title: A puromycin selectable cell line for the enrichment of mouse embryonic stem cell-derived V3 interneurons.
abstract: BACKGROUND
  Spinal V3 interneurons (INs) are a commissural, glutamatergic, propriospinal neuron population that holds great potential for understanding locomotion circuitry and local rewiring after spinal cord injury. Embryonic stem cells hold promise as a cell source. However, the inevitable heterogeneity resulting from differentiation protocols makes studying post-mitotic stem cell-derived neuron populations difficult because proliferative glia quickly overtake a culture. Previously, an induction protocol for V3 INs was established. However, because of the heterogeneous population resulting from the induction protocol, functional characterization of the induced cells was not possible.
  
  
  METHODS
  A selectable murine transgenic embryonic stem cell (ESC) line (Sim1-Puro) was generated by recombineering. The expression of the puromycin resistance enzyme, puromycin N-acetyl-transferase (PAC), was knocked into the locus of a post-mitotic V3 IN marker (Sim1), allowing Sim1 gene regulatory elements to control PAC expression. The resulting cell line was characterized for Sim1 expression by in situ hybridization, for glutamatergic marker expression by immunocytochemistry and quantitative real time polymerase chain reaction (qRT-PCR), and for functional maturation by electrophysiology.
  
  
  RESULTS
  Puromycin selection significantly enriched the population for V3 INs, allowing long-term characterization. The selected population expressed the neuronal marker β-III tubulin and the glutamatergic neuron marker VGluT2. The selected V3 INs also exhibited appropriate functional maturation, as assessed by electrophysiology, and remained glutamatergic for 2 weeks.
  
  
  CONCLUSIONS
  The Sim1-Puro cell line provides a simple, high throughput method for generating large numbers of V3 INs from mouse ESCs for future in vitro and cell transplantation studies.
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis, 1 Brookings Drive, Box 1097, St. Louis, MO, 63130, USA. hao.xu@wustl.edu.
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis, 1 Brookings Drive, Box 1097, St. Louis, MO, 63130, USA. nisha.iyer@wustl.edu.
authors.forename: Nisha
authors.initials: N
authors.lastname: Iyer
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA. jhuettner@wustl.edu.
authors.forename: James E
authors.initials: JE
authors.lastname: Huettner
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis, 1 Brookings Drive, Box 1097, St. Louis, MO, 63130, USA. sakiyama@wustl.edu.
authors.forename: Shelly E
authors.initials: SE
authors.lastname: Sakiyama-Elbert
mesh_terms: D000123:Acetyltransferases / Q000235:genetics; D000943:Antigens, Differentiation; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism; D003470:Culture Media; D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000166:cytology*; D055879:Gene Knock-In Techniques; D007395:Interneurons / Q000166:cytology*; D016254:Mutagenesis, Insertional; D011691:Puromycin / Q000494:pharmacology*; D012097:Repressor Proteins / Q000235:genetics
chemical_list: D000123:Acetyltransferases; D000943:Antigens, Differentiation; D051792:Basic Helix-Loop-Helix Proteins; D003470:Culture Media; D017077:Culture Media, Conditioned; D011691:Puromycin; D012097:Repressor Proteins; C101750:Sim1 protein, mouse; C045336:puromycin N-acetyltransferase