Genetically Encoding Unnatural Amino Acids in Neural Stem Cells and Optically Reporting Voltage-Sensitive Domain Changes in Differentiated Neurons§

Authors

  • Bin Shen,

    1. The Jack H. Skirball Center for Chemical Biology and ProteomicsThe Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Zheng Xiang,

    1. The Jack H. Skirball Center for Chemical Biology and ProteomicsThe Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Barbara Miller,

    1. Laboratory of GeneticsThe Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Gordon Louie,

    1. The Jack H. Skirball Center for Chemical Biology and ProteomicsThe Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Wenyuan Wang,

    1. The Jack H. Skirball Center for Chemical Biology and ProteomicsThe Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Joseph P. Noel,

    1. The Jack H. Skirball Center for Chemical Biology and ProteomicsThe Salk Institute for Biological Studies, La Jolla, California, USA
    2. Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Fred H. Gage,

    1. Laboratory of GeneticsThe Salk Institute for Biological Studies, La Jolla, California, USA
    Search for more papers by this author
  • Lei Wang

    Corresponding author
    1. The Jack H. Skirball Center for Chemical Biology and ProteomicsThe Salk Institute for Biological Studies, La Jolla, California, USA
    • CBPL-W, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, California 92037, USA
    Search for more papers by this author
    • Telephone: 858-453-4100, ext. 1974; Fax: 858-597-0855


  • Author contributions: B. S.: experimental design, collection of data, data analysis and interpretation, manuscript writing; Z. X.: collection of data, data analysis and interpretation; B. M., G.L., and W.W.: collection of data; J.P.N.: provision of study material, manuscript writing; F.H.G.: provision of study material, manuscript writing; L.W.: conception and design, data analysis and interpretation, financial support, manuscript writing.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS June 16, 2011.

Abstract

Although unnatural amino acids (Uaas) have been genetically encoded in bacterial, fungal, and mammalian cells using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs, applications of this method to a wider range of specialized cell types, such as stem cells, still face challenges. While relatively straightforward in stem cells, transient expression lacks sufficient temporal resolution to afford reasonable levels of Uaa incorporation and to allow for the study of the longer term differentiation process of stem cells. Moreover, Uaa incorporation may perturb differentiation. Here, we describe a lentiviral-based gene delivery method to stably incorporate Uaas into proteins expressed in neural stem cells, specifically HCN-A94 cells. The transduced cells differentiated into neural progenies in the same manner as the wild-type cells. By genetically incorporating a fluorescent Uaa into a voltage-dependent membrane lipid phosphatase, we show that this Uaa optically reports the conformational change of the voltage-sensitive domain in response to membrane depolarization. The method described here should be generally applicable to other stem cells and membrane proteins. STEM CELLS 2011;29:1231–1240

Ancillary