Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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Tools • P element – a versatile transposon.

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  • ToolsP element a versatile transposon.

  • P-element structureFigure 14-16

  • Types of vectorsGeneral transformation.Reporter vectors.Regulated expression vectors.Gateway cloning system vectors.FRT containing vectors.C31 site specific recombination vectors.Fold back RNA vectors.

  • Transposon mutagenesisMany variants:SimpleEnhancer trapsFusion proteinOver/ectopic expression

  • A screen using the P element as a mutagen.w; P[w+lacW] X w;TMS, 23 Sb/Drw; P[w+lacW]/TMS, 23 Sb X fz th st inLook for flies that are phenotypically fz or in.These are likely to be due to a P insertion into fz or in.

  • P as a mutagenHow to test if a mutation is due to a P insertion.Can 23 induce reversion? Frequency usually 30-.1%.Reversion is often imprecise can lead to deletion.

  • Reversion of a P insertion (in this case P carries w+):w; fryP/TM6 X TMS, 23, Sb/Drw; fryP/TMS, 23, Sb X w; TM6/DcxFScreen 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.

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

  • Cis acting sequencesGFP reporterPosition independent expression++++-Assay of cis acting regulatory sequences

  • figure 5.16

  • ToolCollection of transposon insertions.Goal: an insertion in every gene.

  • Gal4Gal80UAS No galactose Gal4 does not activate transcriptionGalactoseIn presence of Galactose, Gal80 does not bind Gal4, GAL4 activates transcription

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

  • figure 5.17

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

  • Genetic MosaicsProvides 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.

  • Autonomous vs non-autonomous

  • Lineage Restrictions

  • Developmental TimeLog of clone frequencyLog of clone size

  • Lineage RestrictionsEarly 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.

  • Lineage restrictionsHow to get around the clone size vs clone frequency problem.Minute technique.

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

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