International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 77 - USING TRANSPOSON INSERTIONS IN LARGE DNA CLONES TO ANALYZE GENE EXPRESSION

Dr. Timothy Jinks
MRC-NIMR
Division of Dev. Neurobiology
The Ridgeway
Mill Hill
London NW7 1AA
UK

Co-Authors: 1) Martinez MT, 2) Sedgwick S, 3) Krumlauf R.
Institutions: 1)MRC-NIMR, Division of Dev. Neurobiology, 2)MRC-NIMR, Division of Yeast Genetics, 3) Stowers Institute for Medical Research

Large insert clones of genes and gene complexes are increasingly being used to analyze gene regulation and gene function.  These large insert clones are often difficult to manipulate due to the size and complexity of the sequences in the clone.  New molecular biology techniques are needed to modify these clones for use in experimentation.  We have taken advantage of transposon-mediated integration to create a series of individual random inserts of a reporter gene in Hoxb BACs.  The series of modified BACs were analyzed by mouse transgenesis to reveal the reporteršs expression pattern.  We have analyzed the reporteršs expression for each insertion site and have correlated expression with the precise location in the Hoxb complex.  This has allowed us to establish the range over which enhancers influence gene expression across the complex and also the limit of action of the enhancers beyond the 3š end of the cluster.  This technique is useful for the systematic analysis of large clones to scan for gene regulatory activity.  In addition we have engineered a new transposon-based reporter that provides further utility for analysing large clones by introducing loxP and FRT recombination sites.  These sites permit precise rearrangement of the BACs to be made as well as targeted integration of other DNA sequences.  These new tools are proving useful for our analysis of Hoxb gene regulation and should prove useful more generally for the analysis of large clones.


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