Biol 466 Toll-7 Project 1 Determining the role of Toll-7 in Drosophila melanogaster through RNAi Biol466, Spring 2004 Cassandra Kleve
Jan 08, 2016
Biol 466 Toll-7 Project 1
Determining the role of Toll-7 in Drosophila melanogaster through RNAi
Biol466, Spring 2004
Cassandra Kleve
Biol 466 Toll-7 Project 2
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.
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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
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Extra-cellular
Domain
Cytosol
TIR
domainTauszig et al. 2000
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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 18-wheeler on the chromosome make Toll-7 a candidate for a compensating gene.
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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
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Preliminary Data
-The GAL4/UAS system can be utilized to control the production of the dsRNA
-Three lines of flies with the P-element vector, pWIZ, with the Toll-7 insertion will be used for these experiments
UAS Promoter intron
GAL4
Toll 7 Toll 7
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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
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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!
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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
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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
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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
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Toll-7 insertionSP6 T7
Using the SP6 or T7 promoters on either end of the Toll-7 insertion will allow us to make sense and antisense probes
For the transcription of run-off transcripts the vector is digested on either end of the insertion
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The antisense probes will hybridize to Toll-7 mRNA produced in the cell
Toll-7 insertionSP6 T7
Cleaved by restriction enzyme
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Sense probes will be a control for nonspecific staining
Toll-7 insertionSP6 T7
Cleaved by restriction enzyme
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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.
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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
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Toll-7 appears to be expressed in the CNS
- 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
Kambris et al.
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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.
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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
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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