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Ismael Badillo-Vargas

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Ismael Badillo-Vargas

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    1. Ismael Badillo-Vargas Fall 2006 Gen 875 Class Presentation

    2. Morin et al, 2001 A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

    3. To understand the mechanisms of development of an organism we need study:

    4. Use of GFP, as a fluorescent tag, to fuse with genomic fragments, which encode GFP-tagged proteins.

    5. Previous Studies Generation of cDNA-GFP fusion libraries and their ectopic expression in cultured mammalian cells and plants.

    6. Protein Trap Transposon (PPT) is a P-element which carries a mobile artificial exon encoding GFP design to randomly tag proteins with an enhanced GFP.

    8. Constructed 3 vectors within each reading frame.

    9. Collected embryos for 24h were screened (and larvae).

    10. Confocal Imaging of Living Embryos and Tissues Dissected muscle fibers from adult thoracic indirect flight muscles.

    11. Berkeley Drosophila Genome Project

    12. Recover genomic sequences flanking the P-element insertion site using inverse PCR and the set of oligonucleotides used for EP constructs by www.fruitfly.org.

    13. They recovered the sequences flanking the insertion point of 102 independent lines.

    14. 20 of the 102 insertions for which they obtained sequence data have double or triple insertions.

    15. Known and Predicted Genes

    16. Unpredicted genes

    17. Some of the cDNA sequence flanking GFP do not matched with any EST or predictions, but some correspond to parts of EST sequences located downstream of the insertion.

    18. What this figure represent?

    19. Dynamics of GFP fusion distribution A) The distribution of the protein fusions observed at different times during cell division in syncitial embryo. B) The distribution of the protein fusions observed at different times during the zipper-like cell movements of dorsal closure.

    20. What was their main limitation to identify more positive events?

    21. Discussion >50% of the insertions occurs in long introns.

    22. Discussion The protein trap method might be useful in identifying unsuspected novel genes and functions.

    23. In general GFP reveals in real time the dynamics of protein’s distribution in the whole live organism.

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