Download mRNA over-expression/rescue

Microinjection of mRNA and DNA
Mary Mullins, UPenn
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?
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.
A BMP Activity Gradient in D-V Pattern Formation
BMP antagonists
V
V
D
Wild type
D
Dorsalized
mutants
Dorsalization and Zebrafish Fate Map
Class 4 and 5
dorsalizations
tail
V
D
yolk
A BMP Signaling Pathway
/Lost-a-fin
/
Alk8/Laf
Microinjection of mRNA
Examine mutant
phenotype
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)
Tail bud stage after lecture tonight
(cross your fingers)
•wild type
•rescued somitabun
•somitabun (smad5)
•chordin overexpression in wild type
Circumferential somites in class 4 and 5 mutant embryos
Wild type
Class 5 dorsalization
Dorsalized mutant phenotypes, 1 dpf
somitabun/smad5
somitabun/smad5
Inject mutant embryos
with smad5 mRNA to
rescue the phenotype
Dorsalized mutant phenotypes, 1 dpf
somitabun/smad5
somitabun/smad5
Partial
rescue
Inject mutant embryos
with smad5 mRNA to
rescue the phenotype
Complete rescue
BMP antagonists
D
V
V
D
bmp4 ectopic expression
Wild type
V
D
Non- and partial-rescued somitabun mutants,
chordin ectopic expression
Zebrafish Fate Map
tail
V
D
yolk
Ventralization
Ventralized and Dorsalized Phenotypes at
Bud Stage/ End of Gastrulation
Ventralized
Wild type
Dorsalized
James Dutko
Ventralized Phenotypes at 1 dpf
bmp4 mRNA overexpression phenotypes
James Dutko
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
Agar troughs
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.
Ventralized phenotypes
Hammerschmidt and Mullins, 2002
Overexpression phenotypes of bmp4