Sustained in vivo inhibition of protein domains using single-chain Fv recombinant antibodies and its application to dissect RGMa activity on axonal outgrowth.
Antibodies are powerful tools for delineating the specific function of protein domains, yet several limitations restrict their in vivo applicability. Here we present a new method to obtain sustained in vivo inhibition of specific protein domains using recombinant antibodies. We show that long term in vivo expression of single-chain Fv (scFv) fragments in the developing CNS can be achieved through retroviral transduction. Moreover, specific scFvs generated against the N- and C-terminal domains of the repulsive guidance molecule, RGMa, prevent proper axon targeting in the visual system. This work reveals a previously unappreciated role for the RGMa N-terminal domain in axon guidance, and provides a novel, broadly applicable and rapid procedure to functionally antagonize any protein domain in vivo.
Tassew
Nardos G
NG
Genetics and Development Division, Toronto Western Research Institute, Toronto, Ontario, Canada M5T 2S8.
Charish
Jason
J
Chestopalova
Larisa
L
Monnier
Philippe P
PP
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000494:pharmacology; D001369:Axons / Q000187:drug effects*; D002490:Central Nervous System / Q000166:cytology* / Q000196:embryology; D002642:Chick Embryo; D036386:Ephrin-A5 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology
D000906:Antibodies; D036386:Ephrin-A5; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins