Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines.
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.
Ma
H
H
Institute for Medicine and Engineering, 1024 Vagelos Research Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA.
Zhu
J
J
Maronski
M
M
Kotzbauer
P T
PT
Lee
V M-Y
VM
Dichter
M A
MA
Diamond
S L
SL
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*
D008081:Liposomes; D021382:Protein Sorting Signals