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Microinjection of mRNA and DNA Why inject mRNA? 1.Gain-of-function experiments (today): over & ectopic expression 2.Dominant negative (pseudo loss of function)

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Presentation on theme: "Microinjection of mRNA and DNA Why inject mRNA? 1.Gain-of-function experiments (today): over & ectopic expression 2.Dominant negative (pseudo loss of function)"— Presentation transcript:

1 Microinjection of mRNA and DNA Why inject mRNA? 1.Gain-of-function experiments (today): over & ectopic expression 2.Dominant negative (pseudo loss of function) expts 3.Mutant (today) or morpholino (tomorrow) rescue 4.Epistasis: what genes can and cannot rescue your mutant? Where does your mutant gene lie in a pathway? When mRNA and when DNA? 1. mRNA gives reliable widespread expression. HOWEVER, its short-lived:8-14h. 2. DNA persists much longer, can be tissue-specific, BUT gives mosaic exp. 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). Mary Mullins, UPenn

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

3 Dorsalization and Zebrafish Fate Map tail DV yolk Class 4 and 5 dorsalizations

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

5 Microinjection of mRNA Examine mutant phenotype

6 + ALL progeny mutant-- class 4 to 5 dorsalizations somitabun dtc24, a dominant maternal mutation somitabun dtc24 is an antimorphic mutation (dominant negative) of smad5 (Hild et al., 1999)

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

8 Circumferential somites in class 4 and 5 mutant embryos Wild typeClass 5 dorsalization

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

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

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

12 Zebrafish Fate Map tail DV yolk Ventralization

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

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

15 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 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. Agar troughs Keep agar plate for tomorrow. Seal with parafilm and put into refrigerator. NOTE orientation of wells relative to needle: straight side to left, needle to right.

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


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