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Lipid polymer and preparation method thereof, and method for introducing gene editing plasmid into eukaryotic cells by adopting lipid polymer

A eukaryotic cell and gene editing technology, applied in the field of biomedicine, can solve the problems of cationic liposome intolerance to serum, high eukaryotic cell toxicity, and low eukaryotic cell survival rate, achieving low toxicity, high import rate, The effect of reducing damage rate

Pending Publication Date: 2019-11-05
成都朴名生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cationic liposomes usually have high toxicity to eukaryotic cells, and gene editing plasmids themselves are highly toxic. Coupled with the toxicity of cationic liposomes, the survival rate of eukaryotic cells may be very low
Moreover, cationic liposomes are not resistant to serum and are also difficult to use clinically.

Method used

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  • Lipid polymer and preparation method thereof, and method for introducing gene editing plasmid into eukaryotic cells by adopting lipid polymer
  • Lipid polymer and preparation method thereof, and method for introducing gene editing plasmid into eukaryotic cells by adopting lipid polymer
  • Lipid polymer and preparation method thereof, and method for introducing gene editing plasmid into eukaryotic cells by adopting lipid polymer

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preparation example Construction

[0051] The invention provides a kind of preparation method of lipopolymer, specifically comprises the following steps:

[0052] S1: Take amphoteric phospholipid solution, anionic phospholipid solution and cholesterol solution;

[0053] S2: mixing the amphoteric phospholipid solution, the anionic phospholipid solution and the cholesterol solution and stirring evenly to obtain a mixture A;

[0054] S3: Dry the mixture A under the protection of an inert gas at a drying temperature of 50-60° C. until a lipid film is formed to stop drying;

[0055] S4: adding buffer to the lipid film to hydrate the suspension to form suspended liposomes;

[0056] S5: The suspended liposomes pass through a filter with a fixed pore size while being heated, and then extruded into liposomes with uniform diameter by one of the ultrasonic method, microporous extrusion method, dialysis method or ethanol injection method. The diameter of the plastid is 50nm-2000nm, and the extrusion temperature is 45-55°...

Embodiment 1

[0065] Used instrument and reagent among the embodiment 1 are as follows:

[0066] The electrophoresis apparatus was Mini-Sub Cell GT from Bio-Rad. Avanti mini extruder is produced from Avantipolar lipids. The microplate reader was iMark from Bio-Rad. The microscope was a DM IL LED Fluo from Leica.

[0067] Primers were synthesized by Shanghai Sangong. The gene editing plasmid pX330 was from Addgene. Phospholipids are from Avanti polarlipids. Polyethyleneimine was from Basf. Cationic liposome Lipo8000 is from Biyuntian. WST and 1-methoxy-PMS in CCK8 staining solution were from SIGMA. HEK293 eukaryotic cells were from ATCC. T7E1 enzyme was from NEB. Other conventional reagents are of high purity. High-purity water meets the MilliQ standard.

[0068] 1. Preparation of gene editing plasmids

[0069] The gene editing plasmids pX330 / sgCLTA, pX330 / sgVEGF and pX330 / sgAAVS1 targeting the human CLTA gene, VEGF gene and AAVS1 gene were constructed using the gene editing plas...

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Abstract

The invention discloses a lipid polymer and a preparation method thereof, and a method for introducing a gene editing plasmid into eukaryotic cells by adopting the lipid polymer, and belongs to the technical field of biomedicine, wherein the lipid polymer comprises 20%-100% of amphoteric phospholipid, 0-50% of anionic phospholipid and 0-20% of cholesterol by mass; the lipid polymer is used for packaging the gene editing plasmid; the lipid polymer can show toxicity only when the concentration of the lipid polymer is high. Therefore, the toxicity to the eukaryotic cells is small and the damage rate of the eukaryotic cells is reduced. The introduction rate of the packaged gene editing plasmid introduced into the eukaryotic cells is high, the gene editing ratio is much higher than the introduced gene editing ratio after the cationic liposome is packaged, and the lipid polymer is resistant to serum, and can be used in clinic; the lipid polymer can pack different gene editing plasmids, and has little toxicity to the eukaryotic cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a lipopolymer and a preparation method thereof, and a method for introducing a gene editing plasmid into eukaryotic cells by using a lipopolymer. Background technique [0002] Gene editing technology was invented by Jennifer A. Doudna and Emmanuelle Charpentier et al. in 2012, and applied to eukaryotic eukaryotic cells by Zhang Feng et al. in 2013. The most classical and widely used gene editing system comes from Streptococcus pyogenes and consists of two components. One is Cas9 protein, and the other is sgRNA, a small RNA of about 100nt. Cas9 protein and sgRNA can form complex RNP and bind to the specific site of double-stranded DNA, that is, the target site. There are two factors that determine the binding site. One is that the 20 bases at the 5' end of the sgRNA are complementary to the target site sequence, and the other is that the 3 bases immediately follow...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 赖兵周洲杨莉
Owner 成都朴名生物科技有限公司
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