Engineering CHO Cell Lines to Stably Express B4GALT1,ST6GAL1,and GnTⅢ with Site-directed Integration
10.13865/j.cnki.cjbmb.2025.03.1516
- VernacularTitle:定点整合β1,4半乳糖基转移酶、α-2,6-唾液酸转移酶1和N-乙酰氨基糖基转移酶Ⅲ的CHO工程细胞株构建
- Author:
Xian-Hong LI
1
;
Run-Qing JIA
;
You-Liang WANG
;
Wei-Ling MAN
;
Tian-Hao ZHU
;
Xin-Long YAN
;
Yan-Li LIN
Author Information
1. 北京工业大学化学与生命学院生物系,北京 100124;军事科学院军事医学研究院前沿生物技术实验室,北京 100071
- Publication Type:Journal Article
- Keywords:
β-1,4 galactosyltransferase(B4GALT1);
α-2,6-sialyltransferase 1(ST6GAL1);
N-acet-aminoglycosyltransferase Ⅲ(GnT Ⅲ);
clustered regularly interspaced short palindromic repeats technolo-gy(CRISPR/Cas9);
recombinase-mediated cassette exchange technology;
Chin
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(4):576-585
- CountryChina
- Language:Chinese
-
Abstract:
Glycoengineering was carried out in the mammalian cell line CHO for the production of pro-tein-based drugs.Firstly,the genome sequence of the Rosa26 locus of CHO cells was determined,the gRNA sequences were designed,and the landing pad was integrated into the Rosa26 locus of CHO cells by CRISPR/Cas9 technology.Three targeting vectors co-expressed by glycosyltransferases,which are β-1,4 galactosyltransferase(B4GALT1),α-2,6-sialyltransferase 1(ST6GAL1)and N-acetaminoglycosyl-transferase Ⅲ(GnT Ⅲ),were constructed by overlapping PCR and seamless ligation technology,and the three glycosyltransferase genes were integrated into the CHO Rosa26 locus by Cre enzyme-mediated cassette exchange technology.PCR confirmed that three glycosyltransferases had been successfully site-directed integrated into the Rosa26 site.The mRNA expression levels of the three glycosyltransferases were more than 50 000-fold by qRT-PCR,and the protein expression levels of the three glycosyltrans-ferases were more than 4-fold via western blotting(P＜0.001).A CHO-engineered cell line with three glycosyltransferases integrated into Rosa26 site was successfully constructed.
