title: Development of a low-pressure microtargeting biolistic device for transfection of retinal explants.
abstract: Biolistic transfection offers a key experimental method for molecular perturbation of bona fide, postmitotic neurons within their native local environment in explanted tissues. However, current, commercially available biolistic devices unavoidably deliver traumatic injury to surface layers of explanted tissues because of helium co-emission with DNA-coated gold particles during the shooting process. This makes such methods unsuitable for use with the delicate tissue layers of the mammalian retina. Here, we report the development of a novel and inexpensive microtargeting biolistic device that avoids the trauma associated with conventional entrainment biolistic methods, permitting rapid and efficient transfection of retinal ganglion cells in the adult mammalian retina without significant damage to their local microenvironment. By using low helium inflow pressures and vacuum diversion to eliminate helium emission during the transfection process, we found that the current method allowed efficient transfection as well as morphological and functional preservation of retinal ganglion cells and their local glial microenvironment in transfected retinal explants from adult rats. The use of an ethanol-gold suspension further supported rapid and extended shooting sequences and reduced shot-to-shot variation during transfection compared to existing tubing-based devices. This new biolistic device should be useful not only in the retina, but also in other tissue explant settings in which preservation of local cellular and tissue integrity is a priority.
authors: Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27704, USA. gottron@neuro.duke.edu
authors: Melissa G
authors: MG
authors: Christianson
authors: Donald C
authors: DC
authors: Lo
mesh_terms: D000818:Animals; D019470:Biolistics / Q000295:instrumentation / Q000379:methods; D015415:Biomarkers / Q000032:analysis; D002772:Cholera Toxin / Q000032:analysis; D000431:Ethanol / Q000737:chemistry; D005453:Fluorescence; D006046:Gold / Q000737:chemistry; D006371:Helium; D009457:Neuroglia / Q000166:cytology / Q000502:physiology; D011312:Pressure; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000502:physiology; D014162:Transfection / Q000295:instrumentation / Q000379:methods; D014618:Vacuum
chemical_list: D015415:Biomarkers; D006371:Helium; D000431:Ethanol; D006046:Gold; D002772:Cholera Toxin