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Find video protocols related to scientific articles indexed in Pubmed.
Toward product attribute control: developments from genome sequencing.
Curr. Opin. Biotechnol.
PUBLISHED: 04-24-2014
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Chinese hamster ovary (CHO) cells are important hosts for the production of therapeutic proteins. Recent genome sequencing studies provide an initial baseline of information useful for understanding cell line performance in terms of product quality attributes. However, the lack of a well-established reference genome together with concerns about genome stability have not yet permitted the community to define the detailed relationship between the genome and cell line performance. Emerging efforts to define a new reference genome, together with new data on genome stability, herald an era where cell line's with defined genomes can be combined with defined process parameters to yield product quality attribute control.
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miRNA expression in CHO: nature knows best.
Biotechnol J
PUBLISHED: 02-12-2014
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Improvement of Chinese hamster ovary (CHO) cells for therapeutic protein production has great potential for the manufacturing of biopharmaceuticals. This commentary by Baik and Lee discusses the study by Klanert et al., which describes an improved tool that will allow greater control over the design of CHO cells.
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Bioengineering murine mastocytoma cells to produce anticoagulant heparin.
Glycobiology
PUBLISHED: 12-09-2013
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Heparin, an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of heparin are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into the details of this pathway. As reported previously, MST cells produce a highly sulfated heparin-like polysaccharide that lacks anticoagulant activity (Montgomery et al. 1992. Proc Natl Acad Sci USA 89:11327). Here we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase, and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated heparin. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of HS/HP and CS/DS glycosaminoglycans. The cell associated HS/HP closely resembles heparin with 3-O-sulfo group containing glucosamine residues and shows anticoagulant activity. This study contributes towards a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.
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Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site.
J. Biol. Chem.
PUBLISHED: 11-18-2013
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HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.
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Ultra-performance ion-pairing liquid chromatography with on-line electrospray ion trap mass spectrometry for heparin disaccharide analysis.
Anal. Biochem.
PUBLISHED: 01-26-2011
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A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities.
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Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.
Metab. Eng.
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Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.
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Toward a bioengineered heparin: challenges and strategies for metabolic engineering of mammalian cells.
Bioengineered
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Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Since Chinese hamster ovary (CHO) cells are capable of producing heparan sulfate (HS), a related polysaccharide naturally, and heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. We developed stable human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) expressing cell lines based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells. Both activity assay and disaccharide analysis showed that engineered HS attained heparin-like characteristics but not identical to pharmaceutical heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.