W3L1 Thu Phenotyping techniques, neural development case study

Question 1

Spinal cord regeneration

Answer 1

• Zebrafish can regenerate their spinal cord, cut out and observe
• New cells are generated, incorporate BrdU to incorporated to track. Label dividing cell within a pulse

Question 2

Dominant negative

Answer 2

-Mutant version that outcompetes WT (lead to loss of function, hard to design).Heat inducible dominant negative Fgf receptor to block Fgf signalling.

Question 3

Spinal cord regeneration study

Answer 3

-Heat-induced dominant negative Fgf straight after injury has less BrdU proliferation. And also no GFAP:GFP bridge
-Even delay heat shock doesn’t form GFAP GFP bridge

Question 4

Role of Fgf

Answer 4

Multiple roles of Fgf distinguished by timing of loss of function by inducible dominant negative
Proliferation
Glial bridge formation

Question 5

Myostatin introduction

Answer 5

Myostatin is released by muscle cells to inhibit further muscle overgrowth. Same gene leads to same phenotype in many species (conservation)

Question 6

Epistatic relationship of MD proteins

Answer 6

• double mutant is not worse than the individual mutant (doesn’t add up independently)
  Key proteins anchor the extracellular matrix to the structural
  -proteins inside the cell to allow contractile force to develop:
  Laminin (extracellular link to dystroglycan and integrin complexes) Dystrophin (intracellular protein linking dystroglycan to actin)

Question 6

Epistatic relationship of MD proteins testing

Answer 6

-Using complementation to understand how epistasis work. Epistasis can be dominant or recessive. Dominant would have a 1;2;3;1 ratio whereas recessive are 9;3;4 ratio

Question 7

Duchenne / Becker’s muscular dystrophy

Answer 7

Example of different MDs resulting from different mutations in the SAME protein
* Duchenne and Becker’s MD both due to mutations in the dystrophin gene (2.5 Mb)
* Main difference NOT mutation type, but whether the result is:
out of frame - severe DMD or in frame - less severe BMD

Question 8

Zebrafish models of DMD

Answer 8

• 3 different nonsense zebrafish mutant DMD models
• In vivo observation - can follow disease progression and test therapeutic approaches

Question 9

Zebrafish models of DMD: sapje

Answer 9

• Histology phenocopies human DMD
• Birefringence allows in vivo assessment of phenotype
  Severity in DMD results from loss of functional rest of the protein.
  Milder BMD results from affecting a portion, but restoring the next exon.
  -skip the affected exon can restore the reading frame turn DMD to BMD due to exon skipping

Question 10

Exon skipping using splice MO

Answer 10

Exon skipping can be driven by blocking a normal splice site using a splicing morpholino. However, because cryptic splice sites cannot always be predicted, the MO can have different results.
-detection using PCR at known location to distinguish posibility

Question 11

Zebrafish models of DMD Exon skipping in sapje

Answer 11

Use of the (+133+157) MO results in a cryptic exonic splice site and stop codon.
But use of both MO successfully causes skipping of exon 32 with no other alterations to the protein coding DNA.

Question 12

Exon skipping trials in human DMD

Answer 12

Treatment with AVI-4658 causes skipping of exon 51 and an increase in dystrophin protein as seen by immunostaining in the treated muscle biopsy.

Question 12

Zebrafish models of DMD Exon skipping in sapje

Answer 12

Use of the (+133+157) MO results in a cryptic exonic splice site and stop codon.
But use of both MO successfully causes skipping of exon 32 with no other alterations to the protein coding DNA.