Subject: FlyBase query
Hi Amy,
I am curating your paper on eRF1 and eRF3 in Genetics:
Chao et al., 2003, Genetics 165(2): 601--612
..
UAS-wgPE4 and UAS-wgPE13. I just wanted to check that these two
constructs weren't originally made with an FRT cassette \- the reason I
ask is that we have an HA tagged UAS-wgPE4 construct from:
Hays et al., 1997, Development 124(19): 3727--3736
but that was made with an FRT cassette between the UAS and wg sequences
to start with \- so I wanted to check that the UAS-wgPE4 construct
described in your Genetics paper is a different construct.
..
thanks,
Gillian
Subject: Re: FlyBase query
Hi Gillian,
>Hi Amy,
>
>I am curating your paper on eRF1 and eRF3 in Genetics:
>
>Chao et al., 2003, Genetics 165(2): 601--612
>
..
>
>UAS-wgPE4 and UAS-wgPE13. I just wanted to check that these two
>constructs weren't originally made with an FRT cassette \- the reason I
>ask is that we have an HA tagged UAS-wgPE4 construct from:
>
>Hays et al., 1997, Development 124(19): 3727--3736
>
>but that was made with an FRT cassette between the UAS and wg sequences
>to start with \- so I wanted to check that the UAS-wgPE4 construct
>described in your Genetics paper is a different construct.
All of the wg constructs that we make have FRT sites flanking a polyA
sequence, otherwise it is impossible to recover transformants. The
UAS promoter suffers transient activation when the pUAST
transformation vector is injected, and with something as potent as
wingless downstream, any promoter leakiness will kill the embryo.
The FRT cassette is flipped out in all the lines we use for our
experiments: once a stably transformed line is recovered, it is
crossed to the testis-specific beta-tubulin-promoter driven flippase.
This flips out the cassette in every construct passed through the
male germline (we recover individual lines and test each for
transgene expression to make sure, but so far 100% of all our
constructs have had the FRT cassette removed).
This wgPE4 construct is different from the Hays et al. line because
that line introduced the stop codon in a much more primitive way, by
including the mutation in a PCR primer, and then recloning the PCR
product back into the HA-tagged wild-type transgene. In the process,
some of the sequences downstream of the stop were altered because of
cloning issues. The new wgPE4 construct introduced the stop using a
newer PCR-based methodology, and introduces the single nucleotide
change with no other changes to the wingless sequence. The wgPE13
change was introduced in exactly the same way, so that these two
transgenes could be compared directly.
..
Amy Bejsovec, Ph.D.
Duke University
Dept. of Biology/DCMB Group
Box 91000
B336 LSRC, Research Dr.
Durham, NC 27708-1000