Abstract:
The regenerating amphibian limb provides a useful system for
studying genes involved in the establishment of positional
information. While a number of candidate genes that may playa role
in pattern formation have been identified, their function in vivo is
unknown in this system.
To better ascertain the role of these genes, it would be useful
to be able to alter their normal patterns of expression in vivo and to
assess the effects of this misexpression on limb pattern. In order
to achieve this, a method of introducing a plasmid containing the
eDNA of a gene of interest into a newt blastema (a growth zone of
mesenchymal progenitor cells) is needed. Unfortunately, most
commonly used transfection techniques cannot be used with newt
blastema cells.
In this study, I have used the techniques of lipofection and
direct gene transfer to introduce plasmid DNA containing reporter
genes into the cells of a regenerating newt limb. The technique of
lipofection was most effective when the blastema cells were
transfected in vitro. The optimal ratio for transfection was shown
to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA
present in the mixture (1:3 ratio maintained) resulted in a
corresponding increase in gene expression.
The technique of direct gene transfer was used to transfect
newt blastema cells with and without prior complex formation with
Lipofectin. Injection of plasmid DNA alone provided the most
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promising results. It was possible to introduce plasmid DNA
containing the reporter gene ~-galactosidase and achieve significant
gene expression in cells associated with the injection site. In the
future, it would be interesting to use this technique to inject
plasmid DNA containing a gene which may have a role in pattern
formation into specific areas of the newt blastema and to analyze
the resulting limb pattern that emerges.
