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

Construction method of neutrophil deletion mouse model

A neutrophil and mouse model technology, applied in the field of genetic engineering and genetic modification, can solve the problems of slow neutrophil function research, inability to clear neutrophils for a long time, and short existence time

Active Publication Date: 2022-01-11
XINXIANG MEDICAL UNIV
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Neutrophils play a very important role in the maintenance and functioning of the immune system. After the peripheral blood enters the tissue, it can be replenished in a short period of time through the differentiation and development of bone marrow hematopoietic stem cells. The purpose of eliminating neutrophils for a long time cannot be achieved by injecting drugs or specific antibodies. Therefore, although neutrophils have Very important function, but due to the lack of genetic models of neutrophil-deficient mice, research on the function of neutrophils in various diseases has been slow

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 neutrophil deletion mouse model
  • Construction method of neutrophil deletion mouse model
  • Construction method of neutrophil deletion mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1R26

[0044] The construction of embodiment 1R26-sgGfi1 gene knock-in mouse model

[0045] This example provides gene editing of the mouse Rosa26 site to obtain the sgRNA tandem expression unit (U6-Guide1-sgRNA scaffold-pT-U6-Guide2-sgRNA scaffold-pT) site-specific insertion (knock -in, KI) the method for mouse model, specifically comprises the following steps:

[0046] (1) Determine the targeting sequence: In order to insert the sgRNA tandem expression unit (U6-Guide1-sgRNA scaffold-pT-U6-Guide2-sgRNA scaffold-pT) specifically targeting the mouse gfi1 gene into the mouse Rosa26 site, so targeting Mouse Rosa26 locus design targeting sequence. Referring to previous published literature, paste the genomic DNA sequence of about 1000 bases in length in intron1-2 of the mouse Rosa26 site into the online design website CRISPOR (http: / / crispor.tefor.net / ), and according to the specificity in the output results, According to the high and low performance scores, two sgRNAs were finally sel...

experiment example 1

[0071] Experimental example 1 Cas9: sgGfi1 mouse gfi1 targeting site cleavage detection results in different tissues and organs

[0072] The Cas9:sgGfi1 mice obtained in Example 1 were collected from different tissues and organs, and genomic DNA (rat tail, peripheral blood, bone marrow, liver, brain) was extracted (the method was the same as in Example 1-(5)-A), and This genomic DNA was used as a template, and specific primers (SEQ ID NO: 6 and 7) were used for PCR amplification. The PCR system was as follows: 0.2 μL of each primer, 5 μL of 2×Taq Master Mix (Vazyme P112-01), 1 μL of genomic DNA, Supplement H 2 O to a total volume of 10 μL; the PCR reaction program is as follows: 95°C for 5 min; 94°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, 35 cycles; 72°C for 10 min; PCR products were detected by capillary electrophoresis.

[0073] The specific primer sequences used for PCR amplification are: F-TGAAGGAGCGGCACATTTCT (SEQ ID NO: 6); R-GCACAGCTGTTGACATAGAGGA (SEQ ID NO: 7)....

experiment example 2Ca

[0075] Experimental Example 2 Detection of neutrophils in peripheral blood and bone marrow of Cas9:sgGfi1 mice

[0076] (1) Detection of neutrophils in peripheral blood

[0077] The peripheral blood of wild-type B6 mice, R26-sgGfi1 gene knock-in mice expressing only sgRNA, and R26-Cas9 gene knock-in mice expressing only Cas9 were obtained as control groups, and Cas9:sgGfi1 expressing both sgRNA and Cas9 were collected. The peripheral blood of mice was used as the experimental group, and the cells were labeled with specific antibodies, and then the percentage of neutrophils in the peripheral blood was detected by flow cytometry, the steps were as follows:

[0078] Take 10 μL blood sample and add 10 μL antibody mixture (antibodies include: anti-CD45 APC-eFluor780, anti-CD5 PE-Cyanine7, anti-CD11b Super Bright 600, anti-CD3e FITC, anti-CD19 PE and anti-Ly6G Alexa Fluor 700), Mix well, place on ice and incubate in the dark for 30 min; then add 250 μL 1× erythrocyte lysing solutio...

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 belongs to the technical field of genetic engineering and genetic modification, and particularly relates to a construction method of a neutrophil deletion mouse model. An sgRNA tandem expression unit of a specific targeting mouse Gfi1 gene is knocked into a mouse Rosa26 site through a CRISPR / Cas9 genome editing technology, and then the gene expressing the sgRNA is knocked into a mouse homozygote and the gene overexpressing Cas9 protein is knocked into the mouse homozygote for hybridization, so that the neutrophil deletion mouse model can be obtained. The immunophenotype detection is performed on the peripheral blood of the mouse by using the flow cytometry, and the result shows that the neutrophil removal efficiency of the mouse is up to 100%, such that the brand new genetic model is provided for the research of the effect of the neutrophil in diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and genetic modification, and in particular relates to a method for constructing a neutrophil-deficient mouse model. Background technique [0002] Neutrophils are an important part of the body's innate immune cells, and their cells contain a large number of various types of lysosomes. When foreign pathogenic microorganisms invade the body, they constitute the body's first line of defense against foreign pathogenic microorganisms. It plays an important role in protecting the health of the body. [0003] Neutrophils play a very important role in the maintenance and function of the immune system. Since neutrophils exist in the peripheral circulation system for a short time, once they enter the tissue from the peripheral blood, they can produce blood through the bone marrow in a short time Stem cell differentiation and development are supplemented in time, and the purpose of eliminating n...

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): A01K67/027C12N15/85C12N15/90C12N15/12
CPCA01K67/0275C12N15/8509C12N15/907C07K14/47A01K2217/072A01K2227/105A01K2267/03
Inventor 梁银明张黎琛卢燎勋黄蓉谷妍蓉晁天柱李天函周斌辉
Owner XINXIANG MEDICAL UNIV
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