JP2540734Y2 - Cell-poration high-voltage pulse generator - Google Patents

Description

[Detailed description of the invention]

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for applying electric stimulus to a high molecular substance such as a gene to take it into a cell such as a microorganism.

[0002]

2. Description of the Related Art It is well known that high-molecular substances such as genes can be incorporated into cells by electrical stimulation, and external genes are incorporated into cells in breeding of animals and plants and research on malignant tumors. Experiments to see the transformation and expression are proceeding.

[0003] When an electric stimulus is applied to a cell, the electric double layer of the cell membrane is temporarily disturbed, and a hole is formed. Through this hole, genes and the like outside the cell are taken into the cell.

[0004] The electrical stimulation applied to create temporary disturbances in the cell membrane must be appropriately applied according to the strength of the cell membrane and the size of the cell. For example, in order to introduce a gene into E. coli microorganism cells, Several tens of seconds at a high voltage electric field of 10 to 20 KV / cm. Although the addition of moderate attenuation pulses are appropriate, and the high breakdown voltage switching element is a power supply equipment required therefor was required expensive constructed from the condenser or the like of the high pressure large capacity.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a device which can provide effective electrical stimulation to microbial cells using relatively inexpensive components.

[0006]

The electrostimulation circuit of the present invention comprises a low-voltage DC power supply of about several hundred volts, a capacitor of several hundred μF charged from the power supply, and a charged electric charge to a primary winding of a step-up transformer. A thyristor switch circuit for discharging, a capacitor for rectifying a high-voltage current generated in the secondary winding of the transformer due to the induction of the discharge current, storing the DC charge, a resistor connected in parallel to the capacitor, and a sample cell. Consisting of
The width of the high-voltage pulse applied to the sample cell is changed by changing the parallel resistance value.

[0007]

Conventionally, this type of circuit required a high voltage power supply of several thousand volts, several sets of capacitors for changing the pulse width, and a thyristor with a high withstand voltage and a large current capacity. But relatively inexpensive number 100
It can be composed of electrical components with a withstand voltage of V.

The pulse applied to the sample is obtained by charging and discharging an induction pulse to and from a high-voltage capacitor of several μF. The charging time is 1 msec. ~ Several tens msec. Can be set to

[0009]

FIG. 1 shows an embodiment of the present invention. Where INP
DC voltage applied to UT terminal is 0-300V, 100mA
For example, it can be constituted by a power supply unit used for an electrophoresis apparatus or the like. This DC voltage switch 1
, A switch 2 is turned off, and the charge of this capacitor is transferred to a thyristor 3.
Is connected to the primary coil of the step-up transformer 6 of about 1: 8.

[0010] Between the thyristor 3 and the primary coil is 1
There is a protection resistor 4 of Ω, and a diode 5 for reverse surge protection is connected in parallel with the primary coil.

When a trigger voltage is applied to the gate of the thyristor via the transformer 7, the charge of the capacitor flows at once into the primary coil of the step-up transformer, and the secondary current induced by this current is rectified by the high voltage diode 8. The DC voltage is instantaneously charged into a 2.2 μF capacitor 9 and discharged through a pulse width limiting resistor 10 connected to the capacitor and an electrode cell containing a cell suspension connected between output terminals 11 and 12. . This discharge opens a hole in the lipid bilayer of the cell placed in the electrode cell, and a high molecular substance such as a gene in the suspension is taken into the cell.

[0012]

[Effects of the Invention] The pulse generator of the present invention has an electrode spacing of 1 mm.
Cells of E. faecalis. And Escherichia coli (E. coil.), And try to introduce external genes. Enterococci (E. faecalis.) Using a pulse voltage of 1200 V (12 KV / cm), a conversion efficiency of 0.9 × 10 ⁵ / μg DNA was obtained. In this E. coli (E. coil.), × 10
The conversion efficiency of ⁹ / μg DNA was confirmed. When a parallel resistance of 10 KΩ was connected, a constant of 9 msec. At the time, the highest efficiency was obtained.

[Brief description of the drawings]

FIG. 1 is a basic circuit diagram of a pulse generator according to the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Switch 2 Capacitor 3 Thyristor 4 Protective resistor 5 Diode 6 Step-up transformer 7 Transformer 8 High voltage diode 9 Capacitor 10 Pulse width limiting resistor 11, 12 Output terminal

Claims (1)

(57) [Scope of request for utility model registration] 1. A charge stored in a relatively large-capacity capacitor is transferred to the primary winding of a step-up transformer by a thyristor switch, and a high-voltage pulse on the secondary side generated by a change in the current is converted into a DC charge. A pulse voltage applied between the opposite electrodes of the electrode cell containing the cell suspension by storing the voltage in a capacitor with a withstand voltage and changing the resistance of a resistor connected in parallel with the capacitor
A high-voltage pulse generator for applying electrical stimulation to microbial cells, wherein the pulse width is changed .