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Method for editing large white pig CD163 gene by using CRISPR/Cas9

A large white pig and gene technology, applied in the biological field, can solve the problems of persistent infection, lack of good vaccines and drugs, and antibody-dependent enhancement.

Inactive Publication Date: 2018-11-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2006, pig high fever broke out in my country, which caused serious economic losses to the pig industry. Later, this strain was defined as Highly pathogenic porcinereproductive and respiratory syndrome virus (HP-PRRSV). Pathogenic PRRS (Highly pathogenic porcine reproductive and respiratory syndrome, HP-PRRS) is one of the most damaging diseases to the pig industry. Due to the characteristics of long-term maintenance of PRRS, there are no good vaccines and drugs to prevent and control PRRS at present.

Method used

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  • Method for editing large white pig CD163 gene by using CRISPR/Cas9
  • Method for editing large white pig CD163 gene by using CRISPR/Cas9
  • Method for editing large white pig CD163 gene by using CRISPR/Cas9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Editing CD163 gene by CRISPR / Cas9 to prepare gene-edited cells

[0026] 1. Obtaining isolated porcine fetal kidney cells

[0027] Pig fetal kidney cells are isolated from large white pig fetal kidneys, and the pig fetal kidney cells are separated in an ultra-clean bench. Use scissors and tweezers to remove the kidney tissue of the fetus, wash the removed tissue repeatedly in 75% (v / v) alcohol and PBS with antibiotics in sequence, and cut the tissue block to a size of 1 cubic millimeter with small scissors, 1600rpm Centrifuge for 5 minutes to remove PBS, then add 20% FBS DMEM with antibiotics, gently pipette evenly, and culture in a 37°C cell culture incubator. After putting it into the cell culture box, do not move the culture dish. Three days later, it can be observed that the pig fetal kidney cells have covered the entire culture dish, and then the digestion and culture of ordinary passaged cells can be carried out.

[0028] 2. Obtaining cells containing edited CD16...

Embodiment 2

[0044] 1. Somatic cell nuclear transfer to obtain CD163 gene-edited pigs

[0045] Select ovaries with appropriate developmental stages from healthy Large White sows, extract the contents of follicles with a diameter of 3-5 mm on the surface of the ovary with a syringe, dilute the contents in TL-PVA and resuspend to form a suspension. The suspension was left standing at 37°C until the oocytes were completely precipitated, and the oocytes were sucked out and placed under a stereoscope with a pipette or a suction pipette to pick out oocytes with complete pericytes. The selected healthy oocytes were put into TCM-199 containing 10% (weight percent) follicular fluid, FSH, LH, EGF and cultured for 22 hours. The oocytes were then transferred to TCM-199 containing 10% (weight percent) follicular fluid and EGF with a pipette or a mouth pipette to continue culturing for 22 hours. After 44 hours of culture and maturation, the healthy mature oocytes that have discharged the second polar b...

Embodiment 3

[0052] Example 3: In vivo challenge experiment of anti-PRRS cloned pigs

[0053] 1. 16 piglets aged 4-6 weeks (8 gene-edited pigs + 8 wild-type pigs, earmarked) were divided into two challenge rooms (for two different PRRSV strains (JXA1 strain and MY strain) virus challenge), 8 pigs in each challenge room (4 gene-edited pigs + 4 wild-type large white pigs, 8 pigs mixed together).

[0054] 2. After adapting to the environment for 1 week, the dose of nasal drop per pig: 3ml cell culture solution containing 2×10 5 TCID 50 Virus.

[0055] 3. Blood collection, body temperature measurement, weighing, clinical symptom observation (respiratory, nervous and other symptoms, scoring and statistics), serum Freeze at -80°C for later use. During the virus attack period, if any pig dies, it needs to be counted. The mental state of the wild-type pigs and the gene-edited pigs after challenge was compared: the wild-type pig JXA1 had loose hair, cough, and poor spirit after challenge, whil...

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Abstract

The invention discloses a method for editing a large white pig CD163 gene by using CRISPR / Cas9. The large white pig CD163 gene is edited by using the CRISPR / Cas9, the extracellular domain SRCR5 of a CD163 receptor is destroyed, the 7th exon DNA fragment of the CD163 gene is knocked out, and the nucleotide sequence of the 7th exon of the CD163 gene is represented by SEQ ID NO.1. The edited gene obtained by adopting the method can completely resist infection of PRRSV including a highly pathogenic strain HP-PRRSV, allows the cell surface CD163 receptor expression to be normal, and has normal other biological functions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for editing the CD163 gene of large white pigs by using CRISPR / Cas9. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). PRRS mainly causes abortion and stillbirth in pregnant sows , mummified fetuses, weak piglets, and pigs of all ages, especially piglets, have respiratory symptoms. The characteristic lesion is interstitial pneumonia, and the mortality rate is extremely high. It is a highly contagious global important infectious disease. In 2006, pig high fever broke out in my country, which caused serious economic losses to the pig industry. Later, this strain was defined as Highly pathogenic porcinereproductive and respiratory syndrome virus (HP-PRRSV). Pathogenic PRRS (Highly pathoge...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/113C12N15/85
CPCC07K14/70596C12N15/113C12N15/8509C12N15/907C12N2310/10C12N2310/20
Inventor 何祖勇郭春和刘小凤丛佩清王敏刘小红陈瑶生
Owner SUN YAT SEN UNIV
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