lecture_Xenopus_handouts

lecture_Xenopus_handouts - 1 Functional genomics in lower...

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Unformatted text preview: 1 Functional genomics in lower modelorganisms 1. Xenopus • Functional genomics (RNA, DNA en Morpholino injections, transgenesis) • Bioinformatics 2. Zebrafish • Inleiding • Forward Genetics • Reverse Genetics • Bioinformatics 1. Drosophila • Forward genetics (chemical mutagenesis, P-element insertional mutagenesis, screens) • Reverse genetics (P-element excision, RNA interference) • Bioinformatics 2. C. elegans • Forward genetics (chemical mutagenesis, screens) • Reverse genetics (RNA interference, PCR-based knockouts) • Bioinformatics I. Xenopus Functional genomics in lower modelorganisms 2- embryonic development occurs entirely outside the mother animal- embryogenesis proceeds rapidly- precise maps of embryonic cell specification and cell fate- embryos show a remarkable ability to recover after graft transplantation and micro-surgery- Vertebrate - tetrapod- embryos have a large size and a high degree of transparancy during organogenesis- tissue explants from embryos can be readily grown in vitro Suitability of Xenopus as a model system for embryonic development- embryos can be obtained in very high quantities (ideal for biochemical applications, compound and mutagenesis screens) 3 1.Xenopus, a long time favorite to explore early vertebrate development 4 5 The Wnt pathway 6 β catenine dorsal view ventral view V D dominant inactive GSK 7 8 9 10 1. Strategies to Perturb Development in Xenopus Administration of Reagents Administration via the Medium ( only for small molecules) Microinjection Larger compounds (RNA, DNA and proteins) At 4-cell stage the axes (D-V, A-P) can be distinguished => target specific tissues/organs Other Delivery Methods Electroporation and sperm-mediated transfer Reagents RNA Capped RNA is synthesized in vitro from a cDNA template Very easy, but 2 limitations (1) starts to degrade after gastrulation (2) translation is starting immediately – may generate undesired side effects Oligonucleotides antisense approaches to target specific mRNAs – necessary to inhibit maternal expression (->injection into oocytes) Morpholinos = antisense, chemically modified oligonucleotides that inhibit translation in a specific way by interfering with ribosome-binding. Alternatively, Morpholinos are targeted to exon-intron boundaries and prevent splicing (advantage – effectivity can be checked by RT-PCR) Proteins and Peptides Neutralizing or activating antibodies or peptides DNA/Transgenesis Plasmid DNA injection (mosaic expression) or transgenesis Initial succesful method : REMI (Restriction Enzyme Mediated Integration) via nuclear transfer (stable and uniform) Recent alternatives : use of transposase (Sleeping Beauty, Tol2) , bacteriophage integrase (see Allen and Weeks, Nature Methods 2005) or I-SceI Meganuclease (Ogino et al., MOD 2006, see also Pan et al., Dev. Dyn. 2006) ⇒ use of specific promoters and strategies for inducible gene expression Xenopus life cycle mid-blastula transition no transcription no transcription...
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This note was uploaded on 05/28/2010 for the course WE BIBI000000 taught by Professor Johangrooten during the Spring '10 term at Ghent University.

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lecture_Xenopus_handouts - 1 Functional genomics in lower...

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