Measurements of vector-derived neurotrophic factor and green fluorescent protein levels in the brain.
Demonstrating consistently reliable levels of expression is a critical part of any gene transfer study in order to assess variability and determine effective gene dosages. This article highlights some of the key methods for studying the expression levels of green fluorescent protein and neurotrophic factors after injections of adeno-associated virus (AAV) vectors into the brain. The data demonstrate greater spread and higher levels of expression using the cytomegalovirus/chicken beta-actin (CBA) promoter coupled with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), compared to earlier AAV serotype 2 vectors. Injections of either CBA-nerve growth factor (NGF)-WPRE or CBA-glial cell line-derived neutrotrophic factor-WPRE AAV vectors into the nucleus basalis of the basal forebrain led to clear and consistent elevation of the respective trophic factor as measured by enzyme-linked immunosorbent assay, with NGF vectors affecting the size and number of cholinergic neurons. AAV serotype may also be important for the spread of expression, since injecting an AAV-5 vector into the hippocampus led to higher-frequency transfection of dentate gyrus granule neurons, suggesting altered tropism relative to AAV-2.
Klein
Ronald L
RL
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA. RonKlein88@hotmail.com
Hamby
Mary E
ME
Sonntag
Christopher F
CF
Millard
William J
WJ
King
Michael A
MA
Meyer
Edwin M
EM
D000199:Actins / Q000235:genetics; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002645:Chickens; D002799:Cholinergic Fibers / Q000821:virology; D003587:Cytomegalovirus / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D005809:Genes, Regulator / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D051100:Glial Cell Line-Derived Neurotrophic Factor; D049452:Green Fluorescent Proteins; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D006624:Hippocampus / Q000166:cytology / Q000821:virology*; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections; D020932:Nerve Growth Factor / Q000235:genetics*; D009414:Nerve Growth Factors / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D000199:Actins; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D049452:Green Fluorescent Proteins; D020932:Nerve Growth Factor