XB-ART-51846J Vis Exp January 30, 2016; (107): e53518.
Functional Cloning Using a Xenopus Oocyte Expression System.
Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.
PubMed ID: 26862700
PMC ID: PMC4781710
Article link: J Vis Exp
Species referenced: Xenopus
Genes referenced: foxe3 ldb1
References [+] :
Amin, RNA-seq in the tetraploid Xenopus laevis enables genome-wide insight in a classic developmental biology model organism. 2015, Pubmed, Xenbase