Dr. Michael A. Welte
Professor

Department of Biology
University of Rochester
Hutchison 317
michael.welte@rochester.edu

Papers

An improved method for analyzing transgenes

If transgenes cannot be targeted to specific sites in the genome, newly observed phenotypes in transgenic organisms might be due to either the action of the transgene or mutations caused by the transgene insertion.  To be able to confidently attribute observed phenotypes to the transgenes, we developed a method to create an allelic series of insertions that differ in the presence or copy number of the transgene, but share the same integration site.  Any phenotypic differences between organisms carrying different members of this series should then be due to the transgene alone.

To create such a series, we employed the site-specific FLP recombinase from Saccharomyces cerevisiae to remodel chromosomes in Drosophila, a technique pioneered by Kent Golic.  In vitro , the transgene (plus an eye-color marker) is flanked by two FRTs (the target sites for FLP recombinase) in the same orientation.  This construct is then introduced into flies via a P-element vector.   When FLP is provided in trans, it can catalyze unequal sister chromatid exchange and yield two new arrangements: one that has lost the transgene and one that has the transgene duplicated.


Transgene Diagram

Key:
orange: transgene (eye-color marker plus gene of interest)
light blue: FRT (Flip Recombination Target)
yellow: P-element sequences
gray: genomic DNA

These rearrangements can be recognized by the eye-color marker: absence of the marker yields white eyes, one copy orange eyes, and two copies red eyes.  Because these rearrangements retain the P-element sequences, they share the same genomic DNA disruption and any mutation that might be associated with it.  They differ only in the copy number of transgenes.

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