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Keywords {🔍}

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Topics {✒️}

lentiviral plasmid fuw-teto-ng2-p2a-egfp-t2a-puromycin α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid n-methyl-d-aspartate pluripotency reporter eos-gfp/puror previous high-coverage rna-seq autism spectrum disorders related subjects hrp-conjugated secondary antibodies human ipsc-derived neurons brain-derived neurotrophic factor 10 μm single-stranded oligodeoxynucleotide reverse tetracycline-controlled transactivator tata-box binding protein magnetic-activated cell sorting cell patch-clamp electrophysiology article  google scholar ipsc-derived control neurons multi-electrode array analysis gamma-aminobutyric acid type /dead-box helicase 53 beta-prototype droplet reader detected rna-seq transcripts gov/tools/primer-blast/ article download pdf autism spectrum disorder broad x-linked region neural progenitor cells receptor antagonist 6-cyano-7-nitroquinoxaline-2 pluripotent stem cells rapid single-step induction stem cell reports 20 μm cas9-2nls nuclease coding rna ptchd1-antisense ddx53-3tc ipsc lines opaque-bottom mea plates x-linked ptchd1 locus human ddx53-3tc ipscs ddx53-3tc cell lines ipsc-derived neurons multi-electrode array stem cell models developmental transcriptome full access 50b-ddx53-3tc neurons ddx53-3tc neurons compared isolate ddx53-mutated cells eos lentiviral vectors 50b-ddx53-3tc lines identify pre-selected potential human protein atlas

Schema {🗺️}

WebPage:
      mainEntity:
         headline:Disruption of DDX53 coding sequence has limited impact on iPSC-derived human NGN2 neurons
         description:The X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA (PTCHD1-AS)/DEAD-box helicase 53 (DDX53) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS/DDX53 to the next gene, PTCHD1, which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons. We used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53-3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed. Isogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53-3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed. DDX53-3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.
         datePublished:2023-01-12T00:00:00Z
         dateModified:2023-01-12T00:00:00Z
         pageStart:1
         pageEnd:12
         license:http://creativecommons.org/publicdomain/zero/1.0/
         sameAs:https://doi.org/10.1186/s12920-022-01425-3
         keywords:
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            iPSCs
             DDX53
             PTCHD1-AS
            CRISPR
            NGN2
            Human Genetics
            Microarrays
            Gene Expression
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      headline:Disruption of DDX53 coding sequence has limited impact on iPSC-derived human NGN2 neurons
      description:The X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA (PTCHD1-AS)/DEAD-box helicase 53 (DDX53) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS/DDX53 to the next gene, PTCHD1, which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons. We used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53-3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed. Isogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53-3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed. DDX53-3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.
      datePublished:2023-01-12T00:00:00Z
      dateModified:2023-01-12T00:00:00Z
      pageStart:1
      pageEnd:12
      license:http://creativecommons.org/publicdomain/zero/1.0/
      sameAs:https://doi.org/10.1186/s12920-022-01425-3
      keywords:
         ASD
         iPSCs
          DDX53
          PTCHD1-AS
         CRISPR
         NGN2
         Human Genetics
         Microarrays
         Gene Expression
      image:
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            type:ImageObject
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      author:
            name:Muhammad Faheem
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                     type:PostalAddress
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            name:Eric Deneault
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      name:Genetics & Genome Biology Program, Hospital for Sick Children, Toronto, Canada
      name:The Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada
      name:Genetics & Genome Biology Program, Hospital for Sick Children, Toronto, Canada
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      name:McLaughlin Centre, University of Toronto, Toronto, Canada

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