Gupta, Rajeev; (1997) The use of mRNA differential display in the analysis of somitogenesis in mouse embryos. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes an investigation of differential gene expression, comparing dissected somites and the presomitic mesoderm of early mouse embryos. The method employed was mRNA differential display which uses arbitrarily primed PCR with short oligonucleotide primers to generate reverse transcription PCR fingerprints of the RNA species under study, with in situ hybridisation as a secondary screen of differential bands in the fingerprints. Initial studies revealed that although the original technique was able to generate reproducible fingerprints with nanogram quantities of material, there was a high rate of false positive clones, with multiple clones of identical size arising from any one band. Usually these clones, which are short, make poor in situ hybridisation probes. In order to show the utility of the method, it was used to screen representative RNA made in vitro from two directionally cloned embryonic cDNA plasmid libraries. Using plasmid Southern blot hybridisation as a secondary screen, at least one clone generated from any single band was always truly differential. The dual problems of high background and short products were overcome by using longer arbitrary primers at higher annealing temperatures in the PCR. Using this modified protocol, I have shown that the differential display principle can be used to compare the RNAs derived from singly dissected murine embryonic structures in the in situ hybridisation secondary screen. I report the identification of a murine homologue of the Drosophila gene tartan, recently cloned from the adult brain, as a marker of early somites. Using a 5' RACE approach, I have isolated a novel relative of this gene which is expressed in a different somitic compartment.

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