Nucleic acid molecule for cultivating short-tailed sheep based on HDR gene editing method, kit, method and application

A gene editing, kit technology, applied in the field of cell engineering and genetic engineering

Active Publication Date: 2021-05-11
XINJIANG BIOTECH INST OF ACADEMY OF ANIMAL SCI CHINA & AUSTRALIA SHEEP BREEDING CENT OF XINJIANG ACADEMY OF ANIMAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no efficient way to breed short-tailed sheep breeds

Method used

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  • Nucleic acid molecule for cultivating short-tailed sheep based on HDR gene editing method, kit, method and application
  • Nucleic acid molecule for cultivating short-tailed sheep based on HDR gene editing method, kit, method and application
  • Nucleic acid molecule for cultivating short-tailed sheep based on HDR gene editing method, kit, method and application

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Experimental program
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Effect test

Embodiment 1

[0045] Preparation of sgRNA and ssODN

[0046] 1. Select the first 21 bases located at positions 331-333 of the second exon of the TBXT gene (ie, the PAM sequence) as the sgRNA template sequence for TBXT gene editing fine-wool sheep, which is predicted to have low off-target effects. According to the selected sgRNA template sequence, synthesize complementary DNA Oligos;

[0047] The TBXT gene edited fine-wool sheep sgRNA sequence is located in the first 21 bases of the second exon 331-333, and the homologous recombination template ssODN is added at the same time, that is, the base at the 334th position of exon 2 of the TBXT gene is changed from C to A (C334A), the 333rd base is changed from C to G (C333G), so that the second exon 334 and 333 of the TBXT gene can achieve the purpose of precise editing when the TBXT gene is recombined and repaired ( figure 1 , TBXT gene editing position, sgRNA sequence ( figure 1 Shown as TBXT-sgRNA) and ssODN sequence diagram).

[0048] 2. D...

Embodiment 2

[0058] Preparation of Cas9 mRNA

[0059]Using the px330 (Addgene 42330) sequence as a template, primers Cas9-TF (lowercase letters are the T7 promoter sequence) and Cas9-TR were designed using Oligo6.0 software. The px330 plasmid was used as a template, and Cas9-TF and Cas9-TR were used as primers for PCR amplification according to the following system. PCR amplification system: 10×Buffer 5 μL, dNTP (2.5mM) 4 μL, upstream and downstream primers 1 μL (10 μmol / L) (Table 2), high-fidelity DNA polymerase 1.0 μL, template DNA 1.0 μL, water up to 50 μL; Reaction program: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 55°C for 45s, extension at 72°C for 5min, 35 cycles; final extension at 72°C for 10min. PCR products were detected by 1% agarose gel electrophoresis.

[0060] Take 2 μg of the PCR product from the above step after purification, and use the in vitro transcription kit mMESSAGE mMACHINE T7 ultrakit (ambionAM1345) for in vitro transcription....

Embodiment 3

[0064] Production of TBXT gene-edited sheep

[0065] 1. Estrus Synchronization and Superovulation in Sheep

[0066] (1) Selection of donor and recipient sheep: Xinjiang fine-wool sheep with good body condition, no reproductive diseases and 2-4 years old were selected as donor ewes. Aletai sheep with a body weight of more than 50kg, an age of 2 to 4 years, good body condition and no reproductive diseases were selected as recipient ewes. Xinjiang fine-wool sheep with a body weight of 70-85kg, excellent semen detection and 1-3 years old were selected as rams for semen collection.

[0067] (2) Simultaneous estrus and superovulation: the donor ewe was put into the vagina of the CIDR vaginal suppository as day 0, and the follicle-stimulating hormone was injected continuously in a decreasing manner on the 10th day after the CIDR vaginal suppository was put into the vagina, every 12 hours. , a total of 3 days, the total dose of 240 units / only, take out the CIDR suppository on the ...

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Abstract

The invention relates to a nucleic acid molecule for cultivating short-tailed sheep based on an HDR gene editing method, a kit, a method and application, and belongs to the technical field of cell engineering and gene engineering. The invention provides sgRNA for cultivating short-tailed sheep based on the HDR gene editing method, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1. The sgRNA can perform accurate mutation editing on sheep TBXT genes, the obtained gene edited sheep and offspring thereof have shortened tails and remarkably reduced caudal vertebra number, and meanwhile, the original production performance is kept unchanged.

Description

technical field [0001] The invention relates to the technical fields of cell engineering and genetic engineering, in particular to nucleic acid molecules, kits, methods and applications for breeding bobtail sheep based on homologous sequence-mediated double-strand DNA repair (Homology-directed repair, HDR) gene editing method. Background technique [0002] Tail length is one of the distinguishing characteristics of sheep breeds. The vast majority of special-purpose sheep breeds have thin and long tails, such as fine-wool sheep mainly producing wool, Suffolk sheep and Texel sheep mainly producing meat. These breeds have good wool or meat production performance and can bring huge economic benefits to the breeders. Therefore, they have become the main breeds of sheep produced in developed countries in the sheep industry. In my country, these sheep breeds are also widely promoted and used as improved breeds. Because the long tail is easily contaminated with feces, it is not on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/55C12N15/87C12N15/10C12N15/12A01K67/027
CPCC12N15/113C12N9/22C12N15/87C12N15/102C07K14/4702A01K67/0275C12N2310/20A01K2217/07A01K2227/103A01K2267/02C12Q2521/327C12Q2525/161
Inventor 刘明军李文蓉贺三刚彭新荣刘晨曦刘金瑞张雪梅韩冰李忠慧玛依拉
Owner XINJIANG BIOTECH INST OF ACADEMY OF ANIMAL SCI CHINA & AUSTRALIA SHEEP BREEDING CENT OF XINJIANG ACADEMY OF ANIMAL SCI
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