Kit and method for constructing ApoC2 gene knockout hamster model

A technology of gene knockout and kit, which is applied in the field of disease animal models and its preparation, can solve the problem that mouse models cannot well simulate human HTG-related AS disease conditions, and achieve low lethality, high efficiency, and simple operation Effect

Active Publication Date: 2019-01-18
北京华阜康生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the lack of ApoC2 gene knockout animal models in the prior art, and the technical problem that mouse models cannot wel...

Method used

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  • Kit and method for constructing ApoC2 gene knockout hamster model
  • Kit and method for constructing ApoC2 gene knockout hamster model
  • Kit and method for constructing ApoC2 gene knockout hamster model

Examples

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Effect test

Embodiment 1

[0058] The kit for constructing the ApoC2 gene knockout hamster model includes sgRNA and cas9mRNA, and the sgRNA is obtained by PCR amplification and in vitro transcription of the artificial sequences shown in SEQ ID NO:2 and SEQ ID NO:3.

[0059] The preparation steps of the sgRNA are:

[0060] The artificial sequences shown in SEQ ID NO:2 and SEQ ID NO:3 are used as primers and templates for PCR amplification. The method of PCR amplification is: carry out in a 50 μl system, and the reaction conditions are 98°C, 30s; 98°C, 10s, 56°C, 30s, 72°C, 15s, a total of 35 cycles; 72°C for 10min. The PCR product was subjected to agarose gel electrophoresis, and a 124bp DNA band was recovered with a gel recovery kit (TAKARA kit), and then treated with proteinase K for 30 minutes to remove as much as possible the RNase in the sample, and then extracted with phenol-chloroform , ethanol precipitation to obtain the DNA template of the purified sgRNA. The template was transcribed in vitro ...

Embodiment 2

[0065] In this example, the wild-type hamster purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. is taken as an example to illustrate the method for constructing an ApoC2 gene knockout hamster model using the kit in Example 1. The experimental plan and process of the preparation and analysis of the hamster model in this example have passed the ethical review of experimental animals by the Ethics Committee.

[0066] The wild-type hamsters were raised according to clean-grade standards. Keep the humidity at 50-60% and the temperature at 22-24°C. The photoperiod is light from 7:00-19:00 and dark from 19:00-7:00.

[0067] The concrete method of preparing hamster model with wild-type hamster comprises the following steps:

[0068] Step 1, determine the ApoC2 gene information (GeneID: 101839353) in the database in NCBI (National Center for Biotechnology Information, American National Center for Biotechnology Information), design the hamster ApoC2 gene-sp...

test example 1

[0077] After the F0 generation hamsters were born one week old, the genomic DNA extracted from the toe tissue was collected, and the target fragment was amplified by PCR. The upstream primer was 5'-GGCATTACTCGCCCTTTAGC-3'(SEQ ID NO:4), and the downstream primer was 5'-CCGCAGGTCACAGACAACAT-3'(SEQ ID NO:5). The specific band was detected by electrophoresis after PCR and then sent for sequencing. Sequence and chromatograms to determine the mutation position.

[0078]Further sequence verification of the mutated hamster: link the PCR product to the T vector, transform it into competent cells, inoculate it on the LB medium plate with ampicillin, incubate at 37°C for 12 hours, and pick 20 single clones at random Bacterial colonies were inoculated into LB liquid medium, incubated on a shaker at 37°C for 12 hours, and then the bacterial liquid was sent for sequencing, and the sequences and chromatographic peaks were analyzed to determine the number, position and possible mutation types...

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Abstract

The invention discloses a kit and a method for constructing an apolipoprotein C2 (ApoC2) gene knockout hamster model, wherein the kit comprises sgRNA, which is obtained by PCR amplification of an artificial sequence as shown in SEQ ID NO: 2 and SEQ ID NO: 3, and in vitro transcription. The method comprises the following steps: designing the hamster ApoC2 gene specific targeting sequence, and preparing the cas9 mRNA and the sgRNA; Collection and culture of hamster fertilized eggs; Microinjection: co-injecting the sgRNA and cas9 mRNA into the cytoplasm of a hamster fertilized egg; Finally, the fertilized eggs were transplanted into the surrogate hamsters and the F0 generation hamsters were born. The ApoC2 gene knockout hamster model was successfully constructed by this method.

Description

technical field [0001] The invention relates to the technical field of disease animal models and preparation thereof, in particular to an animal model of cardiovascular disease and a construction method thereof, especially a kit and a method for constructing an ApoC2 gene knockout hamster model. Background technique [0002] Cardiovascular diseases caused by atherosclerosis (AS) seriously endanger human health. It is now clear that hypertriglyceridemia (HTG) is closely related to AS and other cardiovascular diseases, and is an independent risk factor for the occurrence of AS. Extreme hypertriglyceridemia can also induce acute pancreatitis, leading to multiple organ dysfunction. Familial hereditary hypertriglyceridemia is mainly caused by loss of function of genes related to triglyceride (TG) metabolism, the most important of which is loss of function of lipoprotein lipase (LPL). LPL is a hydrolytic enzyme in cell lysosomes, anchored on the surface of capillary endothelial ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N9/22A01K67/027
CPCA01K67/0275A01K2217/075A01K2227/105A01K2267/0375C12N9/22C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 冼勋德
Owner 北京华阜康生物科技股份有限公司
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