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Method for targeted editing of buffalo 18S rDNA gene by virtue of adenovirus system

A technology using adenovirus and adenovirus, applied in the direction of double-stranded DNA virus, recombinant DNA technology, application, etc., can solve problems such as non-disclosure, and achieve the effects of improving efficiency, high-efficiency infection, and high virus titer

Inactive Publication Date: 2017-04-26
GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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Problems solved by technology

[0005] For example, in the article "Comparison of Effects of Adenoviral Vectors Transfecting Different Primary Somatic Cells of Buffaloes" in "Chinese Animal Husbandry and Veterinary Medicine" 2016,43(10):2661-2665, the inventors have about transfecting buffaloes with adenoviral vectors Mammary gland epithelial cells, cumulus cells, and fibroblasts. However, this technology does not disclose any descriptions about the use of artificially constructed adenoviral vectors to target and edit buffalo 18S rDNA. Because the ribosomal gene 18S rRNA gene sequence and The secondary structure is highly conserved and has an important function in protein synthesis. It is the most conserved type of DNA sequence discovered so far. It is generally believed that if a gene editing method can edit 18S rDNA, it proves that the method can be edited. It can also have good gene editing ability for other genes

Method used

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  • Method for targeted editing of buffalo 18S rDNA gene by virtue of adenovirus system
  • Method for targeted editing of buffalo 18S rDNA gene by virtue of adenovirus system
  • Method for targeted editing of buffalo 18S rDNA gene by virtue of adenovirus system

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Embodiment

[0056] The present invention mainly performs gene editing of buffalo 18S through the following methods:

[0057] 1. Construction of sn468-18S-1 artificial vector and sn468-18S-2 artificial vector containing buffalo 18S gene fragment: the construction method includes the preparation of sn468 vector and the construction of sn468-18S-1 and sn468-18S-2 artificial vectors 2 steps;

[0058] (1) Wherein, the preparation method of the sn468 carrier is as follows:

[0059] 1. Digest the pUC57-U6 plasmid with Sal Ⅰ and Nhe Ⅰ enzymes, perform agarose gel electrophoresis on the digested products, and recover by cutting the gel to obtain the U6-Bsmb Ⅰ-gRNA target gene fragment, U6-Bsmb Ⅰ-gRNA The gene sequence of the enzyme is shown in Sequence Table 13; wherein, the enzyme digestion system is as follows: the enzyme digestion system is 3 μg of pUC57-U6 plasmid, 5 μL of 10×FastDigest, 3 μL of Sal Ⅰ, 3 μL of Nhe Ⅰ, added water to 50 μL, and incubated at 37°C for 3 hours; The digested produ...

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Abstract

The invention relates to the technical field of targeted editing of a buffalo 18S rDNA gene, in particular to a method for targeted editing of the buffalo 18S rDNA gene by virtue of an adenovirus system. According to the method provided by the invention, an autonomously constructed 18S gene editing vector is used for editing the buffalo 18S rDNA gene; a constructed adenovirus vector consists of Cas9 protein and sgRNA; and then, the vector is subjected to adenovirus packaging. The buffalo 18S rDNA gene is edited on the basis of the accurate and efficient performances of a Cas9 editing gene, a strong bearing capacity of adenovirus on exogenous genes as well as a strong infection capacity of the adenovirus on primary cells, so that a bottleneck that the buffalo primary cells are difficult for transfection; and the packaged adenovirus can efficiently infect buffalo primary mammary epithelial cells and can efficiently edit the buffalo 18S rDNA gene.

Description

[0001] 【Technical field】 [0002] The invention relates to the technical field of targeted editing of buffalo 18S rDNA gene, in particular to a method for targeted editing of buffalo 18S rDNA gene using an adenovirus system. [0003] 【Background technique】 [0004] Buffalo has the biological characteristics of high temperature and high humidity resistance, disease resistance, rough feeding resistance, and easy feeding, and is suitable for breeding in southern China. Buffaloes are widely bred in Guangxi. Since buffaloes have the characteristics of low lactation and low litter rate, in recent years, in the field of molecular biology, genetic engineering techniques have been used to edit buffalo-related genes, thereby achieving the goal of increasing milk production and litter rate of buffaloes. There are more and more researches on the purpose, and in the field of molecular biology, the gene transfer vectors are mainly divided into viral vectors and non-viral vectors. However, a...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66
CPCC12N15/86C12N2710/10043C12N2800/107C12N2800/80
Inventor 梁贤威朱鹏段安琴庞春英邓廷贤陆杏蓉马小娅梁莎莎
Owner GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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