Using oocytes for Wnt signaling assays: paracrine assays and Wnt-conditioned medium.
Xenopus oocytes have been widely used as a simple protein expression system particularly for the characterization of ion channels and membrane receptors. However, less attention has been given to their use as a means of synthesizing and analyzing secreted signaling molecules. In this review, we describe two assays that address this use of Xenopus oocytes. In the first, the paracrine assay, the oocytes secrete the signal and juxtaposed animal cap explants receive it. This provides an easy and efficient way to manipulate the signaling source since different doses of mRNA for the secreted ligand can be injected into the oocyte. Also the signaling response in the receiving cells can be read in several ways: in vivo by monitoring the localization of GFP-tagged signaling mediators, after fixation by immunostaining, or by monitoring changes in the transcriptional readout by RT-PCR. In a second approach, the oocyte is used to secrete a ligand, here the Wnt family members Wnt5a and 11, into the surrounding medium. This conditioned medium is then used to treat other cell lines to monitor their physiological changes in response to various combinations of Wnt proteins. Only a few recombinant Wnt proteins are commercially available and these are predominantly of mouse origin. Since Xenopus oocytes translate foreign RNA efficiently, this method provides an alternative source of Wnt protein derived from other model species.
Cha
Sang-wook
SW
Division of Developmental Biology, Department of Pediatrics, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA.
Heasman
Janet
J
D000465:Algorithms; D000818:Animals; D002460:Cell Line; D017077:Culture Media, Conditioned; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D009865:Oocytes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051153:Wnt Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D017077:Culture Media, Conditioned; D008024:Ligands; D011994:Recombinant Proteins; D051153:Wnt Proteins; D049452:Green Fluorescent Proteins