scholarly journals Golgi engineering of CHO cells by targeted integration of glycosyltransferases leads to the expression of novel Asn-linked oligosaccharide structures at secretory glycoproteins

2013 ◽  
Vol 7 (S6) ◽  
Author(s):  
Tobias Reinl ◽  
Nicolas Grammel ◽  
Sebastian Kandzia ◽  
Eckart Grabenhorst ◽  
Harald S Conradt
Keyword(s):  
Cho Cells ◽  
Targeted Integration ◽  
Secretory Glycoproteins ◽  
Oligosaccharide Structures

scholarly journals Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

2021 ◽  
Vol 333 ◽  
pp. 07001
Author(s):  
Ryusei Iwao ◽  
Yoshinori Kawabe ◽  
Mai Murakami ◽  
Akira Ito ◽  
Masamichi Kamihira
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Transgene Expression ◽  
Chinese Hamster ◽  
Genomic Locus ◽  
Precise Integration ◽  
Target Chromosome ◽  
Targeted Integration ◽  
Producer Cell ◽  
Cell Genome

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Hydroxyurea selection for enhancement of homology-directed targeted integration of transgenes in CHO cells

2021 ◽  
Vol 62 ◽  
pp. 26-31
Author(s):  
Jin Myeong Kwak ◽  
Youngsik Lee ◽  
Sung Wook Shin ◽  
Jae Seong Lee
Keyword(s):  
Cho Cells ◽  
Targeted Integration ◽  
Selection For

scholarly journals Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2407
Author(s):  
Sung Wook Shin ◽  
Dongwoo Kim ◽  
Jae Seong Lee
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Cell Line Development ◽  
Cho Cell Line ◽  
Vector Systems ◽  
Targeted Integration

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

Targeted Integration into Pseudo attP Sites of CHO Cells Using CRISPR/Cas9

2021 ◽  
Author(s):  
Sana Pourtabatabaei ◽  
Samaneh Ghanbari ◽  
Narges Damavandi ◽  
Elham Bayat ◽  
Mozhgan Raigani ◽  
...  
Keyword(s):  
Cho Cells ◽  
Targeted Integration

Glycosylation of GD4 and Thy-1

1993 ◽  
Vol 342 (1299) ◽  
pp. 43-50 ◽  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Truncated Form ◽  
Site Specific ◽  
Glycosylation Sites ◽  
Oligosaccharide Processing ◽  
Domain 3 ◽  
Protein Cell ◽  
Oligosaccharide Structures

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with m utant molecules with only single glycosylation sites and with a truncated form containing only the two NH 2 -terminal domains, indicate that independent processing occurs at each glycosylation site and that dom ain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 255-261 ◽  
Author(s):  
A Hotchkiss ◽  
C J Refino ◽  
C K Leonard ◽  
J V O'Connor ◽  
C Crowley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Sodium Periodate ◽  
Glycosylation Site ◽  
Tissue Type ◽  
Carbohydrate Structure ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
High Mannose ◽  
Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

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