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.
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.
<< Back to Hsp70 paper
<< Back to Papers |
