Characterization and evaluation of NGF antisense oligonucleotides: inhibition of NGF synthesis in transfected COS cells.
We present a system for the assessment of the inhibiting capacity of antisense oligonucleotides. The aim of this study was to identify an oligonucleotide that can inhibit chicken nerve growth factor (NGF) synthesis. Five antisense chicken NGF phosphorothioate oligonucleotides, AS1-5, were designed and were tested for their capacity to inhibit NGF expression in COS cells. COS cells that transiently expressed chicken NGF were treated with the oligonucleotides, and NGF expression was analyzed using a bioassay and Western blotting for NGF protein. Two oligonucleotides, AS 1 and AS 5, were more capable than the others of inhibiting expression compared with nonsense oligonucleotide, and they targeted the translational initiation and stop sites. The chicken NGF is expressed at a high level from an adenovirus major late promoter, and AS 1 was capable of inhibiting more than 80% of the NGF expression as determined using the bioassay and Western blotting. Expression of another member of the NGF gene family, neurotrophin-4, was not affected by treatment of the antisense oligonucleotides. A 10-fold lower concentration of the AS 1 oligonucleotide could be used to inhibit NGF synthesis if the cellular uptake was facilitated using lipofectin compared with addition of oligonucleotide directly to the culture medium. The amount of oligonucleotide taken up by the cells was similar in the lipofectin-treated cells as in the cells treated by a 10-fold higher concentration of medium-supplemented nucleotide. This system based on COS cells can facilitate evaluation of the capacity of inhibiting antisense oligonucleotides, particularly targeting those genes in which endogenous products are present in low levels and are difficult to analyze.
Hallböök
F
F
Department of Developmental Neuroscience, Uppsala University, Sweden.
Sahlén
A
A
Catsicas
S
S
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002645:Chickens; D018387:Codon, Initiator; D018388:Codon, Terminator; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D005562:Formazans; D015870:Gene Expression / Q000187:drug effects; D009414:Nerve Growth Factors / Q000037:antagonists & inhibitors* / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology*; D010714:Phosphatidylethanolamines; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013873:Thionucleotides / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D018387:Codon, Initiator; D018388:Codon, Terminator; D005562:Formazans; D009414:Nerve Growth Factors; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013873:Thionucleotides; C079782:MTT formazan; C020888:1,2-dielaidoylphosphatidylethanolamine; C073922:neurotrophin 4