Neuronal Screening and Analysis of Gene Traps

Jarvis, Christopher I. (2003). Neuronal Screening and Analysis of Gene Traps. PhD thesis The Open University.



Large scale mutagenesis programmes are crucial for both the identification of new genes and for understanding their function. Gene trapping provides identifiable mouse mutations and mutant phenotypes and the use of embryonic stem (ES) cells allows for large scale screens to be undertaken and several have been carried out (Skames et al., 1995; Chowdury et al., 1997; Hicks et al., 1997; Zambrowicz et al., 1998). Many of these studies employ promoterless plasmid vectors which are dependent upon the transcriptional regulation of the genes in which they integrate to drive their expression. As such, they are limited to trapping those genes whose level of expression at the ES cell stage of development is sufficient for their selection. The data presented here demonstrate that gene trapping dependent upon gene expression in ES cells does allow for the discovery of genes expressed in the adult brain. Using the secretory trap vector pGTl.Stm (Skames et al., 1995), six out of 37 genes trapped (16%) were found to be expressed in the adult brain. Thus a significant fraction of genes involved in adult brain function can be accessed by gene trapping. The complete adult brain mRNA expression patterns of six genes ɑ-mannosidase II, APLP2, SDR-1, laminin-y, glypican-4 and one protein of unknown function, have been analysed.. Comparative studies of the ß-galactosidase staining patterns and in situ hybridisation data for the gene encoding APLP2, confirm that P-galactosidase expression in gene trap lines is a faithful representation of mRNA expression for splice variants incorporating the exon fused to the marker gene. The insertion into the gene encoding laminin-y1 has also proven lethal when breed to homozygosity, in concurrence with targeted disruptions of this gene (Smith et al, 1999; Murray and Edgar, 2000). Altogether these data provide evidence that gene trapping with promoterless vectors in embryonic stem cells is able to produce mutations of interest in highly differentiated tissue such as the brain.
An insertion into the gene encoding glypican-4 was then selected for more detailed study. GPC4 was originally cloned from kidney cells (Watanabe et al., 1995) and is a member of a family of heparan sulfate cell surface proteoglycans. This insertion into GPC4 lies within the first intron of the gene and ß-galactosidase activity in the transgenic embryo matches that of the previously published in situ patterns. The expression pattern of GPC4 in the adult brain is highly limited, with high levels of ß-galactosidase activity observed in the dentate gyrus and in specific layers of the cerebral cortex. This mutation has proven viable and fertile and shows no behavioural abnormalities in the open field test or in the Morris water maze. RT- PCR analysis of heinizygous tissue, however, shows that this gene trap insertion has not provided a null allele; production of properly spliced endogenous transcript continues despite the insertion of the vector into intron 1 of the gene. Such incomplete disruptions of endogenous mRNA have been found in other gene trap insertions (eg-McClive et al., 1998; Voss et al., 1998). The Exl94 mutations most likely represents a hypomorphic allele of the gene GPC4. The full disruption of this gene has yet to be reported, and the line remains of value as an expression marker and may yet reveal subtle phenotypes.

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