transgene expression

Nobody nobody at hgmp.mrc.ac.uk
Wed Nov 22 07:24:39 EST 2000


Dear Colleagues,

I=B4m pretty new in the field of C. elegans and I=B4ve observed an interesti=
ng,
but unexpected phenomenom and would like to hear your comments.

I microinjected a genomic gene sequence of a parasitic nematode (under the
control of the C. elegans let-858 promotor) into C. elegans worms and
integrated this transgene into the genome. Under the experimental
conditions of interest this transgenic line shows differences to the wild
type worms, as expected.
RT-PCR with specific primer and RNA from the parasitic nematode revealed
three different transcripts corresponding to the genomic sequence. These
transcripts differ in their intron/exon assembly so that I postulated
alternative splicing for this gene in the parasite.
To demonstate the transcription of the transgene in the transgenic C.
elegans line, I performed RT-PCR experiments with RNA from transgenic
worms, which were compared to the results obtained with RNA from wild type
worms. In this way I was able to isolate 11 amplicons from the transgenic
worm RNA. The sizes and sequences of these amplicons differ from one
another and are not identical to the parasite RT-PCR amplicons. The
transgenic C. elegans amplicons show alternative splicing that is different
from the splicing events found in the parasite and in some cases even
different exon-intron junctions are used. However, although all identified
transcripts are derived from the microinjected genomic sequence, none of
them codes for the expected transgenic protein.
I=B4m sure that I haven=B4t isolated all of the transcripts produced from th=
e
transgene in C. elegans and I=B4m very convinced that there are also
transcripts encoding the correct transgenic protein since I can observe
differences in the phenotypes of the transgenic and the wild type C.
elegans in my experiments. Unfortunately I haven=B4t got a specific antibody
against the protein of interest so that I can=B4t confirm the transgenic
expression on protein level.

My question is whether it is known that transgene transcripts are spliced
in many alternative ways in C. elegans, or is this phenomenom specific to
my heterologous parasitic nematode gene? If this has been previously
described, what is the molecular basis?

Any comment is welcome. Thank you in advance.
Andreas




Dr. Andreas Kampk=F6tter
Institute of Genetics
Heinrich Heine University
Universit=E4tsstr. 1
40225 D=FCsseldorf

Tel.: (49) 211 81 14808
=46ax: (49) 211 81 14808

andreas.kampkoetter at uni-duesseldorf.de



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