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CHARACTERIZATION OF THE REGULATORY REGION OF THE DISPERSED HOMEOBOX GENE gsh-1

by MCFARLAND, KEVIN LEE

Abstract (Summary)
Gsh-1 , a dispersed homeobox gene, is expressed in a regionally restricted pattern in the developing mouse central nervous system. To better understand the temporal and spatial regulation of gsh-1 , we have isolated mouse genomic regions surrounding the ORF of the gsh-1 gene. This was used for reporter-gene analysis in transgenic mice, and in cell culture. First, a previously isolated 10 kb genomic region surrounding the ORF of the gsh-1 gene was fused to a Lac-Z gene and used for analysis of the cis acting regions of the gsh-1 gene promoter by generating transgenic mice. The expression pattern of the gsh-1/?-gal ;5.5/4.5 transgene construct was localized to the hypothalamus, thalamus, and pons, therefore the transgene showed a similar but distinct pattern compared to the gsh-1 pattern seen earlier by in situ hybridization. A 21 kb piece of the mouse genomic gsh-1 gene was then isolated, containing 14.5 kb of 5' and 6.5 kb of 3' the gsh-1 gene. A gsh-1/?-gal ;14.5/0 reporter construct was expressed diffusely throughout the midbrain and medial gaglionic eminence. Again this pattern was similar to, but did not completely recapitulate the distinct pattern of gsh-1 seen by in situ hybridization in the developing mouse embryo. Second, a SV-40 LTAg gene was inserted into the first exon of the gsh-1 gene, and this construct was used to generate transgenic mice. The mice developed brain tumors, which were developed into two cell lines that expressed gsh-1 mRNA. Wild type gsh-1 /SV-40 LTAg mice were bred to heterozygous gsh-1 knockout mice to create a gsh-1 null cell line. Third, in order to perform analysis of the cis acting regions of the gsh-1 promoter, gsh-1 constructs with a luciferase reporter gene were generated. These were then transfected into the gsh-1/SV-40 LTAg cell line clones. Transient transfection of these constructs showed a ~ 350 fold increase in activity in a gsh-1 expressing cell lines over a non-neuronal cell line. Also, luciferase activity for the gsh-1/luc ;14.5/6.5 construct is 4 fold higher than luciferase activity for the gsh-1/luc ;5.5/4.5 construct. Fourth, a test of the autoregulation of gsh-1 , showed no increase in luciferase expression.
Bibliographical Information:

Advisor:

School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:gsh i homeobox promoter transient transfections reporter genes

ISBN:

Date of Publication:01/01/2002

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