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Morpholinos
Reverse genetics, the ability to inactivate a given gene in an entire
animal, is just beginning to be addressed in the zebrafish. Established
techniques and materials required for generating both germline and somatic
gene inactivation in mammals and lower organisms have only recently become
available to the zebrafish community. Two approaches have been attempted
in order to develop somatic gene inactivation in zebrafish embryos. The
use of morpholino oligonucleotides has consistently phenocopied gene mutations,
whereas RNAi has not been successful.
While germline gene inactivation generates very important data that cannot
always be obtained through somatic gene inactivation, the latter approach
is often much cheaper and faster. Morpholino oligos, first developed for
clinical applications, have been successful in inducing antisense effects
in zebrafish embryos. These 25 bp DNA analogs operate by blocking mRNA
translation or splicing. They only operate when complementary to a sequence
between the 5’ UTR through the first 25 bases 3’ of the AUG
start site or to splice junctions. The principal difference between the
two is that AUG morpholinos also affect maternal transcripts, and can
therefore create more severe phenotypes than a homozygous gene inactivation.
Morpholinos are typically injected into zebrafish embryos at the 1–8
cell stage at a final concentration range of 0.1–1.0 nM. The DNA
analogs are immune to DNAse degradation and are thus stable in the embryo
for extended periods (up to 5 days). Furthermore their small size allows
for even distribution to all cells in the developing embryo at concentrations
sufficient for inhibition. The main downsides of morpholinos are genetic
variation between individual zebrafish, leading to unforeseen mismatches
and therefore inconsistent results, and non-specific toxicity.
The Figure shows an example of a GATA-1 morpholino directed to the 5’
AUG sequence, that inactivates blood production as evidenced by globin
in situ hybridization. In this case, red blood cells re-appear starting
at 5 days post fertilization.
More information 1
and 2.
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