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Method for acquiring gene editing sheep by RNA-mediated specific FGF5 gene knockout and special sgRNA for method

A species-specific, sheep-like technology, applied in DNA/RNA fragments, recombinant DNA technology, artificial cell constructs, etc., which can solve the high cost and technical requirements of large animal genome modification and transformation, the difficulty of cultivating new varieties, and the low selection efficiency. and other problems, to achieve more accurate targeting efficiency, avoid biosafety problems, and simple construction steps.

Active Publication Date: 2015-12-09
新疆畜牧科学院生物技术研究所
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the conventional breeding cycle is long, the predictability is poor, and the selection efficiency is low. The genetic gain obtained by conventional breeding methods in variety improvement is gradually flattening, especially in the breeding of varieties with multiple excellent traits such as high yield, high quality, and stress resistance. It is becoming more and more difficult to breed new species, and the cost and technical requirements for modification and transformation of large animal genomes are still high

Method used

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  • Method for acquiring gene editing sheep by RNA-mediated specific FGF5 gene knockout and special sgRNA for method
  • Method for acquiring gene editing sheep by RNA-mediated specific FGF5 gene knockout and special sgRNA for method
  • Method for acquiring gene editing sheep by RNA-mediated specific FGF5 gene knockout and special sgRNA for method

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, preparation sgRNA and Cas9mRNA

[0035] 1. Design the target sequence of sheep FGF5 gene and the sgRNA that recognizes the target sequence

[0036] The full-length sequence of the sheep FGF5 gene is shown in Sequence 1 of the sequence listing. The 268th to 270th nucleotides from the 5' end are the start codons, the 21108th to 21110th nucleotides are the stop codons, and the 328th to 629th nucleotides are the stop codons. The first nucleotide is exon 1. The protein encoded by the sheep FGF5 gene is shown in sequence 2 of the sequence listing.

[0037] Two sgRNAs were designed for the target sequence of the sheep FGF5 gene (the target sequence is on exon 1 of the sheep FGF5 gene), see the italic part in FGF5-TF1 and FGF5-TF2 in Table 1.

[0038] 2. Preparation of sgRNA FGF5

[0039] 1. Use restriction endonuclease BbsI enzyme single digestion px330 plasmid, then carry out 1% agarose gel electrophoresis (the 1% agarose gel electrophoresis figure of restri...

Embodiment 2

[0054] Example 2, sgRNA / Cas9mRNA mutation efficiency detection

[0055] 1. Obtaining fertilized sheep eggs

[0056] 1. Oocyte maturation

[0057] Collect sheep ovaries from the slaughterhouse (the ovaries are from Kazakh sheep), wash them with normal saline for 3-4 times, extract the oocytes, wash them with maturation solution for 3-4 times, and then drop them into well-balanced maturation solution (maturation solution The volume of the drop is 75-78μl, and each drop is filled with 25-30 oocytes), and placed in a solution containing 5% CO 2 cultured in a 38.6°C incubator (the following incubator cultures are all under the same conditions).

[0058] Equilibrate the maturation liquid: place the maturation liquid droplet in the incubator for 2h. Maturation solution: TCM199 culture solution + 10% FBS by volume + 0.05IU / mlFSH+0.05IU / mlLH+1μg / mlestradiol+24.2μg / ml sodium pyruvate+0.1mM / L cysteine+10ng / mlEGF+100IU / ml penicillin+100IU / ml streptomycin.

[0059] 2. In vitro fertil...

Embodiment 3

[0086] Example 3, Production of gene-edited sheep

[0087] 1. Selection of experimental sheep

[0088] Xinjiang fine-wool sheep with good body condition, no reproductive diseases and 2-4 years old were selected as donor ewes. Select the Altay sheep with a weight of more than 50kg, an age of 2-4 years, good body condition and no reproductive diseases as recipient ewes. Select purebred Xinjiang fine-wool sheep with a body weight of 70-85kg, excellent semen detection and 1-3 years old as semen collection rams.

[0089] 2. Synchronous estrus and superovulation

[0090] During the estrous cycle of the sheep, the donor ewes were put into the vagina of the CIDR vaginal suppository, and on the 10th day after the CIDR vaginal suppository was put in, FSH (Ningbo Sansheng Company, China) was injected continuously in a decreasing manner, once every 12 hours, for a total of 3 days , the total dose is 240 units / only, take out the CIDR suppository in the morning of the 12th day, wash the ...

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Abstract

The invention discloses a method for acquiring gene editing sheep by RNA-mediated specific FGF5 gene knockout and special sgRNA for the method. The sgRNA provided by the invention can realize specific targeted modification of sheep FGF5 gene, is RNA shown in the second to the 21st nucleotides from the sequence 4 to 5' end tail of a sequence table or RNA with the second to the 21st nucleotides from the sequence 4 to 5' tail end of the sequence table. The invention provides an effectively technical tool for sheep functional gene researches.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and relates to CRISPR / Cas9 technology, in particular to a method for obtaining gene-edited sheep by RNA-mediated specific knockout of the FGF5 gene and its special sgRNA. Background technique [0002] Genome manipulation technology is a cutting-edge technology developed in recent years based on genome and genetic information technology to achieve precise editing of specific genes or genome target sites through artificial design. It has become a research hotspot in the fields of biomedicine, agricultural animal breeding, and model animals. . In the field of animal breeding, since the potential of traditional breeding methods to increase production has reached its limit, using efficient and stable genome manipulation technology to innovate breeding methods and improve the efficiency and technical level of modern animal breeding is crucial for the creation of new breeding materials and breed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N5/071
Inventor 李文蓉张雪梅刘明军彭新荣刘晨曦林嘉鹏贺三刚吴阳升
Owner 新疆畜牧科学院生物技术研究所
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