title: Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons.
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.
authors.affiliation: The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Shen
authors.forename: Zheng
authors.initials: Z
authors.lastname: Xiang
authors.forename: Barbara
authors.initials: B
authors.lastname: Miller
authors.forename: Gordon
authors.initials: G
authors.lastname: Louie
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Wang
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: Fred H
authors.initials: FH
authors.lastname: Gage
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D008564:Membrane Potentials; D058953:Neural Stem Cells / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D059008:Time-Lapse Imaging; D014448:Tyrosine-tRNA Ligase / Q000235:genetics*
chemical_list: C512587:2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid; C488060:4-benzoylphenylalanine; D001577:Benzophenones; D003619:Dansyl Compounds; D010649:Phenylalanine; C509671:voltage-sensor-containing phosphatase, Ciona intestinalis; D010744:Phosphoric Monoester Hydrolases; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D000409:Alanine