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

Jurkat-ki-r5 cell line and its construction method and application

A construction method and cell line technology, applied in applications, animal cells, vertebrate cells, etc., can solve problems such as unstable expression of transgenes

Active Publication Date: 2021-05-14
SHENZHEN CHILDRENS HOSPITAL
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even with previously established CCR5 transgenic T cell lines, the expression vector was randomly integrated into the genome, resulting in unstable expression of the transgene

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
  • Jurkat-ki-r5 cell line and its construction method and application
  • Jurkat-ki-r5 cell line and its construction method and application
  • Jurkat-ki-r5 cell line and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Establishment of Jurkat-KI-R5 cells

[0034] 1.1 Cell culture

[0035] Jurkat cells ( Number:TIB-152 TM ) culture density maintained at 0.5-2x10 6 cells / mL, 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, 100 μg / mL streptomycin and 2 mmol / L L-glutamine were added to the basal medium RPMI1640 for culture.

[0036] 1.2 DNA vector construction

[0037] The DNA vector encoding humanized Cas9 protein was purchased from Addgene (Cat. No. 51142). The DNA vector pU6-CCR5-gRNA that produces gRNA targeting the CCR5 gene locus is constructed from the pU6-gRNA plasmid (Addgene Cat. No. 53062) by ligating the annealed DNA duplex of the relevant targeting sequence to two BbsI restriction sites or other IIS Type IIS restriction enzyme (Type IIS restriction enzyme) sites were constructed. The DNA site targeted was GTGCACAGGGTGGAACAAGA. There are two homology arms of the CCR5 gene on both sides of the repair template plasmid, wherein the upstream hom...

Embodiment 2

[0071] Embodiment 2: Jurkat-KI-R5 cells are analyzed and detected

[0072] 2.1 Flow cytometry analysis

[0073] BD LSR-II flow cytometry was used to analyze the expression of human CCR5 and CD4 proteins on the surface of Jurkat-KI-R5 cells. Data were further analyzed by FACS Diva (Becton Dickinson Company) and FlowJo software. 1x10 6 Cells were washed with PBS and stained with DAPI, APC anti-hCCR5 (BD antibody) and PE anti-CD4 (BD antibody) for 30 minutes at room temperature. After staining, cells were washed twice with PBS, resuspended in FACS buffer, and analyzed on a machine.

[0074] join figure 2 (e)CCR5 in CD4 + Highly expressed on the surface of Jurkat-KI-R5 cells.

[0075] Depend on figure 2 (e) It can be seen that the Jurkat-KI-R5CCR5 peak of the APC channel in the left picture shifts to the right, while the CD4 of the PE channel in the right picture has no obvious shift. The results showed that the cell surface CCR5 expression of Jurkat-KI-R5 was significan...

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 present invention relates to a Jurkat-KI-R5 and its construction method and application. The present invention knocks a powerful enhancer / promoter combination (CAG) into CD4+Jurkat T cells through CRISPR / Cas9 gene editing technology homologous recombination The promoter region of the CCR5 gene, and the genetically modified cell line was named Jurkat‑KI‑R5 cells. The precise recombination of the CAG promoter of Jurkat-KI-R5 cells according to the present invention to the specified position of the CCR5 promoter leads to stable and high expression of CCR5. In contrast to maternal Jurkat cells, Jurkat‑KI‑R5 cells are highly susceptible to HIV‑1 infection. Therefore, Jurkat‑KI‑R5 cells provide a much-needed cell platform for AIDS research.

Description

technical field [0001] The invention relates to biotechnology, in particular to Jurkat-KI-R5 cell line and its construction method and application. Background technique [0002] Despite tremendous efforts in the development of antiretroviral therapy to combat HIV over the past few decades, AIDS remains one of the most important infectious diseases globally. According to the UNAIDS report, the global AIDS epidemic in 2013, the estimated number of people living with HIV rose from 8.1 million in 1990 to 35.3 million in 2012. In addition, drug-resistant HIV strains have recently been reported. Therefore, it is very important to develop new treatment methods for clearing HIV in patients. Immortalized HIV host cell lines are important for AIDS research and the development of treatments. [0003] HIV primarily targets macrophages and CD4+ T cells in vivo. Additionally, the CCR5 and CXCR4 coreceptors are required for HIV to infect T cells. Therefore, establishing CD4+ T cells t...

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 Patents(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10
CPCC07K14/7158C12N5/0646C12N15/85C12N2510/00C12N2800/107C12N2800/30
Inventor 付雪梅
Owner SHENZHEN CHILDRENS HOSPITAL
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