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- de la Rosa, Andrés, 1988-
(author)
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Updated transient gene expression (TGE) protocol and the development of a small-scale TGE protocol : evaluated by testing longevity molecules as potential protein expression enhancers
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In: Biological Procedures Online. - 1480-9222.
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Journal article (other academic/artistic)abstract
- Transient gene expression (TGE) is commonly used to quickly produce protein-based drugs, such as antibodies, that require post-translational modifications. We have previously published a protocol for efficient and inexpensive TGE of multispecific and multivalent antibodies, which we have improved upon and described in the first part of this paper; by replacing the expensive Expi293 expression medium with BalanCD HEK293 medium, the medium cost was decreased by approximately 90%, and in addition, the harvesting procedure was shortened from 2.5 hours to 15 minutes by mixing the harvested cell medium with the mineral compound diatomaceous earth that effectively absorbs and sequester cells and cell debris. The cell medium can then be quickly filtered without the need to exchange obstructed filters and without the previously required 1-hour centrifugation step. In the second part of this paper, a small-scale TGE protocol was developed to surmount the cost limitation of testing many culture conditions. The small-scale TGE protocol uses 6-well plates, which is the cheapest alternative for scaling down the protein expression, and consumes 83% less material compared to transfections done with the smallest available shaking flasks for cell culture. To test the small-scale TGE protocol, we evaluated substances belonging to a category called longevity molecules as potential protein expression enhancers. Though the longevity molecules failed to increase protein expression in the conditions tested, our results corroborate the functionality of the small-scale TGE protocol and provide a straightforward methodology to expediently evaluate factors that can lead to improved protein production protocols in the future.
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- Fang, Xiaotian T., et al.
(author)
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Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells
- 2017
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In: Biological Procedures Online. - : Springer Science and Business Media LLC. - 1480-9222. ; 19
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Journal article (peer-reviewed)abstract
- Background: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.Methods: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost).Results: The protocol has been used to generate multiple variants of tetra-and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (A beta) aggregates in vivo.Conclusions: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.
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- Hermansen, Russell A., et al.
(author)
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Extracting functional trends from whole genome duplication events using comparative genomics
- 2016
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In: Biological Procedures Online. - : Springer Science and Business Media LLC. - 1480-9222. ; 18
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Research review (peer-reviewed)abstract
- Background: The number of species with completed genomes, including those with evidence for recent whole genome duplication events has exploded. The recently sequenced Atlantic salmon genome has been through two rounds of whole genome duplication since the divergence of teleost fish from the lineage that led to amniotes. This quadrupoling of the number of potential genes has led to complex patterns of retention and loss among gene families. Results: Methods have been developed to characterize the interplay of duplicate gene retention processes across both whole genome duplication events and additional smaller scale duplication events. Further, gene expression divergence data has become available as well for Atlantic salmon and the closely related, pre-whole genome duplication pike and methods to describe expression divergence are also presented. These methods for the characterization of duplicate gene retention and gene expression divergence that have been applied to salmon are described. Conclusions: With the growth in available genomic and functional data, the opportunities to extract functional inference from large scale duplicates using comparative methods have expanded dramatically. Recently developed methods that further this inference for duplicated genes have been described.
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- Weishaupt, Holger, et al.
(author)
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A Method to Study the Epigenetic Chromatin States of Rare Hematopoietic Stem and Progenitor Cells; MiniChIP-Chip
- 2010
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In: Biological Procedures Online. - : Springer Science and Business Media LLC. - 1480-9222. ; 12:1, s. 1-17
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Journal article (peer-reviewed)abstract
- Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP-chip), we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions.
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