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Tools

Tools. P element – a versatile transposon. Figure 14-16. P -element structure. Types of vectors. General transformation. Reporter vectors. Regulated expression vectors. Gateway cloning system vectors. FRT containing vectors. φC31 site specific recombination vectors.

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Tools

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  1. Tools • P element – a versatile transposon.

  2. Figure 14-16 • P-element structure

  3. Types of vectors • General transformation. • Reporter vectors. • Regulated expression vectors. • Gateway cloning system vectors. • FRT containing vectors. • φC31 site specific recombination vectors. • Fold back RNA vectors.

  4. Transposon mutagenesis • Many variants: • Simple • Enhancer traps • Fusion protein • Over/ectopic expression

  5. A screen using the P element as a mutagen. w; P[w+lacW] X w;TMS, Δ23 Sb/Dr w; P[w+lacW]/TMS, Δ23 Sb X fz th st in Look for flies that are phenotypically fz or in. These are likely to be due to a P insertion into fz or in.

  6. P as a mutagen • How to test if a mutation is due to a P insertion. • Can Δ23 induce reversion? Frequency usually 30-.1%. • Reversion is often imprecise – can lead to deletion.

  7. Reversion of a P insertion (in this case P carries w+): w; fryP/TM6 X TMS, 23, Sb/Dr w; fryP/TMS, 23, Sb X w; TM6/DcxF Screen for white eyed flies with normal bristles. These will be w; fryRV/TM6 (or w; fryRV/DcxF) . Establish a stock that is w; fryRV/TM6 and test for fry function.

  8. Screen fry- chromosomes molecularly to identify cases where imprecise excision lead to a deletion of part or all of fry.

  9. Assay of cis acting regulatory sequences Position independent expression Cis acting sequences GFP reporter + + + +  -

  10. figure 5.16

  11. Tool • Collection of transposon insertions. • Goal: an insertion in every gene.

  12. Gal4 Galactose Gal80 UAS No galactose – Gal4 does not activate transcription In presence of Galactose, Gal80 does not bind Gal4, GAL4 activates transcription

  13. Gal4 enhancer trap insertions provide a wide range of cell type/tissue pattern/developmental stage specific gene expression drivers.

  14. figure 5.17

  15. A temperature sensitive GAL80 transgene is available that can be used to temporally control GAL4.

  16. Genetic Mosaics • Provides a cell marker that cannot be diluted out. Very valuable for tracing cell lineage. • Can use to study gene function. • Gets around some aspects of pleiotropy. • Allows additional functional tests of genes and pathways.

  17. Autonomous vs non-autonomous

  18. Lineage Restrictions

  19. Log of clone size Log of clone frequency Developmental Time

  20. Lineage Restrictions • Early attempts to identify lineage restrictions were hampered by the difficulty in obtaining large clones. • Data did show that clone shape was not random. For example, clones on the leg are long and thin (extended along the proximal distal axis.

  21. Lineage restrictions • How to get around the clone size vs clone frequency problem. • Minute technique.

  22. Lineage Restrictions • Anterior - Posterior Compartment boundary. • Also a compartment for the expression of regulatory genes. • E.g. hedgehog and DPP are expressed along AP boundary.

  23. Ways to generate clones that express a gene that neighboring cells do not. • Flip out

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