Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange

2017 ◽  
Vol 114 (11) ◽  
pp. 2616-2627 ◽  
Author(s):  
Martina Baumann ◽  
Elisabeth Gludovacz ◽  
Natalie Sealover ◽  
Scott Bahr ◽  
Henry George ◽  
...  
Keyword(s):  
Cho Cells ◽  
Recombinant Gene ◽  
Cassette Exchange ◽  
Integration Sites ◽  
Gene Integration

Improved recombinant gene expression in CHO cells using matrix attachment regions

2004 ◽  
Vol 107 (2) ◽  
pp. 95-105 ◽  
Author(s):  
Jong-Mook Kim ◽  
Jung-Seob Kim ◽  
Doo-Hong Park ◽  
Ho Sung Kang ◽  
Jaeseung Yoon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cho Cells ◽  
Matrix Attachment Regions ◽  
Recombinant Gene Expression ◽  
Recombinant Gene

scholarly journals A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells

Methods ◽  
2016 ◽  
Vol 95 ◽  
pp. 3-12 ◽  
Author(s):  
Bahar Baser ◽  
Johannes Spehr ◽  
Konrad Büssow ◽  
Joop van den Heuvel
Keyword(s):  
Cho Cells ◽  
Genomic Integration ◽  
Expression Of Genes ◽  
Integration Sites

Accumulative gene integration technique using recombinase mediate cassette exchange

2009 ◽  
Vol 108 ◽  
pp. S18
Author(s):  
Yujiro Kameyama ◽  
Yoshinori Kawabe ◽  
Akira Ito ◽  
Masamichi Kamihira
Keyword(s):  
Integration Technique ◽  
Cassette Exchange ◽  
Gene Integration

scholarly journals Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ngan T. B. Nguyen ◽  
Jianer Lin ◽  
Shi Jie Tay ◽  
Mariati ◽  
Jessna Yeo ◽  
...  
Keyword(s):  
Cho Cells ◽  
Structure And Function ◽  
Glycan Structure ◽  
Wild Type ◽  
Complex Type ◽  
Cassette Exchange ◽  
Glycosylation Pathway ◽  
And Function ◽  
First Time ◽  
Gene Expression Levels

AbstractTherapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

scholarly journals The Chicken β-Globin 5′HS4 Boundary Element Blocks Enhancer-Mediated Suppression of Silencing

1999 ◽  
Vol 19 (5) ◽  
pp. 3714-3726 ◽  
Author(s):  
Mark C. Walters ◽  
Steven Fiering ◽  
Eric E. Bouhassira ◽  
David Scalzo ◽  
Scott Goeke ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Integration Site ◽  
Position Effects ◽  
Chromatin Insulator ◽  
Cassette Exchange ◽  
Erythroleukemia Cells ◽  
Integration Sites ◽  
Dnase I Hypersensitive Site ◽  
Insulator Elements ◽  
Chs4 Insulator

ABSTRACT A constitutive DNase I-hypersensitive site 5′ of the chicken β-globin locus, termed 5′HS4 or cHS4, has been shown to insulate a promoter from the effect of an upstream enhancer and to reduce position effects on mini-white expression in Drosophilacells; on the basis of these findings, it has been designated a chromatin insulator. We have examined the effect of the cHS4 insulator in a system that assays both the level of gene expression and the rate of transcriptional silencing. Because transgenes flanked by insulator elements are shielded from position effects in Drosophilacells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector with the cHS4 insulator in a colony assay did not increase the number of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the effect of cHS4 on the silencing of an integrated β-geo reporter at three genomic sites in K562 erythroleukemia cells. In this assay, enhancers act to suppress silencing but do not increase expression levels. While cHS4 blocked enhancement at each integration site, the strength of the effect varied from site to site. Furthermore, at some sites, cHS4 inhibited the enhancer effect either when placed between the enhancer and the promoter or when placed upstream of the enhancer. These results suggest that the activity of cHS4 is not dominant in all contexts and is unlikely to prevent silencing at all genomic integration sites.

CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells

2019 ◽  
pp. 213-232 ◽  
Author(s):  
Daria Sergeeva ◽  
Jose Manuel Camacho-Zaragoza ◽  
Jae Seong Lee ◽  
Helene Faustrup Kildegaard
Keyword(s):  
Genome Editing ◽  
Cho Cells ◽  
A Genome ◽  
Gene Integration ◽  
Targeted Gene Integration

scholarly journals Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system

2011 ◽  
Vol 64 (3) ◽  
pp. 267-279 ◽  
Author(s):  
Yoshinori Kawabe ◽  
Hirokatsu Makitsubo ◽  
Yujiro Kameyama ◽  
Shuohao Huang ◽  
Akira Ito ◽  
...  
Keyword(s):  
Cho Cells ◽  
Specific Gene ◽  
Chromosomal Locus ◽  
Integration System ◽  
Site Specific ◽  
Gene Integration

scholarly journals Exploring new gene integration sites for gene knock-in by gene-trapping strategy

2015 ◽  
Vol 24 (3) ◽  
pp. 549-559 ◽  
Author(s):  
Isamu Nanchi ◽  
Yuki Yoshimura ◽  
Kazuomi Nakamura ◽  
Yusaku Masago ◽  
Tetsuya Ohbayashi ◽  
...  
Keyword(s):  
Gene Trapping ◽  
Integration Sites ◽  
New Gene ◽  
Gene Integration
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