[12] aneN3 cationic lipid containing targeted group and fluorescent group, transgenic vector, and preparation methods of cationic lipid and transgenic vector
A technology of cationic lipids and transgenic carriers, applied in other methods of inserting foreign genetic materials, gene therapy, organic chemistry, etc., can solve the problems of carcinogenicity, poor cell selectivity, lack of targeting groups, etc., and achieve good selectivity , simple preparation method and high transfection efficiency
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[0046] A [12]aneN containing a targeting group and a fluorescent group 3 Class cationic lipid, the structural formula of described cationic lipid is as follows:
[0047]
[0048] The method for cationic lipid, its synthetic route is as follows:
[0049]
[0050] The specific steps of the method include:
[0051] Step 1. Weigh the compound of formula 3 (1.28g, 3.13mmol), compound of formula 2 (1.0g, 3.13mmol), 1-hydroxybenzotriazole catalyst (1.15g, 0.57mmol), 1-ethyl- (3-Dimethylaminopropyl) carbodiimide hydrochloride condensation agent (0.60g, 3.13mmol) and N,N-diisopropylethylamine (0.80g, 6.26mmol), add dimethyl sulfoxide Dissolve 40mL of solvent and react under nitrogen atmosphere for 8-10 hours. After the reaction, evaporate the solvent under reduced pressure, add 35mL of water, extract with dichloromethane (40mLx2), combine the organic phases, wash with saturated brine, and dry over anhydrous sodium sulfate , the solvent was removed, and silica gel column chroma...
Embodiment 1
[0082] The preparation of the transgenic carrier containing cationic lipid formula 1 compound in embodiment 1
[0083] The raw materials include cationic lipid compound 1 prepared in Embodiment 1, dioleoylphosphatidylethanolamine DOPE (dioleoylphosphotidylethanolamine) and anhydrous chloroform. In a high-temperature sterilized flask, the synthesized cationic lipid formula 1 compound (0.005mmol) and DOPE were dissolved in 2.5mL anhydrous chloroform at a molar ratio of 1:2, and after fully mixing and dissolving, the solvent was spin-dried under reduced pressure at room temperature to obtain Liposome membrane, the mixture was put into a vacuum drying oven for drying (drying time 12 hours, temperature 25°C) to remove residual chloroform, and the dried liposome membrane was preheated to 70°C in advance, trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer (10 mM, pH 7.4) was mixed and added in such an amount that the final lipid concentration was 1.0 mM. Finally, the ...
Embodiment 2
[0085] The transgenic carrier prepared in embodiment 2 was tested in the following aspects:
[0086] (1) Agarose gel electrophoresis experiment of transgene vector and pUC18-DNA complex
[0087] Preparation of transgenic vector and pUC18-DNA complex
[0088] At room temperature, add the transgene carrier into the PE tube respectively. The concentration of the transgene carrier substance is 0, 1, 5, 10, 20, 50 μM, and the concentration of pUC18-DNA is 9 μg / mL. Add the corresponding volume of ultrapure water to make The final volume of the reaction solution was kept at 20 μL, and centrifuged to mix evenly. Put it in a constant temperature water bath at 37°C, and after incubating for 1 hour, add 2 μL of 10×LoadingBuffer loading buffer to terminate the reaction.
[0089] Preparation of agarose gel
[0090] Weigh 280 mg of agarose, add 40 mL of 1×TAE (Tris-acetic acid) buffer solution, and heat with microwave to completely dissolve the agarose particles to obtain a colorless tra...
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