Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Preparation and application of porcine embryo fibroblast cell line edited by CD163 gene

A fibroblast, gene editing technology, applied in the direction of cells modified by introducing foreign genetic material, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problem of genetic biological function loss, respiratory system collapse, death and other problems , to achieve the effect of improving efficiency and specificity and reducing major economic losses

Inactive Publication Date: 2019-04-23
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Porcine reproductive and respiratory syndrome, also known as PRRS, swine fever, etc., is a rapidly evolving common disease in pigs. Young pigs are often susceptible to infection, which can lead to respiratory system collapse and eventually death. Pregnant sows are infected The cubs will be lost, and gene editing can prevent the spread of this nasty disease. The existing treatment method uses the plasmid vector system of CRISPR-Cas9 genome editing to knock out the entire CD163 gene, and obtain CD163 gene knockout pig embryos Fibroblast cell lines, cell lines established by existing methods may carry foreign genetic material, and knocking out the entire gene of CD163 will lead to loss of other biological functions of the gene

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Expression and purification of Cas9 protein:

[0023] The plasmid containing the Cas9 gene was transformed into E.coli Rosetta, and a single clone was picked and cultured overnight. The overnight bacteria were transferred to a large bottle of LB medium at a ratio of 1:100, and cultivated to OD at 200rpm at 37°C 600 When =0.6, add IPTG to a final concentration of 0.5mM, continue to culture at 160 rpm at 22°C for 16 hours, and then harvest the bacteria to extract protein. The following steps are all carried out at low temperature: collect the bacteria at 6000rpm, centrifuge for 15min, remove the supernatant, suspend the precipitate with molecular sieve buffer (20mM Tris, 50mM NaCl, pH 7.5, 1mM TCEP), collect it in a vial, and ultrasonically lyse it on ice ( Ultrasound 6s, interval 12s, 120 times, about 300W). Centrifuge at 15000 rpm for 20 min to remove the precipitate, and filter the supernatant with a 0.22 μm filter to sterilize. The supernatant was separated with Ni...

Embodiment 2

[0040] Expression and purification of Cas9 protein:

[0041] The plasmid containing the Cas9 gene was transformed into E.coli Rosetta, and a single clone was picked and cultured overnight. The overnight bacteria were transferred to a large bottle of LB medium at a ratio of 1:100, and cultivated to OD at 200rpm at 37°C 600 When =0.6, add IPTG to a final concentration of 0.5mM, continue to culture at 160 rpm at 22°C for 16 hours, and then harvest the bacteria to extract protein. The following steps are all carried out at low temperature: collect the bacteria at 6000rpm, centrifuge for 15min, remove the supernatant, suspend the precipitate with molecular sieve buffer (20mM Tris, 50mM NaCl, pH 7.5, 1mM TCEP), collect it in a vial, and ultrasonically lyse it on ice ( Ultrasound 6s, interval 12s, 120 times, about 300W). Centrifuge at 15000 rpm for 20 min to remove the precipitate, and filter the supernatant with a 0.22 μm filter to sterilize. The supernatant was separated with Ni...

Embodiment 3

[0058] Expression and purification of Cas9 protein:

[0059] The plasmid containing the Cas9 gene was transformed into E.coli Rosetta, and a single clone was picked and cultured overnight. The overnight bacteria were transferred to a large bottle of LB medium at a ratio of 1:100, and cultivated to OD at 200rpm at 37°C 600 When =0.6, add IPTG to a final concentration of 0.5mM, continue to culture at 160 rpm at 22°C for 16 hours, and then harvest the bacteria to extract protein. The following steps are all carried out at low temperature: collect the bacteria at 6000rpm, centrifuge for 15min, remove the supernatant, suspend the precipitate with molecular sieve buffer (20mM Tris, 50mM NaCl, pH 7.5, 1mM TCEP), collect it in a vial, and ultrasonically lyse it on ice ( Ultrasound 6s, interval 12s, 120 times, about 300W). Centrifuge at 15000 rpm for 20 min to remove the precipitate, and filter the supernatant with a 0.22 μm filter to sterilize. The supernatant was separated with Ni...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical field of cell genetic engineering, in particular to preparation and application of a porcine embryo fibroblast cell line edited by a CD163 gene. The pig embryonic fibroblast cell line can reduce carrying of exogenous hereditary materials and can reduce other biological function loss; the preparation comprises the following steps of (1) expression of Cas9 protein, (2) purification of Cas9 protein, (3) design of an sgRNA leader sequence, (4) in vitro transcription, (5) porcine embryo fibroblast nuclear transformation, (6) cell dilution, (7) separated screening, (8) expanded culture and (9) collection and identification. The cell line can be used as a donor of somatic cell nuclear transplantation for the preparation of transgenic pigs.

Description

technical field [0001] The invention relates to the technical field of cell genetic engineering, in particular to the preparation and application of a CD163 gene edited porcine embryonic fibroblast cell line. Background technique [0002] Porcine reproductive and respiratory syndrome, also known as PRRS, swine fever, etc., is a rapidly evolving common disease in pigs. Young pigs are often susceptible to infection, which can lead to respiratory system collapse and eventually death. Pregnant sows are infected The cubs will be lost, and gene editing can prevent the spread of this nasty disease. The existing treatment method uses the plasmid vector system of CRISPR-Cas9 genome editing to knock out the entire CD163 gene, and obtain CD163 gene knockout pig embryos Fibroblast cell line, the cell line established by the existing method may carry exogenous genetic material, and knocking out the entire CD163 gene will lead to loss of other biological functions of the gene. Contents ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor 朱庆锋刘文华陈庄陈中健吴秀菊刘圣杰何丽云张群洁戴彰言
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com