Gene transfer into rat brain using adenoviral vectors.
Viral vector-mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose-efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described.
Puntel
Mariana
M
Board of Governors Gene Therapeutics Research Institute, Departments of Medicine and Biomedical Sciences, Cedars-Sinai Medical Center and Departments of Medicine, and Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Kroeger
Kurt M
KM
Sanderson
Nicholas S R
NS
Thomas
Clare E
CE
Castro
Maria G
MG
Lowenstein
Pedro R
PR
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D001921:Brain / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D005455:Fluorescent Antibody Technique / Q000379:methods; D018014:Gene Transfer Techniques / Q000009:adverse effects; D005822:Genetic Vectors / Q000009:adverse effects; D007150:Immunohistochemistry / Q000379:methods; D015213:Neuroimmunomodulation; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D013238:Stereotaxic Techniques