Antisense inhibition of low-affinity nerve growth factor receptor in kidney cultures: power and pitfalls.
1. Antisense inhibition of gene expression implies that the expression of the target protein is selectively inhibited at either the translational or the transcriptional level by complementary DNA or RNA constructs that are antiparallel to the target sequence. The antisense inhibition strategy provides means to study the roles of individual proteins and has, in spite of its limitations, gained a wide range of both therapeutic and experimental applications. 2. In developmental biology, protein expression has been selectively inhibited by the use of antisense gene transfection and by antisense deoxyoligonucleotides. The transfectability of embryonic tissues is variable, but in general fetal and embryonic cells take up foreign DNA relatively efficiently, in particular, short deoxyoligonucleotides that penetrate mesenchymal cells within a few hours without any manipulation. 3. We have now evaluated the advantages and pitfalls of antisense inhibition by deoxyoligonucleotides in organ culture and describe our experience from the inhibition of low-affinity nerve growth factor receptor expression in embryonic mouse and rat kidneys. 4. The expression of nerve growth factor receptor can be specifically inhibited by deoxyoligonucleotides, but the target sequence-dependent window of, in particular, phosphorothioate-modified oligonucleotides is quite narrow. The culture conditions affect the response to the oligonucleotides and their cellular incorporation is variable with respect to the cell type and stage of differentiation.
Sainio
K
K
Department of Pathology, University of Helsinki, Finland.
Saarma
M
M
Nonclercq
D
D
Paulin
L
L
Sariola
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016477:Artifacts; D001483:Base Sequence; D015153:Blotting, Western; D003470:Culture Media / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects*; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000187:drug effects* / Q000196:embryology; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D009024:Morphogenesis; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology*; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013873:Thionucleotides / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003470:Culture Media; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D017475:Receptors, Nerve Growth Factor; D013873:Thionucleotides