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Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism

A magnetic nanometer and gene carrier technology, applied in the direction of introducing foreign genetic material using carriers, other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve problems such as poor biocompatibility, low transformation efficiency, and low carrier capacity , to achieve the effects of easy promotion, improved conversion efficiency, and simple operation

Active Publication Date: 2013-08-07
INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many literatures have reported high-quality, well-dispersed Fe 3 o 4 Nanoparticle preparation methods, but most of these materials cannot be directly applied to animal cell gene transformation, because these Fe 3 o 4 The magnetic nanoparticles generally have problems such as poor biocompatibility, low carrier capacity, and magnetic instability, which lead to low conversion efficiency.

Method used

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  • Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism
  • Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism
  • Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of magnetic nano-gene carrier; (1) Preparation of nano-Fe 3 o 4 Sol: Accurately weigh 0.2molFeCl 3 and 0.1molFeCl 2 , added to 1L of 0.1mol / L HCl solution, fully dissolved and stirred evenly. Slowly add 1.4 L of 1.5 mol / L ammonia solution to the above solution at a flow rate of 10 ml / min, and continue stirring for 30 min after the addition is complete. Then slowly add 0.1 mol / L HCl solution to the reaction system, adjust the pH value of the system to 7.0, and continue to stir and react for 30 min. The whole reaction process is at N 2 under protection. Take out the reaction system, centrifuge and filter, discard the supernatant, recover the precipitate, and wash it several times to remove residual ions so that no Cl can be detected. - until. Transfer the solid obtained from the reaction to a flask, add 0.8L of deionized water, stir well, and disperse evenly, then add 0.2L of 30%H 2 o 2 , heated to 95°C, degummed for 8 hours, and obtained nano-Fe 3 o...

Embodiment 2

[0038] Under the condition of an external magnetic field, the magnetic nano-gene carrier transformed porcine fetal fibroblasts: 1×10 5 Pig fetal fibroblasts were inoculated in a 6-well plate one day before transformation and placed at 37°C, 5% CO 2Cultivate overnight in the incubator until the cells adhere to the wall and proliferate to 70-80%. Cells were exchanged before transformation and cultured in serum-free DMEM. Dilute 1 μg of pEGFP-N1 plasmid DNA in 50 μl of culture medium (DMEM medium without fetal bovine serum) and mix gently. 2 μl (concentration of 1 μl / μg) of nano-Fe prepared in Example 1 3 o 4 / PEI carrier was diluted in 50μl culture medium, mixed gently, and incubated at room temperature for 5min. After 5 minutes, mix it with the above plasmid DNA, mix gently, and incubate at room temperature for 20 minutes to obtain nano-Fe 3 o 4 / PEI / pEGFP-N1 complex. The complex was added to each well of a culture plate containing pig fetal fibroblasts, driven by a 0.3T...

Embodiment 3

[0046] Under the condition of an external magnetic field, the magnetic nano-gene carrier transforms pig sperm cells: the pig sperm cells are diluted to 1×10 6 pieces / ml. Dilute 1 μg of pEGFP-N1 plasmid DNA in 50 μl of culture medium (DMEM medium containing 10% fetal calf serum), and mix gently. 2 μl (concentration of 1 μl / μg) of nano-Fe prepared in Example 1 3 o 4 / PEI carrier was diluted in 50 μl culture medium, mixed gently, and incubated at room temperature for 5 minutes; then it was mixed with the above plasmid DNA, mixed gently, and incubated at room temperature for 20 minutes to obtain nano-Fe 3 o 4 / PEI / pEGFP-N1 complex. Add the complex to 1ml of sperm cell solution, shake the culture plate back and forth to make it evenly distributed, drive it under a 0.3T magnetic field, incubate for 30min, and culture it in a 17°C incubator. The efficiency of sperm transformation was detected by immunohistochemical detection and real-time quantitative PCR. In this example, the ...

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Abstract

The invention relates to a magnetic nano-gene vector for transporting a gene, and a preparation technology and application thereof. The invention also provides a method for transforming animal and plant cells by a foreign gene loaded by the magnetic nano-gene vector. In the method, the magnetic nano-gene vector is nano Fe3O4 / PEI particles, and enters the animal and plant cells and / or generative cell with the foreign gene under drive of an external magnetic field; genetic transformation and expression are realized; and a new variety with excellent performance can be obtained through transgenosis cultivation. The preparation method and the transformation method disclosed by the invention are simple to operate, easy to control, and convenient to popularize. The transformation method disclosed by the invention is not limited by species; the receptor cell specificity is not required; and the transformation method can be widely applied to a transgenic technology of animals and plants. In the transformation method disclosed by the invention, the transformation efficiency of the gene can be significantly improved; and the copy number of the transferred foreign gene can be effectively controlled by controlling the quantity of the foreign genes combined with the magnetic nano-gene vector.

Description

technical field [0001] The invention relates to a preparation method and application of a magnetic nano-gene carrier for transgenic organism cultivation. The carrier can mediate exogenous gene transformation into animal and plant cells, and can be applied to the preparation of transgenic germplasm and the cultivation of new varieties. Background technique [0002] Nanoparticles have been widely used in the field of biology due to their unique physical and chemical properties. Nanomaterials have the following advantages as gene carriers: they can wrap, concentrate and protect nucleic acids from degradation by nucleases; they have large specific surface area and have bio-affinity; they have controllable degradability, which can realize the controlled release of genes and effectively prolong Time of action; avoids the potential danger that may be caused by the use of conventional viral vectors. Nano Fe 3 o 4 In addition to the general advantages of nanomaterials, it also has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N15/85C12N15/82
Inventor 崔海信王琰孙长娇孟志刚孙金海李奎姜建芳赵翔
Owner INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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