Gene transfer into neural cells in vitro using adenoviral vectors.
Adenoviral vectors are excellent vehicles to transfer genes into the nervous system due to their ability to transduce dividing and nondividing cells, their ability to be grown to very high titers, and their relatively large insert capacity. Also, adenoviral vectors can sustain very long-term transgene expression in the CNS of rodents and in neurons and glial cells in culture. Successful gene transfer into the nervous system is dependent on the development, production, and quality control of vector preparations, which need to be of the highest quality. This unit provides protocols to clone, rescue, amplify, and purify first-generation adenoviral vectors. Detailed quality control assays are provided to ensure that vector preparations are devoid of contamination from replication-competent adenovirus and lipopolysaccharides. Also included are methodologies related to adenoviral-mediated gene transfer into neurons and glial cells in culture, and the analysis of transgene expression using immunocytochemistry, enzymatic assays, and fluorescence-activated cell sorting (FACS) analysis.
Southgate
Thomas
T
Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and Department of Medicine, University of California at Los Angeles, California, USA.
Kroeger
Kurt M
KM
Liu
Chunyan
C
Lowenstein
Pedro R
PR
Castro
Maria G
MG
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards; D015870:Gene Expression / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D019076:Transgenes / Q000235:genetics