TRANSGENESIS IN THE ZEBRAFISH, DANIO RERIO : EFFICACY OF SPERM-VECTOR AND MICROINJECTION METHODS

Abstract

The present investigation aimed to understand the mechanism involved in sperm-mediated gene transfer and ability of electroporation to enhance DNA uptake by spermatozoa. In this effort the efficacy of sperm-mediated gene transfer was compared with the relatively established technique of generating transgenic fish, the cytoplasmic microinjection. The studies were conducted on the zebrafish Danio rerio, an excellent experimental model which enables germ-line investigations in a relatively short time all together, five gene constructs three with firefly luciferase gene (pMTL, pRSVL, pGEM-luc) are two with chloramphenicol acetyltransferase (CAT; pUSVCAT, FV-2) were used in both supercoiled and linear forms. The aspects studied included expression and fate of the transgenes during early development, adulthood and their germ-line transmission. Scintillation spectroscopy, light-microscopy and ultrastructural in situ hybridization techniques were employed to assess the nature of association between spermatozoa and foreign DNA. ability of electroporation, an efficient bacterial transformation and mammalian cell transfection technique was explored to enhance uptake of foreign DNA by zebrafish spermatozoa.