Jurkat-KI-R5 cell line and construction method and application thereof

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

Active Publication Date: 2017-08-01
SHENZHEN CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Even with previously established CCR5 transgenic T cell lines, the expression vector wa

Method used

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  • Jurkat-KI-R5 cell line and construction method and application thereof
  • Jurkat-KI-R5 cell line and construction method and application thereof
  • Jurkat-KI-R5 cell line and construction method and application thereof

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Experimental program
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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

[0047] Embodiment 2: Jurkat-KI-R5 cells are analyzed and detected

[0048] 2.1 Flow cytometry analysis

[0049] 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.

[0050] join figure 2 (e)CCR5 in CD4 + Highly expressed on the surface of Jurkat-KI-R5 cells.

[0051] 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...

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Abstract

The invention relates to Jurkat-KI-R5 and a construction method and application thereof. A powerful enhancer/starter combination (CAG) is knocked into a starter area of a CCR5 gene of a CD4+Jurkat T cell by a CRISPR/Cas9 gene editing technique in a homologous recombination way; the name of the gene-modified cell system is Jurkat-KI-R5 cell. The Jurkat-KI-R5 cell has the advantages that a CAG starter of the Jurkat-KI-R5 is precisely recombined into the specified location of a CCR5 starter, and the high expression of the CCT5 is stably caused; oppisite to a maternal line Jurkat cell, the Jurkat-KI-R5 cell is easily infected by HIV-1 (human immunodeficiency virus-1); a cell platform urgently demanded by the HIV disease study is provided by the Jurkat-KI-R5 cell.

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

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Application Information

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