Project: Research project

Project Details


Vertebrate gene targeting has so far been possible only in the mouse.
Comparative studies of similar mutations in other vertebrates would
allow a more complete analysis useful for establishing the importance
of conserved genes implicated in fundamental processes of human
development and disease. Because the zebrafish embryo is nearly
transparent and easily observed during development, it is an excellent
model of vertebrate embryogenesis. However, efficient transgenesis of
the zebrafish has not yet been established primarily due to the lack of
vector systems capable of high level integrated reporter gene expression
screenable throughout the life cycle. A non viral vector, pFRMwg, has
been designed which stably expresses the green fluorescent protein (GFP)
almost ubiquitously throughout the zebrafish. In addition, transgenic
techniques have been optimized to allow screening of homologous
recombination events in the living embryo. In the proposed study, this
vector system will be modified so that an integrated tester locus
containing a blue mutation of GFP can be targeted by microinjection of
the 1-cell embryo with green GFP vectors and screened for green GFP
expression during development. The frequency of this in vivo gene
targeting will be tested and optimized as the variables of vector design
and the type of embryos chosen to be microinjected are manipulated. The
goal will be to establish the conditions for efficient germ line
transfer of gene targeting events.

Human gene therapy offers a tremendous potential for both the cure and
prevention of disease. Again, the primary animal model has been the
mouse, and several limitations have become apparent in this system:
expression of gene therapy vectors can not be easily screened in live
mice, and the potential of unintended germ line transfer can only be
tested on a large scale by PCR of male gametes. Additionally, long term
persistence of expression (e.g. 1 year) is yet to be shown. Using the
pFRMwg vector as a marker for gene therapy in zebrafish, expression can
be scored in living fish. In this study, persistence and localization
of this expression will be tested as a function of the variables of DNA
dose, DNA assimilation cofactors and route of administration and finally
correlated with the state of the vector DNA over time. The goals are to
achieve persistent high level expression and eventually to estimate the
potential of germ line transfer after a particular treatment.
Effective start/end date4/1/983/31/01


  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences


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