A PARTIAL SEQUENCE OF VITELLOGENIN ENABLES TO TRANSPORT AND ACCUMULATE FOREIGN PROTEIN INTO EGGS IN MEDAKA Oryzias latipes
In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. Functional analyses of domains of Vtg were carried out with in vitro experiments and biochemical approaches. Therefore, the critical sequence of Vtg that enables the transportation of proteins secreted from the liver into the eggs in vivo remains unknown.
To apply the transport property of Vtg to the bioreactor, it is necessary to identify the smaller effective region precisely in the full length of Vtg, which is designated as the ''Vtg signal'' in this study. Based on the results of in silico analysis showing that medaka Vtg contains the secretory signal peptide (1MRGLILALSLALVAANQ17) and the receptor-binding region (178HLSKTKDL185), we predicted that the 300 aa N-terminal portion of Vtg ("Vtg signal") was sufficient to transport the foreign proteins produced in the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal (Figure. 1).
