A sensitive mutation screening method supporting cell line development for biotherapeutics

2016 ◽  
Vol 505 ◽  
pp. 73-75 ◽  
Author(s):  
Ildana Valisheva ◽  
Reed J. Harris ◽  
Judith Zhu-Shimoni
Keyword(s):  
Cell Line ◽  
Mutation Screening ◽  
Screening Method ◽  
Supporting Cell ◽  
Cell Line Development

scholarly journals Optimising the mutation screening strategy in Marfan syndrome and identifying genotypes with more severe aortic involvement

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Roland Stengl ◽  
András Bors ◽  
Bence Ágg ◽  
Miklós Pólos ◽  
Gabor Matyas ◽  
...  
Keyword(s):  
Marfan Syndrome ◽  
Single Gene ◽  
Mutation Screening ◽  
Screening Method ◽  
Connective Tissue Disorder ◽  
Screening Strategy ◽  
Gene Panel ◽  
Dominant Negative ◽  
Second Phase ◽  
High Detection Rate

Abstract Background Marfan syndrome (MFS) is a systemic connective tissue disorder with life-threatening manifestations affecting the ascending aorta. MFS is caused by dominant negative (DN) and haploinsufficient (HI) mutations of the FBN1 gene. Our aim was to identify mutations of MFS patients with high detection rate and to investigate the use of a gene panel for patients with Marfanoid habitus. We also aimed to examine correlations between genotype and cardiovascular manifestations to predict “malignant” mutations. Methods 136 individuals were enrolled. In the first phase, next-generation sequencing (NGS) and Sanger sequencing were performed for 57 patients to screen the FBN1 gene, followed by multiplex ligation-dependent probe amplification (MLPA) in negative cases. For repeated negative results, NGS gene panel involving 9 genes was used. In the second phase, 79 patients were tested primarily with the same gene panel, negative samples were tested by MLPA. Results 84 pathogenic mutations were detected, out of which 78 affected FBN1, 6 non-FBN1 mutations (2 TGFB2, 1 TGFBR2, 2 TGFBR1, 1 SMAD3) are associated with Loeys-Dietz syndrome (LDS). LDS patients had lower systemic score and they were younger, but their aortic involvement did not differ. MLPA detected 4 multi-exon deletions of FBN1 gene, which could not be identified by our first-step screening method. Aortic involvement (aortic dissection and/or dilation) did not differ significantly among HI and DN mutations (p = 0.061). Combined group of HI and DN mutations eliminating a disulphide-bonding cysteine (DN Cys) had significantly higher aortic involvement rate than DN mutations not eliminating a disulphide-bonding cysteine (DN non-Cys) (p < 0.001). Patients with DN Cys required significantly more aortic surgeries than HI and DN non-Cys mutations (p = 0.042 and p = 0.015, respectively). Conclusions Due to the relevant number of mutations affecting genes other than FBN1, preferred approach for testing individuals with Marfanoid habitus is using a gene panel rather than single-gene analysis, followed by MLPA for negative samples. DN Cys and HI mutations should be considered as risk factors for aortic involvement. Genetic testing for patients with Marfanoid features and a systemic score under 7 is recommended, as LDS patients may have lower scores, but they may have severe cardiovascular manifestations.

scholarly journals Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

2020 ◽  
Author(s):  
Tobias Groß ◽  
Csaba Jeney ◽  
Darius Halm ◽  
Günter Finkenzeller ◽  
G. Björn Stark ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Large Scale ◽  
Single Cells ◽  
Protein Detection ◽  
Cell Line Development ◽  
Cell Printing ◽  
Cell Clones ◽  
The University

AbstractThe homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.Author summaryI completed my undergraduate degree in biochemistry at the University of Ulm and finished my master's degree in pharmaceutical biotechnology at the University of Ulm and University of applied science of Biberach with a focus on biotechnology, toxicology and molecular biology. For my master thesis, I went to the University of Freiburg to the department of microsystems engineering, where I developed a novel workflow for cell line development. I stayed at the institute for my doctorate, but changed my scientific focus to the development of the emulsion coupling technology, which is a powerful tool for the quantitative and highly parallel measurement of protein and protein interactions. I am generally interested in being involved in the development of innovative molecular biological methods that can be used to gain new insights about biological issues. I am particularly curious to unravel the complex and often poorly understood protein interaction pathways that are the cornerstone of understanding cellular functionality and are a fundamental necessity to describe life mechanistically.

scholarly journals High performance CHO cell line development platform for enhanced production of recombinant proteins including difficult-to-express proteins

2013 ◽  
Vol 7 (S6) ◽  
Author(s):  
Pierre-Alain Girod ◽  
Valérie Le Fourn ◽  
David Calabrese ◽  
Alexandre Regamey ◽  
Deborah Ley ◽  
...  
Keyword(s):  
Cell Line ◽  
Recombinant Proteins ◽  
High Performance ◽  
Cho Cell ◽  
Cell Line Development ◽  
Development Platform ◽  
Enhanced Production ◽  
Cho Cell Line

Insect Cell Line Development Using Flp-Mediated Cassette Exchange Technology

2013 ◽  
pp. 15-27 ◽  
Author(s):  
João Vidigal ◽  
Fabiana Fernandes ◽  
Ana S. Coroadinha ◽  
Ana P. Teixeira ◽  
Paula M. Alves
Keyword(s):  
Cell Line ◽  
Insect Cell ◽  
Insect Cell Line ◽  
Cell Line Development ◽  
Cassette Exchange

Mutation Profiling in Haemophilia A

2001 ◽  
Vol 85 (04) ◽  
pp. 577-579 ◽  
Author(s):  
J. Oldenburg
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Mutation Screening ◽  
Screening Method ◽  
Coagulation Factor ◽  
Causative Mutation ◽  
Haemophilia A ◽  
Coding Region ◽  
Systematic Analysis ◽  
Intron 22 Inversion ◽  
Gradient Gel Electrophoresis

SummaryHaemophilia A is a X-linked recessive bleeding disorder caused by deficiency or absence of coagulation factor VIII (FVIII) due to heterogeneous defects in the FVIII gene. The large size of the FVIII gene (26 exons spanning 186 kb) has hampered mutation analysis for many years. In 1991 the first systematic analysis of the complete coding region of the FVIII gene was performed by Higuchi et al. using Denaturing Gradient Gel Electrophoresis (DGGE) as a mutation screening method (1, 2). Notably, the causative mutation was not found in about half of the severely affected patients (1). This mystery was solved in 1993, when the intron 22 inversion was discovered (3, 4) that accounts for about 50% of the severe haemophilia A cases. The inversion mutation can be easily detected by Southern Blot. A recently described PCR-based method is more sophisticated, however once established, it allows rapid and convenient detection of the intron 22 inversion (5).

Methods for Estimating the Probability of Clonality in Cell Line Development

2020 ◽  
Vol 15 (2) ◽  
pp. 1900289 ◽  
Author(s):  
Chun Chen ◽  
Kim Le ◽  
Huong Le ◽  
Kristi Daris ◽  
Neil Soice ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Line Development
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