Procedural Protocol, Survival to Hatching and Plasmid DNA Fate After Microinjection into Tilapia Zygotes

The plasmid vector, pBRd-AK1-BGH4.6.10 (pBGH), containing the bovine growth hormone sequence driven by an avian retroviral long terminal repeat (LTR) was microinjected at 0.1, 0.5, 1 or 5 ng of pDNA/20 nl of physiological saline into tilapia, Oreochromis mossambicus × O. niloticus embryos. In 24 replicates, normally 60 zygotes were microinjected with either the entire linear 8.5 kb pBGH/ClaI vector or a 3.8 kb pBGH/SalI restriction fragment. Overall mean survival to fry hatching was 7.6% in plasmid DNA-microinjected, 15.6% in sham-microinjected and 48.1% in uninjected control embryos. There were no significant ( P > 0.05) differences in survival to hatching between those embryos microinjected with the 3.8 or the 8.5 kb restriction fragment, nor was there a trend toward decreasing survival as the plasmid DNA concentration increased from 0.1 to 5 ng. The significant ( P < 0.05) increase in survival among uninjected control embryos to hatching indicates that microinjection trauma was the major cause of mortality. Large quantities of plasmid DNA were recovered from pooled-embryo samples. Multiple bands (positive signals) were detected usually in the high molecular weight (HMW) genomic DNA regions. Position shifting of these HMW bands upon digesting with various restriction endonucleases provided evidence for plasmid DNA integration into tilapia embryo chromosomal DNA. Otherwise, these positive signah may have been end-twnd ligations of increasingly longer plasmid DNA constructs. Putative transgenic O. mossmbicus × O. niloticus were found among eight of 27 surviving adults.     

Application of High Hybridization Stringency Technology to the Analysis of Putative Transgenic Fish and Their Progeny

Eight putative transgenic hybrid tilapia ( Oreochromis mossambicus × O. niloticus ) and their F, progeny were screened by Southern blot analysis for the presence of the bovine growth hormone transgene (bGH). Transgenic mice known to contain one copy of the bGH gene per genome were used as positive controls allowing for the determination of detection levels under high hybridization stringency conditions. At a sensitivity level of one copy per genome, the bovine growth hormone transgene was not detected in any of the experimental animals. The apparent lack of bGH transgene in the fish DNA at one copy per genome, coupled with no detectable levels of bovine growth hormone being found in either serum or whole blood of the original adults, indicates these fish are either not transgenic, or that they are mosaics and do not express the transgene. Results demonstrate the need for positive controls when conducting Southern blot analysis of potentially transgenic organisms. Mammalian models appear to be suitable positive controls and should be employed until transgenic piscine models are readily available. It is suggested that high hybridization stringency is necessary when analyzing for evolutionarily conserved transgenes such as those controlling growth hormones.