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How do we select the appropriate genes to edit to enhance CHO cell properties? Background literature suggest some that are obvious but in the environment of the cell the multiplicity of interactions makes successful selection unpredictable. Using model CHO cell systems, this presentation will focus on approaches and technologies that screen for genes with greater likelihood of presenting the “drivers” that control CHO cell suitability for production of recombinant therapeutics.
The genomics era of biology has provided the scientific and medical community with an exquisitely-detailed view of the genetic complexity underlying human disease. While technical advances have made genome sequencing cheaper, more robust, and faster than ever, similarly effective technologies that enable basepair-by-basepair manipulation of DNA in human cells to model disease or therapeutic intervention have only recently been described. Specifically, adaptation of CRISPR/Cas9, a bacterial genome defense system, for eukaryotic molecular genetics has ushered in a new phase of the genomics revolution. Here, I will present the framework of a CRISPR/Cas9-based platform for drug target discovery and validation and how this platform can be exploited for single gene to genome-scale experimentation.
1. Modifications of CRISPR/Cas9 enable a wide range of genetic manipulations in human cells.
2. CRISPR/Cas9-mediated cell line engineering can be applied to study one or all human genes within a single experiment.
1. Genome sequencing efforts have provided a parts-list for CHO cells
2. CRISPR has enabled the rapid engineering of cells
3. We have developed cell lines with multiple genetic changes to improve CHO cell growth and product quality
MicroRNAs with their ability to target hundreds of genes have been shown to play an essential role in the behaviour of cells. Therefore, the depletion or overexpression is a vital tool for the improvement of bioprocess attributes of CHO producer cell lines concerning productivity, growth or longevity. With the development of newer and better genome engineering tools we considered the depletion or complete deletion in several cell lines.
Currently, scientists in cell line development and cell engineering struggle with limiting dilutions and FACS for isolating single cells. This is then followed by a separate whole well imaging step to provide assurance of clonality.
The new VIPS system streamlines the workflow to combine single cell printing and whole well imaging into one easy to use system.
Data will be presented with commercial lines to illustrate high seeding and cloning efficiencies.
Additional workflow benefits will also be presented.
Many cellular processes are accomplished through the assembly of multiple proteins acting in concert to catalyze specific activities. Moreover, multiprotein complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Although technologies tremendously improved, production of recombinant multiprotein complexes often remains challenging and requires considerable investments, particularly for large complexes that might be incompletely characterized.
We will illustrate how genome editing technologies (such as the CrispR/Cas9 approach) allow to isolate complexes produced from their natural genomic environment for detailed analysis of their composition and how the baculovirus expression vector system (BEVS) has turned out to be particularly powerful for reconstitution of multisubunit complexes. We will comment on current developments and their potential to accelerate protein complex research: use of Lambda red recombination for improvement of the baculoviral genome, vector development for parallel expression/co-expression screening and Tandem Recombineering by SLIC cloning and Cre-LoxP fusion to generate multigene expression constructs. As model systems, we will use human multi-protein complexes involved in the regulation of gene expression such the pTefb cdk/cyclin pair, nuclear hormone receptor complexes or the 10 subunits transcription/DNA repair complex TFIIH.
CRISP/Cas9, Recombinant protein production, Baculovirus, multi-gene expression, multiprotein complex, synthetic biology
We have developed powerful cell engineering tools from novel hyperactive transposases and their cognate transposons. The system facilitates rapid development of high productivity, stable CHO and other mammalian cell lines in engineered genetic backgrounds. The valuable features of the system, and characterization of its performance will be illustrated with specific cell line development and cell engineering case studies.
Synthetic biology based on the “Design-Build-Test-Learn” cycle offers a new paradigm for CHO cell engineering, where it is possible to engineer the host cell factory in a product specific manner via combinatorial “tuning” of discrete cellular synthetic processes and directed engineering of the synthetic capacity and process performance of the host cell itself. Using this approach permits “one-size-fits-all” genetic vectorology and mechanistically blind screening of transfected cells to be replaced with tailored design and construction of specifically fit-for-purpose cell factories. This engineering design system relies upon a toolbox of synthetic parts with user-defined functionality and platform technologies that work in synchrony to enable product manufacturability. I describe our systematic approach, based on a combination of ‘omic datastreams and modeling, to create new synthetic genetic parts and cell engineering strategies that permit us to control core cellular synthetic processes such as transcription, translation and polypeptide folding/assembly in a high-capacity host cell chassis.
Martina Baumann1, 2, Sabine Vcelar1, Michael Melcher1, 2, Vaibhav Jadhav1, Norbert Auer1, Anja Puklowski3, Till Wenger3, Nicole Borth1, 2
1Austrian Centre of Industrial Biotechnology, Vienna, Austria
2University of Natural Resources and Life Sciences, Vienna, Austria
3Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
Chromosomal rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells. Using Chromosome counting and chromosome painting we show that i) different host cell lines are distinguishable by marker chromosomes ii) there is high variability in chromosome counts as well as karyotype variants within each population, be it host cell line, selected pool or subclone; iii) subcloning does not contribute to a more homogenous karyotype. To conclude, genomic variance is high in all populations of CHO cell lines as it occurs with each division, making subcloning an unsuitable tool to enhance population homogeneity.