Zebrafish - experimental methods

Question 1

Mutagenesis screens

Answer 1

• ENU forward genetic screems
• Any mutation that disables a gene that is essential for early development is likely to be lethal
  
  • homozygous mutants F3 embryos therefore need to be examined and scored before they die at an early stage
• When an interesting phenotype is found among the F3 generation, the F2 parents are outcrossed to WT to reduce the burden of other non-developmental mutations and to set up a permanent line
• The majority of mutations in developmentally important genes are lethal. Therefore, the mutant line needs to be mainted by heterozygotes that are crossed when a mutant embryos are required
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Question 2

What is complementation analysis?

Answer 2

• This is carried out on mutants with similiar phenotypes to see if their mutation is on the same allele or not.
• Done by mating heterozygotes together. If the mutation is on the same gene then 25% of progeny should have phenotype. In this case the mutants are said to have not complementated each other.
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Question 3

Haploid screens and gynogenetic diploids

Answer 3

• No need to generate an F2 family
• recessive mutations will show a phenotype in the F2 progeny
• Haploid syndrome (die after a few days and may have other abnormalities) can be overcome by generating gynogenetic diploids

Question 4

Insertional retrovirus

Answer 4

• Retrovirial insertion has been the method of generating many enhancer trap lines
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Question 5

Micromanipulation in the zebrafish?

Answer 5

• Not as easy to do compared to xenopus
• However, still possible to carry out cell lineage and transplantation experiments
• Cell lineage
  
  • fluorescent dextrans are used
• Transplantation
  
  • Microinjection equipment sucks cells out of the donor and inject them into the host

Question 6

Overexpression methods in the zebrafish?

Answer 6

• Injection of mRNA into embryos
  
  • May or may not fill the entire embryo
  • Accompanied with a GFP RNA so the domain of action cn be visualised
  • Will usually degrade within 24 hours so again is only useful for early developmental stages
• Transgenics
  
  • Transposon, Tol2 can be used to insert DNA
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Question 7

How to do conditional mutagenesis?

Answer 7

• The Gal4 system
  
  • Many driver lines have been generated
  • Majority of these lines are enhancer trap lines in which an endogenous enhancer drives expression of Gal4 in specific cells or tissue
• Heatshock promoter

Question 8

Inhibition/loss-of-function

Answer 8

• AMOs
  
  • need to be injected into fertilised egg or early blastomeres
• To ensure specificity the right controls are required
  
  • poof that translation of the product has been inhibited by Western blotting
  • RNA encoding for same proetin but insensitive to AMO can rescue the phenotype

Question 9

Is RNAi realible in the zebrafish?

Answer 9

NO

Question 10

How is activity of a gene tested?

Answer 10

• Overexpression of mRNA in WT embryos
• and more specifically by overexpression in mutant embryos
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