The fate of injected DNA in tilapia (Oreochromis niloticus)

Sulaiman, Zohrah Haji (1994) The fate of injected DNA in tilapia (Oreochromis niloticus). University of Southampton, Doctoral Thesis.

Abstract

The fate of injected linear pFV1CAT, both circular and linear forms of PV2CAT, FV2β-gal and cβA in host tilapia were investigated.

It was found that transgene expression was occasionally detectable before mid-blastula transition. It appears that for all the constructs studied, the highest level and incidence of transgene expressions are coincident with the host's activity at the stage of development. Mosaic character of spatial expression is a common feature in tilapia injected with FV2β-gal and cβA. This is also true for the distribution of transgene DNA in injected tilapia. The results also appear to suggest that FV2β-gal may be spatially regulated by the host i.e. by integration to a nearby enhancer element. FV2 is not transcribed as efficiently as cβA (P< 0.01 at day 3) but it is a better construct to use than FV1 (P< 0.05). Circular forms show higher level of expression than the linear forms of CAT gene constructs (P< 0.01) but there is no difference in survival (P< 0.05), transgene copy number and replication between the two physical forms of β-gal and CAT gene constructs. Integration appears to be an early event for the linear forms. Degradation of transgenes probably begins immediately following injection, rate of loss being approximately 68-80% at 8 cell and 32/64 cell stages, and 30% at day 1. Replication of the transgene also occurs at day 1 (5-7 fold increase) and a second round of replication is observed at day 7 (10 fold increase). Most of the persisting transgene copies are in head to tail and head to head arrays. Transgene DNA copy number ranges from less than 1 to > 10 per individual tilapia and there is no correlation between copy number and its expression (P> 0.05).

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