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Construction of a sensitive pyrogen‐testing cell model by site‐specific knock‐in of multiple genes
Author(s) -
Hu Ruobi,
Li Hui,
Lei Zhen,
Han Qing,
Yu Xiuyan,
Zhou Na,
Zhang Xuehui,
Mao Yiqing,
Wang Xi,
Irwin David M.,
Niu Gang,
Tan Huanran
Publication year - 2019
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27084
Subject(s) - tlr4 , cd14 , chemistry , microbiology and biotechnology , biology , signal transduction , flow cytometry
A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS‐mediated pyrogen reaction. We established a new TLR4/MD2/CD14‐specific overexpressing knock‐in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock‐in cell line model with LPS leads to the release of the cytokines IL‐6 and TNF‐alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.