Xenopus - experimental methods

Question 1

Fate maps

Answer 1

• Classically done with vital staining
• Recently injectable lineage labels - these can fill whole cells and not diffuse
  
  • e.g. DiI
• Because there is little increase of size in the early embryo, passive labels of this sort does not become diluted
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Question 2

What is needed when establishing the function of any gene product?

Answer 2

1. expression pattern
2. the biological activity
3. and the effect of specific inhibition in vivo

Question 3

How is the expression pattern determined?

Answer 3

• By ISH

Question 4

Why are the biological activity and inhibition experiments easy in the xenopus?

Answer 4

• ease of injecting material into embryos
• and use of ancillary techniques such as microsurgical isolation of explants or UV irradiation

Question 5

How is biological actiity measured? Gain of function.

Answer 5

• Will usually be tested with the injection of mRNA made in vitro
  
  • mRNA is made using plasmids carrying promoters for RNA polymerase and a polyA addition site to ensure a poly A cap in vivo to stabilise the measage
• mRNA can be injected into whole fertilised egg or into a specific location in the blastomere during cleavage to give control over its location
• The mRNA is translated by the protein synthesis machinary of the egg and is likely to persist throughout early development

Question 6

How to do gain-of-function with temporal control?

Answer 6

• For transcription factors:
  
  • Adding the binding domain of the glucocorticoid receptor. This will led protein to be sequestered in the cytoplasm by hsp90 untill a a glucocorticoid is added that will relieve the protein and allow it to move into the nucleus.
  • An example glucocorticoid is dexamethasone which lipid soluble and can therefore penetrate the embryo

Question 7

How to study gain-of-function at later stage of development? When injected RNA may have been degraded?

Answer 7

• Introduction of genes by transgenesis
  
  1. Plasmid DNA is incorporated into sperm which is then used to fertilise the egg to create transgenic zygotes
  2. Injection of plasmid into egg accompanied with a transposase or a estriction endonuclease
• GFP is usually incorporated in the transgene so successful transgenics can be identified

Question 8

What is a problem with transgenics in xenopus?

Answer 8

• long generation time means that pure transgeinic lines are not bred
• Instead transgenic founders are used
• This means that each transgenic will have a different insertion site and copy number which may cause some varaibility in biological behaviour

Question 9

Loss-of-function techniques in xenopus?

Answer 9

1. AMOs
   
   1. the most common method
   2. they hybridise to complementary RNA and block translation.
   3. It is necessary to have an anitbody to the target protein to has been effective.
2. Antisense oligos
   
   1. are used to inhibit a maternally acting gene
   2. injected into the oocyte
   3. Targeted by RNAseH which destroys the message
   4. RT-PCR is required to make sure the RNA is destroyed
   5. To establish later developmental effects, the treated oocyte needs to made into an embryo. DIFFICULT. Done by maturing egg in vitro and stainging with dye. Then reimplantating into a ovulating female.
3. Dominant negative
   
   1. specifically inhibit the function of normal protein
      2.

Question 10

Are target knock-out possible in xenopus?

Answer 10

• Xenopus does not have embryonic stem cells. So there is no equavilent technique for target mutagenesis done in the mouse.
• HOWEVER, new reverse genetic technologies, ZFNs, TALENs, CRISPRs have made this possible
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