Reference : Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived i...
Scientific journals : Article
Life sciences : Genetics & genetic processes
Systems Biomedicine
http://hdl.handle.net/10993/44932
Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson's Disease Point Mutation A30P.
English
Barbuti, Peter mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > >]
Antony, Paul mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Translational Neuroscience]
Rodrigues Santos, Bruno mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > >]
Massart, François mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Translational Neuroscience >]
Cruciani, Gérald mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Translational Neuroscience >]
Dording, Claire mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) >]
Arias, Jonathan mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > >]
Schwamborn, Jens Christian mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Developmental and Cellular Biology]
Krüger, Rejko mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Translational Neuroscience]
2020
Cells
9
9
Yes (verified by ORBilu)
International
2073-4409
2073-4409
Switzerland
[en] A30P ; CRISPR-Cas9 ; Parkinson’s disease (PD), patient-derived iPS ; SNCA ; alpha-synuclein ; fluorescent-activated cell sorting (FACS) ; high-content screening (HCS) ; isogenic cell lines ; single-cell clones
[en] The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson's disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy prior to generating footprint-free isogenic iPSC lines, retaining a normal molecular karyotype, pluripotency and three germ-layer differentiation potential. Directed differentiation into midbrain dopaminergic neurons revealed that SNCA expression is reduced in the gene-corrected clones, which was validated by a reduction at the alpha-synuclein protein level. The generation of single-cell isogenic clones facilitates new insights in the role of alpha-synuclein in PD and furthermore is applicable across patient-derived disease models.
Researchers ; Professionals
http://hdl.handle.net/10993/44932
10.3390/cells9092065
https://www.mdpi.com/2073-4409/9/9/2065
Open Access
FnR ; FNR1271931 > Rejko Krüger > Mamasyn > > 01/10/2018 > 30/09/2021 > 2018

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