Reporter gene expression in Transgenic Tilapia, Oreochromis niloticus(L.)

Razak, Shaharudin Abdul (1999) Reporter gene expression in Transgenic Tilapia, Oreochromis niloticus(L.). University of Southampton, Doctoral Thesis.

Abstract

The purpose of this study was to analyse the expression of a reporter gene, lacZ spliced to a ubiquitous carp β-actin promoter in transgenic tilapia, Oreochromis nilotricus (L). Since lacZ is not diluted by cell division, it would serve as a genetic marker. Cells that express lacZ can be easily identified histochemically by reacting it with X-gal that produces a blue colouration on reaction with the enzyme β-galactosidase (product of lacZ gene expression).

Transient expression assay using the carp β-actin construct in G_O microinjected tilapia embryos revealed that the expression was very mosaic. This is obviously the result of mosaic distribution of the transgene. All the cells/tissues/organs examined are able to express lacZ but do not do so at the same time in any one embryo. The level and extent of lacZ expression varied considerably among individuals.

In the stably integrated germ-line transmitted tilapia (C86, C118 and C58 line), expression level varied between each transgenic line according to their copy numbers.

In the C86 line, it was possible to obtain 100% of lacZ expressing hemizygous progeny from putative homozygous fish when crossed with wild type. However in the C118 line, an average of 80% of lacZ expressing hemizygous embryos were obtained. This could be the outcome of unequal recombination during meiosis.

Apart from investigating the variable expression in embryos of C86 and C118 line, both lines were also used to generate triploid transgenic fish through heat-shock induction. The females were sterile through disruption of gonads while a proportion of the males could still be reproductively functional.

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