchange element that changes the temperature of the liquid in the container, and a temperature control section is connected to the heat exchange element. Introduction device.