Why are zebrafish that have been mutated to model human diseases difficult to identify?
Peter Cattin (Auckland University): You actually wouldn’t know if you had mutant fish here because they would be heterozygotes. Often those mutations that we are interested in, and by mutations we are looking at things like muscle mutants or blood vessel - or blood itself – mutants … They would typically be fatal if they were homozygotes. So they will tend to be present in the population as hets [heterozygous]. You wouldn’t know they were there because they are able to survive with the dominant allele. So any one of these could be mutants.
It’s through doing back crosses that you look. You look then at the phenotype of the developing embryos and take if from there. Then you go to your more genetic, PCR-type things to identify what genes have been affected, and look for upstream/downstream effects, those sorts of things.
Alice Lan (PhD student, Auckland University): When I have a mutant I will have mismatched DNA, and when I hybridise this it will create kinks [DNA from the fish is bound by complementary base pairing to wildtype DNA. Where the DNA is the same as the wildtype it will bind; where it differs because of a mutation it will ‘kink’].
So Cel1 [a restriction enzyme] will cut through the kinks and create a different band. If I run a profile in gel electrophoresis, I will be able to see the extra band. Then I will know that’s the mutant; that’s the interesting fish. Now I want to have a look.
Peter Cattin (Auckland University): You’ve got to very carefully monitor where each of your fish progeny have gone, and the crosses. It’s got to be a very deliberate and carefully managed mathematical and logistical exercise, as well as the genetic side of it.