title: Establishment of Reporter Lines for Detecting Fragile X Mental Retardation (FMR1) Gene Reactivation in Human Neural Cells.
abstract: Human patient-derived induced pluripotent stem cells (hiPSCs) provide unique opportunities for disease modeling and drug development. However, adapting hiPSCs or their differentiated progenies to high throughput assays for phenotyping or drug screening has been challenging. Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and a major genetic cause of autism. FXS is caused by mutational trinucleotide expansion in the FMR1 gene leading to hypermethylation and gene silencing. One potential therapeutic strategy is to reactivate the silenced FMR1 gene, which has been attempted using both candidate chemicals and cell-based screening. However, molecules that effectively reactivate the silenced FMR1 gene are yet to be identified; therefore, a high throughput unbiased screen is needed. Here we demonstrate the creation of a robust FMR1-Nluc reporter hiPSC line by knocking in a Nano luciferase (Nluc) gene into the endogenous human FMR1 gene using the CRISPR/Cas9 genome editing method. We confirmed that luciferase activities faithfully report FMR1 gene expression levels and showed that neural progenitor cells derived from this line could be optimized for high throughput screening. The FMR1-Nluc reporter line is a good resource for drug screening as well as for testing potential genetic reactivation strategies. In addition, our data provide valuable information for the generation of knockin human iPSC reporter lines for disease modeling, drug screening, and mechanistic studies. Stem Cells 2017;35:158-169.
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Meng
authors.initials: M
authors.lastname: Li
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Huashan
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Small Molecule Screening Facility, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Gene E
authors.initials: GE
authors.lastname: Ananiev
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Musser
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Kathryn H
authors.initials: KH
authors.lastname: Ness
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Dianne L
authors.initials: DL
authors.lastname: Maglaque
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Krishanu
authors.initials: K
authors.lastname: Saha
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Bhattacharyya
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Neuroscience, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Xinyu
authors.identifier: 0000-0002-5128-4424
authors.initials: X
authors.lastname: Zhao
mesh_terms: D001374:Azacitidine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry; D058953:Neural Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D001374:Azacitidine; D051860:Fragile X Messenger Ribonucleoprotein 1; D008156:Luciferases; D054852:Small Molecule Libraries
keywords: Drug discovery; FMR1; FMRP; Fragile X syndrome; High throughput; Induced pluripotent stem cells; Luciferase