JPS5876091A - Genetic transformation of eucaryota - Google Patents

Abstract

PURPOSE:To inject DNA securely into cell and to carry out the titled transformation, by suspending an eucaryote in a DNA-containing solution, pricking the cell nucleus with a spicule from the outside, pulling it out. CONSTITUTION:An eucaryote (any of yeast, a fungus such as a mold, animal and plant, and, when one such as plant has a cell wall, the cell wall may be removed and the cell may be processed into protoplast) is placed on the Petri dish 1, a DNA-containing solution having the desired genetic information is added to the dish, the Petri dish 1 is placed on the objective 2, the central part of the cell nucleus is pricked through with the hypodermic needle 3, and it is pulled out, so that DNA is introduced into the cell. DNA is incorporated into chromosome without being modified at all. Consequently, almost in all cases, genetic information except that of injected DNA is not admixtured, and screening of genetic variant is not required. Useful for breeding of animal or plant, improvement of kinds, remedy for animal, etc.

Description

[Detailed description of the invention] This is the real deal! relates to a true 1s organism O genetic transformation method.

Various proposals have been made as a method for converting the genetic traits of living things, and this is one of them! There is a known method of injecting a solution containing 1M silica into cells using a syringe needle, which is referred to as italointubation solution. However, this method not only made it impossible to quantitatively inject DMA into cells, but also the equipment used in this method was complicated and operationally inefficient. .

On the other hand, after incorporating foreign DMA into the vector 1) Imu, this is taken up into cells. Although there are methods to do so, these methods have drawbacks such as the need to develop a vector compatible with the host, or the inability to introduce some vectors into the host. Furthermore, even when using a platform that successfully incorporates DMA into cells, the target substance may not always be able to enter the cells. Therefore, the desired DMA
It must be analyzed through complicated procedures to determine whether or not it has been taken up into cells.

The present invention solves these drawbacks and provides a method for converting the genetic traits of eukaryotes by reliably injecting desired D11 molecules into cells using a simple method. After turning into a prosthetic cell if necessary, the cell is suspended in a solution containing DMA having the desired genetic information, and the nucleus (center of the cell) is extracted from the outside with a needle. The present invention provides a method for genetic transformation of eukaryotes, which is characterized by incorporating a gene into cells.

In the present invention, the cells into which DMA is injected may be fungi such as yeast or mold, as long as they are derived from wing root organisms.

It can be either a plant or an animal. In addition, plants that have cell walls, such as plants, may be decomposed by removing the cell walls and turning into plastids.

On the other hand, the MM injected into the cell can be of any type, such as one integrated into the vector XIN, or one copy of the chromosomal psum7 fragment! Any of the nine complementary 1) Immu, etc., which is transcribed from 1M to 1M, may be used.

DMA is used after being dissolved in a 0jI-tonic solution such as a buffer or physiological saline, and the concentration is sometimes not controlled by one company, but since the amount of the solution taken into the cells is constant, it is natural if the concentration is high. X) The amount of Im injection increases.

To inject D) fm into eukaryotic cells, a needle-like object with a needle diameter of approximately A1 to A511Q1 is usually used, but depending on the purpose, D-fm using a needle other than OSS may be used. I) Suspend eukaryotic cells in a solution containing the protein, pierce (the center of) the nucleus of w4IIAa from the outside using the above-mentioned needle, and then pull out the needle to infuse the cell nucleus with I) ml. Can be injected.

Specifically, this can be carried out using an apparatus as shown in FIG. That is, cells are placed on a vetri dish 1, then a bale containing a DMA-containing solution is placed on the objective lens 2, and the central part of the nucleus of the cell is inserted with the injection needle 5 while pulling down the phase difference W. All you have to do is stab it all the way through. Figure 2 a w
IL Id The procedure of this method is explained in WB.

According to this method, the amount of the D]f solution injected into the cells is a constant amount (approximately L 5 /Am'). Moreover, according to this method, DMA can be injected into the cell nucleus extremely efficiently, for example, at a rate of 5,000 DMA per hour.
~7000 cell membranes can be incorporated. Furthermore, according to the method of the present invention, DMA can be injected quantitatively, so very stable results can be obtained. In addition, the nm itself in the solution containing the anti-inflammatory drug that is taken into cells by this method, and how 1
There is no need to select whether one program has been imported.

Thus, the DMA ('DW) containing the desired genetic information is injected into the cell nucleus and integrated into chromosome 1. In this way, p-alm is not subject to any modification (chemical modification, enzymatic degradation,
It is integrated into the chromosome without undergoing any pre-integration into the vector, etc.).

Therefore, in most cases, there is no contamination of genetic information other than the genetic information possessed by the injected cells.

Therefore, there is no need to screen for genetically transformed strains having the desired genetic information.

As is clear from the above explanation, if this method is used, it is possible to transform the genetic traits of eukaryotes with extremely high efficiency, thereby increasing the production of eukaryotic cells. It can be applied to the creation of new products by introducing new traits into true cover organisms, the breeding of animals and plants, the improvement of varieties, and the treatment of animals.

Next, examples of the present invention will be shown.

As an example cell! Us Ll! I cyst (fibroblast)'! 'to,
, a defect (Tef) was used. In addition, plR5 as DNA
A plasmid containing the herpes simplex virus-derived thymidine kinase (Deri gene) was used.
Adjust the concentration to a concentration that contains the number of 11 molecules shown in Table 1 below. □ First, about 100 of the above cells were scattered in the center of the Petri dish and allowed to adhere, and then the DMA solution 1/-
Add. Place this sticky IJ dish on the objective lens, and in this state insert the injection needle (thickness rl, 1~(L 5 tap)
Using a needle, only the center (nuclear part) of the whole cell was pierced. Thereafter, the DNA solution was removed and selection was carried out in an exfoliating medium.

After 4 days, the number (%) of divided cells was determined.

In addition, cells OI & (cells) that acquired TK as a genetic trait
I looked into it. These results are shown in Table 1.

Chapter 1 Table O00 6’+4　　　　50 Portrait ts.

Top t.　　　　.

1 210 1 41αi% 2 62494 642 tons 01 256 115 0 On the other hand, most of the uptake of labeled thymidine is selective); It turned out to be crowded. I) MAO of thymidine kinase
This means that everything is injected into the cell nucleus and expressed. Just a few out of all the cells injected with psumamu! ! The fact that it was possible to obtain S as a genetic trait and store it as a permanent strain proved that 1) Imu was integrated into the host cell.

Furthermore, even when DImu was similarly injected into the cytoplasm, no expression of thymidine kinase was observed.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the apparatus used to carry out the method of the present invention, No. 21! Ia-a is the theory W14WJ showing the procedure of the method of the present invention. 1... beetle dish, 2... objective lens,! 1ess injection needle, 4... injection needle holder, 5... condensing lens,
6...Phase difference ring filter. M...Cell #1...Cell nucleus, O...Cytoplasm. p ・1 D summer mu

Claims (1)

[Claims] After eukaryotic cells have been transformed into cyprotoplasts (as required), they are suspended in a solution containing X1IlIm carrying the desired O genetic information, and the cells are transfected with external needles. A method for genetic transformation of eutrophic organisms, characterized by incorporating 1M MU into the cells by removing the poached bird.