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Method for high-efficiency introducing exogenous genetic matter into living cell

A genetic material and cell technology, applied to cells modified by introducing foreign genetic material, other methods of inserting foreign genetic material, using vectors to introduce foreign genetic material, etc. The frequency of success and other issues, to achieve the effect of rapid recovery of cell activity, reduction of somatic cell clonal variation, and small cell damage

Inactive Publication Date: 2003-03-12
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

[0005] However, the problem in the above reports is that the frequency of successful transformation has not been mentioned, and there has been no further optimization and improvement in technology; moreover, there have been no continuous reports, which shows that as a transformation method, it has not been systematically studied and is not yet mature enough.
Therefore, many parameters are not yet mature enough for the introduction of foreign genes by laser microbeams.

Method used

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  • Method for high-efficiency introducing exogenous genetic matter into living cell
  • Method for high-efficiency introducing exogenous genetic matter into living cell
  • Method for high-efficiency introducing exogenous genetic matter into living cell

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment one, material and method 1. plant material

[0044] Brassica napus (Brassica napus) variety H165 was used as the recipient material for transformation. This variety was selected by the Economic Crops Genetic Engineering Laboratory of the Institute of Genetics, Chinese Academy of Sciences and its cooperative units. )New varieties. 2. Plasmids and strains

[0045] Plasmid pTBt9 was constructed by myself, which contains the 3.5kb cry1Aa10 insecticidal crystal protein gene of Bacillus thuringiensis. 3. Enzymes and Reagents

[0046] Restriction enzyme, T 4 Ligase and Taq DNA polymerase were purchased from TaKaRa Company, "DIG DNA Labeling and Detection Kit" was purchased from Boehringer Mannhein Company, and the rest were domestic or imported analytical reagents. 4. Test insects and sources

[0047] The insects used in the insect resistance test of the transgenic rapeseed were the second instar cotton bollworm (Heliothis armigera) and the second instar diamon...

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Abstract

A process for effectively introducing the exogenous genetic materials into living cell includes treating the living cell, tissue block, or organ of plant in hyperosmotic cultural liquid for a certaintime, putting it in the culture liquid containing exogenous genetic material, and stabbing by microbeam laser. The exogenous genetic material immediately comes in the cell via the stabbed hole which is then closed in 3 seconds. Its advantages are less damage to cell, the growth-promoting action of laser on cell, and correct location.

Description

technical field [0001] The invention relates to a method for efficiently introducing exogenous genetic material into living cells, specifically treating living cells, tissue pieces or organs in hypertonic culture solution for a period of time (the key technology is to use suitable osmotic agents and suitable processing time), and then put it into the ordinary culture medium containing exogenous genetic material to form a gradient of cell osmotic pressure, which is high inside and low outside, and then immediately punctured with high-frequency pulses of micro-beam laser (the key technology is the best technology of laser micro-beam parameters, such as output energy, wavelength, pulse number, etc.), the exogenous genetic material immediately enters the cell from the hole, and the hole closes in a short time, so that the genetic material remains in the cell and integrates with its genome. Transformants are then selected in selection medium. Background technique [0002] The es...

Claims

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Application Information

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IPC IPC(8): A01H1/00A01H1/06C12N5/10C12N15/82C12N15/87
Inventor 王兰岚陈正华胡赞民宋桂英周奕华刘桂珍徐正平
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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