Assessment 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 expression, pattern of integration and germ-line transmission of the transgene in the host fish. It was possible to detect luciferase expression in vivo, and the pattern of time-course expression was similar in both linearized- and supercoiled-plasmid injected groups. Strong luciferase activity was detected 15-20 hours after injection, coinciding with early somitogenesis. Expression was detectable in a few 1 week-old individuals but was not detectable in all adults and in F1 progeny. In vivo screening for expression of the transgene in the developing embryo using luciferase assay as a method for detecting the presence of the transgenic fish compares favourably, with PCR and Southern blot analysis (SBA). No integration of the introduced DNA into the genome of treated fish and their progeny, was detected, instead it remained in extrachromosomal form. Most of the first generation founders were mosaic. Germline transmission was observed in one individual only. A probable reason for the absence of integration in this study when compared to the varying frequencies reported earlier in the same fish is discussed.
Patil
J G
JG
Department of Zoology, National University of Singapore.
Wong
V
V
Khoo
H W
HW
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015027:Zebrafish
D004247:DNA; D008156:Luciferases