Background

The elucidation of the molecular basis of pattern formation and differentiation in frog embryos has been hindered by the lack of a system for temporal and tissue-specific expression of wild type and mutant forms of developmen-tally important genes. RNA injection, the most common transient expression method in Xenopus, has been effectively used to study maternally expressed genes. However, since RNAs are translated immediately after injection, this method is unfavorable for the study of zygotic gene products that are expressed only after the midblastula transition. Direct injection of DNA can be used to express genes behind temporal and tissue specific promoters after the mid-blastula transition. However, in frog embryos this approach has only been marginally successful for two reasons: (1) injected DNA does not integrate into the frog chromosomes during early cell cycles, and therefore, the embryo expresses the genes in a highly mosaic pattern, and (2) many promoters lack adequate temporal fidelity and tissue specificity of expression when the DNA is not integrated into the genome of the frog. To overcome these technical problems, we have developed the nuclear transplantation-based approach to transgenesis

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

described in this chapter. The approach enables stable expression of cloned gene products in Xenopus embryos, allowing a broader range of feasible experimentation than that previously possible by transient expression methods.

Unlike plasmid injection, transgenesis allows stable, temporally and spatially controlled expression of gene products in desired cells of the Xenopus embryo. We have used transgenesis to express genes of interest ectopically, to direct expression of modified gene products which dominantly interfere with the function of their endogenous, wild-type counterparts, and to analyze the spatial regulation of promoters in the embryo (1). We have been able to obtain large numbers of transgenic embryos readily for these purposes and to interpret reliably the effect of transgene expression without the cell-to-cell variability of expression within an embryo, which plagues many studies using plasmid-injected embryos.

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