Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction method of dwarf syndrome animal model

A technology for constructing methods and animal models, which can be applied in the field of genetic engineering and can solve problems such as greatly limited applications

Pending Publication Date: 2021-12-10
FOSHAN UNIVERSITY
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the function of ABE depends on the PAM sequence of NGG mode, and its editing window is at the 4-8 position of the sgRNA, the probability of having a suitable PAM sequence for the start codon ATG is 62.5% (10 / 16), that is There is a probability of nearly 40% that ABE-mediated gene start codon disruption cannot be used to achieve gene knockout, so the application of ABE in genetic modification of pigs is greatly limited

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
  • Construction method of dwarf syndrome animal model
  • Construction method of dwarf syndrome animal model
  • Construction method of dwarf syndrome animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the construction of pig GHR gene knockout vector

[0037] (1-1) Exon 6 of the porcine GHR gene (NCBI GenBank Gene ID: 397488) was selected as the target of ABE-mediated exon skipping. The length of the exon is 173bp, and if skipping is successfully realized, it will lead to a frameshift mutation, which will result in the knockout of the pig GHR gene.

[0038] (1-2) Use pig GHR gene-specific upstream primer 5'-TCTGACTGTCAAAGCACCTTG-3' (SEQ ID NO.1) and pig GHR gene-specific downstream primer

[0039] 5'-GTTCACCAGCTGATCTCATG-3' (SEQ ID NO.2) was amplified by PCR on the pig genome template, and the wild-type amplified fragment was 410bp, and its sequence composition was determined by gene sequencing.

[0040] (1-3) Sequence analysis found that both the porcine GHR gene exon 6 splice acceptor and the porcine GHR gene exon 6 splice donor have an sgRNA target, which can be used for ABE-mediated conservative sequence mutations to achieve penetrance The sub-jump...

Embodiment 2

[0057] Example 2, Preparation of Gene Knockout Cells

[0058] (2-1) ABE plasmid PX-ABEmaxAW is linearized with BbsI restriction endonuclease (the nucleotide sequence after linearization is shown in SEQ ID NO.9), the gene fragment is split by agarose gel electrophoresis, and the gel is cut After recovery, mixed with the primer-dimer formed by high-temperature annealing of sgRNA1-F and sgRNA1-R, it was reacted overnight at 4°C with DNA ligase to obtain a ligation product.

[0059] (2-2) Culture low-passage Bamaxiang pig fibroblasts, and use Lipofectamine 3000 reagent for cell transfection.

[0060] (2-3) After 24 hours of transfection, the cells were subcultured at a density of 10,000 / 35 mm culture dish.

[0061] (2-4) After subculture of cells for 2 days, add 1 μg / mL puromycin and culture for 7-10 days until single-cell colonies grow, pick out single-cell colonies and culture them in 4-well dishes, and inoculate each single-cell colony in 1 well , spread to 35mm petri dish to...

Embodiment 3

[0068] Example 3, Construction of Gene Knockout Pig Model

[0069](3-1) To produce the GHR gene knockout pig model by somatic cell nuclear transfer technology, collect fresh pig ovaries from the slaughterhouse, wash them with 39°C normal saline several times, and draw out 3-6mm ovaries with a 10ml syringe with a 1.2mm diameter needle. The follicular fluid in the follicles with a diameter of 39°C was mixed with PBS preheated at 39°C, and then the cumulus cell-egg cell complex with compact multi-layered cumulus cells was selected under a stereo microscope, and then washed with oocyte maturation liquid at 38.5 Cultivate in an incubator at ℃, saturated humidity, and 5% CO2 for 38 to 40 hours, remove cumulus cells with 0.1% hyaluronidase, and select under a stereoscope with normal morphology, complete plasma membrane, uniform cytoplasm and discharge of the first polar body The MII stage oocytes used as nuclear transfer recipients were put back into the maturation medium and placed ...

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 discloses a construction method of a dwarf syndrome animal model. According to the invention, an All-in-one improved ABE plasmid is utilized, and the All-in-one improved ABE plasmid comprises ABE for carrying out single-base editing and an sgRNA expression element, and is used for carrying out single adenine base editing on a pig GHR gene, so that a single-cell colony capable of jumping aiming at exon 6 is prepared. According to Western Blotting identification, the single cell colony shows that GHR protein deletion occurs, and no sgRNA-dependent genome off-target is detected through gene sequencing, so that ABE-mediated exon jumping is successfully and accurately triggered in the porcine cells, and gene knockout is realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing an animal model of dwarf syndrome. Background technique [0002] Growth hormone receptor gene (Growth hormone receptor, GHR) is a cell membrane receptor for cells to receive growth hormone. Growth hormone binds to GHR to trigger intracellular signals and exert biological effects that stimulate cell growth and division. Human GHR loss-of-function mutations will cause Laron syndrome (Laron syndrome), and pig GHR loss-of-function mutations will cause phenotypes such as short stature and developmental delay similar to Laron syndrome, so GHR gene knockout pigs are simulated human Laron syndrome Ideal large animal model. [0003] The traditional genetic improvement of pigs is limited by factors such as long breeding cycle and limited genetic resources, highlighting the great potential of genetic modification technology in pig genetic improvement. ...

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): C12N15/85C12N15/877A01K67/027C12N15/12
CPCC12N15/8509C12N15/8778A01K67/0276C07K14/72C12N2800/107A01K2227/108A01K2267/03
Inventor 朱向星唐冬生严爱芬冯娟刘连张晓莉盘家圣林子盛文健聪
Owner FOSHAN UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap 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