title: Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines.
abstract: Gene transfer into CNS is critical for potential therapeutic applications as well as for the study of the genetic basis of neural development and nerve function. Unfortunately, lipid-based gene transfer to CNS cells is extremely inefficient since the nucleus of these post-mitotic cells presents a significant barrier to transfection. We report the development of a simple and highly efficient lipofection method for primary embryonic rat hippocampal neurons (up to 25% transfection) that exploits the M9 sequence of the non-classical nuclear localization signal of heterogeneous nuclear ribonucleoprotein A1 for targeting beta(2)-karyopherin (transportin-1). M9-assistant lipofection resulted in 20-100-fold enhancement of transfection over lipofection alone for embryonic-derived retinal ganglion cells, rat pheochromocytoma (PC12) cells, embryonic rat ventral mesencephalon neurons, as well as the clinically relevant human NT2 cells or retinoic acid-differentiated NT2 neurons. This technique can facilitate the implementation of promoter construct experiments in post-mitotic cells, stable transformant generation, and dominant-negative mutant expression techniques in CNS cells.
authors.affiliation: Institute for Medicine and Engineering, 1024 Vagelos Research Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: H
authors.initials: H
authors.lastname: Ma
authors.forename: J
authors.initials: J
authors.lastname: Zhu
authors.forename: M
authors.initials: M
authors.lastname: Maronski
authors.forename: P T
authors.initials: PT
authors.lastname: Kotzbauer
authors.forename: V M-Y
authors.initials: VM
authors.lastname: Lee
authors.forename: M A
authors.initials: MA
authors.lastname: Dichter
authors.forename: S L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002642:Chick Embryo; D046508:Culture Techniques; D004622:Embryo, Mammalian; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008081:Liposomes; D009474:Neurons / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution; D014162:Transfection / Q000379:methods*
chemical_list: D008081:Liposomes; D021382:Protein Sorting Signals