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Microinjection of mRNA and DNA

Microinjection of mRNA and DNA. Mary Mullins, UPenn. Why inject mRNA? Gain-of-function experiments (today) : over & ectopic expression Dominant negative (pseudo loss of function) expts Mutant (today) or morpholino (tomorrow) rescue

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Microinjection of mRNA and DNA

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  1. Microinjection of mRNA and DNA Mary Mullins, UPenn • Why inject mRNA? • Gain-of-function experiments (today): over & ectopic expression • Dominant negative (pseudo loss of function) expts • Mutant (today) or morpholino (tomorrow) rescue • Epistasis: what genes can and cannot rescue your mutant? Where does your mutant gene lie in a pathway? Why inject DNA? • Gain-of-function experiments: over & ectopic expression • Dominant negative expts • Promoter analysis • Transgenics--in vivo labelling of subsets of cells or subcellular domains, promoters, ectopic expression (UAS/Gal4, heat shock, Cre-lox). When mRNA and when DNA? 1. mRNA gives reliable widespread expression. HOWEVER, it’s short-lived:8-14h. 2. DNA persists much longer, can be tissue-specific, BUT gives mosaic exp.

  2. BMP antagonists A BMP Activity Gradient in D-V Pattern Formation BMP Wild type D V Dorsalized mutants V D

  3. Class 4 and 5 dorsalizations Dorsalization and Zebrafish Fate Map tail V D yolk

  4. A BMP Signaling Pathway /Lost-a-fin / Alk8/Laf

  5. Microinjection of mRNA Examine mutant phenotype

  6. somitabundtc24, a dominant maternal mutation + ALL progeny mutant-- class 4 to 5 dorsalizations somitabundtc24 is an antimorphic mutation (“dominant negative”) of smad5(Hild et al., 1999)

  7. Tail bud stage after lecture tonight (cross your fingers) • wild type • rescued somitabun • somitabun (smad5) • chordin overexpression in wild type

  8. Circumferential somites in class 4 and 5 mutant embryos Wild type Class 5 dorsalization

  9. Dorsalized mutant phenotypes, 1 dpf somitabun/smad5 somitabun/smad5 Inject mutant embryos with smad5 mRNA to rescue the phenotype

  10. Partial rescue Complete rescue Dorsalized mutant phenotypes, 1 dpf somitabun/smad5 somitabun/smad5 Inject mutant embryos with smad5 mRNA to rescue the phenotype

  11. BMP antagonists V D BMP Wild type D V Ventralized Dorsalized V D Non- and partial-rescued somitabun mutants, chordin ectopic expression bmp4 ectopic expression

  12. Zebrafish Fate Map tail V D yolk Ventralization

  13. Ventralized and Dorsalized Phenotypes at Bud Stage/ End of Gastrulation Ventralized Wild type Dorsalized James Dutko

  14. Ventralized Phenotypes at 1 dpf bmp4 mRNA overexpression phenotypes James Dutko

  15. Agar troughs Inject through the chorion --Advantage:embryos remain protected by chorion after injection, good survival --Disadvantage: Need a good needle to penetrate the chorion and not break needle NOTE orientation of wells relative to needle: straight side to left, needle to right. Important--very little media in trough, so chorion exposed above media. The surface tension keeps embryo in well, when pull out needle. Otherwise when try to withdraw needle, embryo comes with it. Keep agar plate for tomorrow. Seal with parafilm and put into refrigerator.

  16. Ventralized phenotypes Hammerschmidt and Mullins, 2002 Overexpression phenotypes of bmp4

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