Method for knocking out p66shc gene in pig embryo

A gene and embryo technology, applied in the field of constructing p66shc gene knockout pig embryos, can solve the problems of embryo apoptosis, affecting the efficiency of embryo development, etc.

Active Publication Date: 2017-05-24
HUNAN UNIV OF HUMANITIES SCI & TECH
View PDF4 Cites 46 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have also shown that the level of ROS produced can cause apoptosis of embryos and affect the efficiency of embr

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

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for knocking out p66shc gene in pig embryo
  • Method for knocking out p66shc gene in pig embryo
  • Method for knocking out p66shc gene in pig embryo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0024] (1) Design Oligo DNA sequence

[0025] First, total DNA was extracted from porcine aborted fetal tissue, by engineering p66 shc Gene upstream primer and reverse downstream primer PCR clone p66 shc The full length of the gene is then subjected to DNA sequencing, the sequence is saved, and its promoter region is analyzed for future use. Next, on p66 shc Design a pair of oligo DNA of about 20bp in the expression DNA region of the gene, which can be designed through the following online tools: CRISPR Design (http: / / crispr.mit.edu / ) of the Massachusetts Institute of Technology or E-Crisp of the German Cancer Research Center (www.e-crisp.org / E-CRISP / designcrispr.html). 1-3 pairs of Oligo synthesis are preferentially selected according to the score. Through online design, select the following three better pairs of Oligo:

[0026] (1).TCAATAAGCCCCACGCGGGGCTGG

[0027] (2).CCGGGGTTTCCTACTTGGTTCGG

[0028] (3).GCGAGGAGTGGACCCGCCACGGG

[0029] According to the requirements...

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 provides a method for knocking out p66shc in a pig embryo by utilizing a CRISPR-Cas9 gene editing method. The method comprises the following concrete operation steps: (1) designing an Oligo DNA sequence; (2) connecting Oligo; (3) transforming and amplifying a constructed plasmid; (4) verifying knock-out efficiency by plasmid transfected cells; (5) constructing a CRISPR-Cas9 system of the p66shc gene; (6) producing the pig embryo with the p66shc gene knocked out; and (7) verifying a p66shc gene knock-out result. By adopting the scheme of the invention, the ROS level in an in vitro embryo culture process is effectively reduced, so that in vitro production efficiency of the pig embryo is improved by reducing damage and the like in the in vitro embryo culture process, and finally, an effective in vitro production system of the pig embryo is constructed.

Description

technical field [0001] The present invention mainly relates to the fields of embryo engineering technology and genetic engineering, in particular, to a method for knocking out p66 by using CRISPR / Cas9 gene editing method shc Methods for genetically porcine embryos. Background technique [0002] The development of early embryos has a great relationship with the level of reactive oxygen species (Reactive Oxygen Species, ROS) in the culture environment. Existing research results have shown that the presence of hydrogen peroxide can induce cyclic arrest, apoptosis, necrosis and other biological events in mouse fertilized eggs during in vitro culture. [13] . Mild oxidative damage initially results in depolarization of the inner mitochondrial membrane, changes in the mitochondrial matrix and intracellular mitochondrial distribution; the study found that with H 2 o 2 After 48 hours of treatment, the fertilized eggs shrank, and after 72 hours, their development was arrested and ...

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
IPC IPC(8): C12N15/89
CPCC07K14/4703C12N15/89
Inventor 胡军和晏娇李甲梅
Owner HUNAN UNIV OF HUMANITIES SCI & TECH
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
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap