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Transcript
P element as a transgenesis tool A P element is a transposition element (able to transpose, or move, around the genome) As a transgenesis tool it generally consists of: Inverted repeats (transposase recognition site) Visible marker (to determine whether your progeny has the P element) Gene of interest Uses of P elements in the lab: Mutagenesis: if a mobilized P element lands in a gene, it can inactivate the gene Insertion of useful genes into the genome, including o Enhancer trapping (see diagram below): if a mobilized P element (that includes a reporter gene under control of a minimal reporter) lands in an enhancer region, reporter expression is controlled by that enhancer (ex. GFP or Gal4) o Gal80 or UAS insertions Enhancer trapping How a useful P element strain is generated 1. Inject P element plasmids containing your gene of interest and a marker (for this example, w+) into a w- 2-3 egg (this egg has already been fertilized and laid) a. 2-3: strain that contains on its third chromosome 1) Kinked (Ki) dominant visible marker and 2) P-element that encodes for a transposase (transposase recognition site is mutated so the P element is stable) 2. Transposase cuts out P-element from the plasmid at the transposase recognition site 3. P-element will insert itself into the genome in a few cells within the embryo; hopefully some of these cells are germ cell precursors 4. Allow egg to hatch and develop; this fly will likely have white eyes (unless P element inserted into eye precursor cells) 5. Mate with w- fly (that lacks the transposase) 6. Select red-eyed progeny that lack Ki, which carry your insert but don’t contain the transposase 7. Determine where gene landed using inverse PCR, or map to chromosome by crossing to balancers 8. Observe phenotype of strain (either suggesting disruption of gene or a useful enhancer trap, if you’re lucky) Other facts about P elements: Restricted to germline (transposase includes intron whose excision suppressed in somatic cells) Once a P element has landed in a genome and is established in a strain, further transposition appears to be repressed via repression of transposase RNA
