Transcript Toll-7

Determining the role of Toll-7
in Drosophila melanogaster
through RNAi
Biol466, Spring 2004
Cassandra Kleve
Biol 466
Toll-7 Project
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What is a Toll gene?
-Toll-related receptors are named for their sequential and
structural similarity with Toll
-Toll was discovered during a mutagenesis screen and found
to have a role in dorsal ventral patterning of the embryo
-It was later learned that it had a role responding to
infection.
Biol 466
Toll-7 Project
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Mammalian TLRs (Toll-like receptors)
-The most conserved region of the genes is the TIR domain
-Stands for Toll/interleukin-1 receptor
-Mammalian Toll-like receptors respond to distinct
microbial patterns
-TLR4: bacterial cell-wall LPS
-TLR3: viral dsRNA
Biol 466
Toll-7 Project
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Extra-cellular
Domain
TIR
domain
Tauszig et al. 2000
Biol 466
Cytosol
Toll-7 Project
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Why Toll-7?
- Mutations in 18-wheeler cause death during
larval development with no obvious
morphological defect
-Sequence similarity and close proximity to 18wheeler on the chromosome make Toll-7 a
candidate for a compensating gene.
Biol 466
Toll-7 Project
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Determining the Role of Toll-7
-A convenient method for creating “knockdown” mutants of
Toll-7 is through RNAi
-RNAi is thought to work through the processing of dsRNA
into 21-23 bp fragments of small interfering RNA (siRNA)
-Catalyze the cleavage of the complementary mRNA
-Cause a functional loss of Toll-7
Biol 466
Toll-7 Project
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Preliminary Data
-The GAL4/UAS system can be utilized to control the
production of the dsRNA
GAL4
UAS Promoter
Toll 7 intron
Toll 7
-Three lines of flies with the P-element vector, pWIZ, with
the Toll-7 insertion will be used for these experiments
Biol 466
Toll-7 Project
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Project goals:
-1- Demonstrate the production of siRNA in the presence of
GAL4
-2- Show Toll-7 mRNA degradation in tissues producing
GAL4
-3- Examine embryos for a defect present in the absence of
Toll-7
Biol 466
Toll-7 Project
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-The RNAi will become activated
when flies with the Toll-7 construct are
crossed with GAL4 lines
- Using flies that produce GAL4 in
specific tissues will allow us to cause
Toll-7 deficiencies in specific tissues
Problem with these crosses: both the
GAL4 and Toll-7 insertions are labeled with
red eyes!
Biol 466
Toll-7 Project
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Balancer Chromosomes
-1- Have a dominant morphological mutation
-2- Have a number of inversions to prevent recombination
-3- Are homozygous lethal
Biol 466
Toll-7 Project
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Sample Cross:
- A minimum of 10 crosses will be set
- This will be repeated with a second insertion of the Toll-7
construct
Biol 466
Toll-7 Project
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Toll-7 RNA probes
- Toll-7 mRNA will be detected using digoxygenin labeled
RNA probes
- These will be synthesized as run off transcripts from the
pSPT19 vector with a Toll-7 insertion
- Two different fragments of Toll-7 will be used
- an extracellular fragment that was inserted into pWIZ
- a fragment from the TIR domain
Biol 466
Toll-7 Project
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Using the SP6 or T7 promoters on either end of
the Toll-7 insertion will allow us to make sense
and antisense probes
SP6
Toll-7 insertion
T7
For the transcription of run-off transcripts the
vector is digested on either end of the insertion
Biol 466
Toll-7 Project
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The antisense probes will hybridize to Toll-7
mRNA produced in the cell
SP6
Toll-7 insertion
T7
Cleaved by restriction
enzyme
Biol 466
Toll-7 Project
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Sense probes will be a control for nonspecific
staining
SP6
Toll-7 insertion
T7
Cleaved by restriction
enzyme
Biol 466
Toll-7 Project
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(1) Test for the production of siRNA
- Cross the Toll-7 construct into the da-GAL4 line
- daughterless is expressed ubiquitously throughout
development
-A northern blot analysis
should reveal the presence of
21-23 bp fragments that
hybridize to both sense and
antisense probes
Roignant et al.
Biol 466
Toll-7 Project
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(2) Toll-7 mRNA degradation
- For flies that emerge from the da-GAL4 cross we will use
northern blots to measure the level of Toll-7 mRNA present
-We would expect to see less Toll-7 mRNA in flies with
the Toll-7/da-GAL4 combination than without
- We can also use tissue specific GAL4 lines
-For these we would perform in situ hybridizations to
evaluate Toll-7 mRNA levels
Biol 466
Toll-7 Project
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Toll-7 appears to be expressed in the CNS
Kambris et al.
- We will cross the Toll-7 construct into flies that
produce GAL4 in the CNS
- If Toll-7 has a role in the the development of the
CNS we would expect to see developmental
abnormalities
Biol 466
Toll-7 Project
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Tissue specific Toll-7 mRNA degradation
- GAL4 can be located using anti-GAL4 antibodies
- Toll-7 will be expressed
normally in tissues that
do not produce GAL4
- Toll-7 should not be
present in tissues
producing GAL4
Kambris et al.
Biol 466
Toll-7 Project
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(3) Do we see a defect?
- Lethality of flies with both the Toll-7 construct and GAL4
driver could be one of the first signs of a defect
- Embryos resulting from the CNS GAL4 crosses will be
stained with antibodies to label the CNS to screen for
developmental abnormalities
- The strongest phenotype would be present in flies crossed
with the da-GAL4 driver
- Abnormalities may be more apparent in flies with multiple
copies of the Toll-7 construct or GAL4 insertion
Biol 466
Toll-7 Project
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Acknowledgements:
- Dr. Eldon and the lab
- Dr. Carthew’s lab at Northwestern University for pWIZ
- Dr. Marsh’s lab at UCI for help with the embryo injections
- Howard Hughes Medical Institute
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Toll-7 Project
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