O-Glc Pathway-based Glyco-engineered Cell Construction Service

Professional Glyco-engineered Cell Construction Based on O-Glc Pathway That Exceeding Your Expectations

CD BioGlyco has engineered O-glycosylation in the endoplasmic reticulum to form highly complex and specific structures with specialized functions. Most O-glycosylations are conserved such as O-Glc, which is abundant and conserved. We control the expression of the O-Glc pathway by regulating its early extension steps. We provide gene-targeting design services and cell line construction services for complex glycosylation pathways. At the same time, we control each step of the service to validate and analyze it. Moreover, we provide high-quality Cell-based Glycan Display Array such as Cell-based O-Glycan Array.

O-Glc Pathway-based Glyco-engineered Cell Construction Service at CD BioGlyco

CD BioGlyco provides regulatory services for the biosynthetic process of the O-Glc pathway. Our researchers provide design and testing services for gRNAs of O-Glc pathway-related genes. Detect gRNAs of glycogenes by high-throughput services of fluorescence-activated cell sorting (FACS) and indel detection by amplicon analysis (IDAA). We target glycogenes to control their expression by gene editing services such as knockout or knockin. We not only provide glyco-engineered cell line construction services but also provide analysis of the effects of gene editing on glycan structure and expression.

We provide construction services for double-knockout cells. Meanwhile, gene editing is assessed by genomic PCR and DNA sequencing with Q5 polymerase.
After FACS enrichment and single-cell cloning, we monitor efficiency by IDAA profiling and immunohistology.
To validate the efficacy of the glycosylation pathway, we use the human embryonic kidney 293 (HEK293) cell line as a model. We use mass spectrometry to analyze the glycan structures generated by the O-Glc pathway.

Fig.1 Flow chart of O-Glc pathway-based glyco-engineered cell construction service. (CD BioGlyco)

Publication

Journal: Academic Accelerator Source Normalized Impact per Paper PNAS

Technology: Mass spectrometric analysis, Amino acid sequence, Gene knockout

Published: 2018

IF: 12.779

Results: In this study, two novel O-glycosyltransferases (POGLUT2 and POGLUT3), were identified in the O-Glc pathway. Gene-specific insertion of gene-specific guide RNA (gRNAs) into vectors was used for transfection in HEK293T cells. Successful gene editing was verified using PCR and DNA sequencing. Mass spectrometry analysis of NOTCH1 produced in HEK293T cells lacking either the two O-glycosyltransferases or both genes showed that either POGLUT2 or POGLUT3 were modified by O-Glc. This modification directionally controls NOTCH1 cell surface expression and affects the fine-tuning mechanism of the Notch signaling pathway.

Fig.2 Factor design results. (Takeuchi, et al., 2018)

Applications of O-Glc Pathway-based Glyco-engineered Cell

Knockout of GTf genes in the constructed glyco-engineered cell lines greatly improves the study and development of basic functions of specific types of glycosylation.
O-Glc pathway-based glyco-engineered cell dissects and identifies specific structure-function relationships in detail.
O-Glc pathway-based glyco-engineered cell predicts the estimation of glycan structures produced by a cell or tissue, which is very useful for understanding cell properties and regulation as well as for engineering cells.

Advantages of Us

To promote and accelerate the development of projects, we provide an integrated service of CRISPR/Cas9 gRNA delivery and screening.
Our researchers provide efficient analysis services for the effects of single and combinatorial targeting of GTf genes on cell surface glycans.
High-throughput screening and validated gRNA improve multiallelic cleavage efficiency.

CD BioGlyco has a comprehensive Glycan Display Platform to meet the needs of clients in glycan display. Our serious service attitude and thoughtful service have been praised by clients around the world. Please feel free to contact us if you are interested in the O-Glc pathway-based glyco-engineered cell construction.

References

Narimatsu, Y; et al. A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome. Glycobiology. 2018, 28(5): 295-305.
Takeuchi, H.; et al. Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling. Proc Natl Acad Sci USA. 2018, 115(36): E8395-E8402.

This service is for Research Use Only, not intended for any clinical use.