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Gene transduction method with nano granule as carrier based on ultrasonic intervention guiding

A nanoparticle and ultrasonic technology, applied in the field of nanobiology and biological nanomaterials, biotechnology, to achieve the effect of cheap, easy to accept and master, easy to operate

Inactive Publication Date: 2005-10-26
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Can nano particles be used as gene carriers to transfer foreign genes into plant cells under the guidance of ultrasound to overcome the shortcomings of these two methods? No one has done research before

Method used

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  • Gene transduction method with nano granule as carrier based on ultrasonic intervention guiding
  • Gene transduction method with nano granule as carrier based on ultrasonic intervention guiding
  • Gene transduction method with nano granule as carrier based on ultrasonic intervention guiding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Ultrasonic treatment method for effective protection of nanoparticle DNA

[0037] 1. Ultrasonic treatment of DNA-nanoparticle complexes. Take six 150μl centrifuge tubes, add nanoparticle solution to the centrifuge tubes, then add plasmid DNA solution, mix well, and let stand at room temperature for 30 minutes. The experiment is divided into two groups. The first group: take three centrifuge tubes, On the generator, under the intensity of 120W and 40KHz, it was subjected to ultrasonic treatment for 30min, 20min and 10min respectively; the second group: the samples in the three centrifuge tubes were treated the same as the first group;

[0038] 2. Add NaOH solution to the second group of samples, shake well, and elute the DNA in the DNA-nanoparticle complex according to the method of extracting DNA;

[0039] 3. Ultrasonic treatment of DNA. Add plasmid DNA to a 150 μl centrifuge tube, and ultrasonicate for 5 minutes on an ultrasonic generator at an intensity of 120W and 40...

Embodiment 2

[0042] Efficient gene transduction of animal cells based on ultrasound using polylysine starch nanoparticles as gene carriers

[0043] 1. Inoculate COS-7 cells in a six-well culture plate in 5% CO 2 , Cultivate in an incubator at 37°C. When the cell coverage reaches 50% to 70%, discard the medium, wash the cells with D-Hanks solution, digest with trypsin for 5 minutes, and add fresh medium to make a cell suspension solution, the cell suspension was treated under different ultrasonic intensities, and the survival rate of the cells was detected by the MTT method. The results are shown in Table 1. Therefore, 120W and 40KHz intensity were selected as the optimal treatment conditions for 2 minutes.

[0044] Ultrasonic intensity processing time

15min

10min

5min

2min

1min

120W, 40KHz

180W, 40KHz

300W, 40KHz

10.6％

0％

0％

49.8％

11.4％

0％

95.7％

51.3％

10.8％

98.1％

97.6％

65.5％

...

Embodiment 3

[0049] Transplantation of Fluorescent Nanoparticles into Plant Cells Based on Ultrasound

[0050] 1. Establishment of Arabidopsis thaliana suspension cell line: Transplant well-growing Arabidopsis callus into fresh medium of MS+BA0.1mg / l+NAA 0.5mg / l. After one week, select the uppermost callus and transfer it to the liquid medium, and cultivate it in the dark for 48 hours on a shaker at 110rpm;

[0051] 2. Take 1ml of the above solution, add polylysine starch nanoparticles modified by ruthenium pyridine, mix evenly on a micro-oscillator, and process it at 120W, 40KHz ultrasonic intensity for 5min;

[0052] 3. Centrifuge the above-mentioned cell suspension at 500rpm / min for 1min, suck off the supernatant, wash the suspended cells repeatedly with MS liquid medium, centrifuge again, and remove the supernatant, repeat this 5 times;

[0053] 4. Cultivate the above-mentioned suspended cells on a shaker at 110rpm in the dark for 48 hours;

[0054] 5. Observed under a fluorescent mi...

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Abstract

A gene transduction method ultrasonic-based, which using the nano particulate as the carrier. It consists of animal transgenic gene method and plant transgenic gene method. The outer gene joins with the particulate with the effect of static form together to form the gene carrier, which can protect the DNA being broken by ultrasonic. The ultrasonic synchronously give effect to particulate gene carrier, cells, organize and apparatus, the carrier can run efficiently through the short period alleyway of cell wall, cell film, core film cell, enables the outer gene constructs with the gene group in cell. This method has a high efficiency, and is provided with convenient manipulation, high efficiency, no request of differential function e.g. other merits. It can be applied widely to these range, animal and plant transgenic technology and genic cure of human being.

Description

technical field [0001] The invention relates to the fields of biotechnology, nanobiology, application of bionano materials and the like, in particular to an animal and plant transgenic method using nano particles as gene carriers. Background technique [0002] Nanoparticles as gene carriers have been successfully used in gene transduction of animals and human cells. Compared with commonly used gene carriers, they have the advantages of high biological affinity, good stability, easy control, and strong targeting. Gene transduction and gene therapy have shown great application prospects. However, when this nanoparticle gene carrier introduces exogenous genes into animal recipient cells, there are still challenges in the following aspects: When the gene-nanoparticle complex is co-cultured with cells, the barrier of the cell membrane makes the exogenous gene integrated into the cell genome. There are many intermediate links, which make it difficult for most nanoparticles carryi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
Inventor 刘选明刘俊肖苏尧童春意杨粤军秦玉芝
Owner HUNAN UNIV
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