Disruption of ephrin-A5 in developing \u3ci\u3eXenopus laevis\u3c/i\u3e embryos by shRNA

Eph receptors are the largest class of receptor tyrosine kinases (RTKs) that participate in bidirectional cell signaling with their cell surface ephrin ligands during the development of vertebrate embryos. Ephrin-A5 is one ligand that interacts with the EphA4 receptor, but is present much earlier than EphA4 in developing Xenopus laevis embryos. This raises the question why ephrin-A5 is expressed so early and suggests that it may have a role in embryonic development and tissue differentiation independent ofEphA4. This project attempted to study ephrin-A5\u27s function using a short hairpin RNA (shRNA) knockdown approach. A vector encoding shRNA of the ephrin-A5 mRNA under the control of a strong eukaryotic promoter was injected into early Xenopus laevis embryos in the attempt to knock down ephrin-A5 expression and determine its function. A protein gel visualized the presence of viable proteins and Western Blots visualized the knockdown attempts. The shRNA vector used (pCAX+571) was determined not to have knocked down the expression of ephrin-A5; thus ephrin-A5\u27s function in embryonic development has yet to be determined.