ASSESMENT OF PMTL CONSTRUCT FOR DETECTION IN-VIVO OF LUCIFERASE EXPRESSION AND FATE OF THE TRANSGENE IN THE ZEBRAFISH, BRACHYDANIO-RERIO

Early embryos of the zebrafish Brachydanio rerio were cytoplasmically microinjected with pMTL plasmid containing firefly luciferase gene, in both linearized- and supercoiled-plasmid forms, to evaluate in vivo e xpression, pattern of integration and germ-line transmission of the tr ansgene in the host fish. It was possible to detect luciferase express ion in vivo, and the pattern of time-course expression was similar in both linearized- and supercoiled-plasmid injected groups. Strong lucif erase activity was detected 15-20 hours after injection, coinciding wi th early somitogenesis. Expression was detectable in a few 1 week-old individuals but was not detectable in all adults and in F-1 progeny. I n vivo screening for expression of the transgene in the developing emb ryo using luciferase assay as a method for detecting the presence of t he transgenic fish compares favourably, with PCR and Southern blot ana lysis (SBA). No integration of the introduced DNA into the genome of t reated fish and their progeny, was detected, instead it remained in ex trachromosomal form. Most of the first generation founders were mosaic . Germline transmission was observed in one individual only. A probabl e reason for the absence of integration in this study when compared to the varying frequencies of integration reported earlier in the same f ish is discussed.