TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

Scientific Reports
Barun MahataKaushik Biswas

Abstract

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding t...Continue Reading

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Citations

Aug 5, 2015·ACS Chemical Biology·Hideaki Mabashi-AsazumaDonald L Jarvis
Sep 26, 2015·Metallomics : Integrated Biometal Science·Kathryn M MarshallOneel Patel
Jul 12, 2017·Current Opinion in Lipidology·Hannah Sunshine, Maria Luisa Iruela-Arispe

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Datasets Mentioned

BETA
GM2

Methods Mentioned

BETA
transfection
PCR
genotyping
FACS
targeted knockout
electrophoresis

Software Mentioned

REAL
TAL Effector Nucleotide Targeter
Paired Target Finder
TALEN
Graphpad Prism
ZiFiT Targeter

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