Insertional Mutagenesis in Zebrafish by SB Transposons

Project: Research project

Project Details

Description

DESCRIPTION: (Applicant's Abstract) The goal of this project is to develop and
use the Sleeping Beauty (SB) Transposon System for delivery of a variety of
gene-trap, enhancer-trap, and poly(A)-trap constructs for delivery of
insertional mutagenesis and gene-tagging in zebrafish. Zebralish are a nearly
ideal vertebrate model system for developmental genetics. However,
sophisticated mechanisms for insertional inactivation of genes are lacking in
zebrafish. Such methods can permit (1) the rapid isolation of genes associated
with phenotypic abnormalities, (2) identification of genes involved with the
normal growth and development of specific tissues and organs(whether a
phenotypic response is evident or not) and (3) development of lines of animals
that have marked genes whose responses to mutations in other genes can be
detected and evaluated. Transposon-mediated insertional inactivation of
chromosomal genes is a promenent genetic tool in lower organisms; however,
until we developed the Sleeping Beauty (SB) Transposon System, there were no
highly active DNA transposons available for fish. In this application ,we
proposed to extend the SB transposon system for insertional mutagenesis and
gene tagging as well as creation of zebrafish that can be used as sensors for
mutations in other genes important to proper development. We will use a variety
of transposons that contain flourescent protein genes with regulatory sequences
that will direct the expression of the flourescent proteins whenever the
transposons insert themselves into genes. As a result, investigators will be
able to pre-determine the tissue and temporal specificity of a gene that has
been tagged by one of our transposons, which will permit more directed gene
screens. Owing to its optical clarity at all times during early development,
the use of flourescent markers in transposon-trap vectors is particularly
suitable for use in zebrafish.

We propose to accomplish the following specific aims in order to achieve the
above goals: 1) Evaluate the efficiencies of trapping' genes with
transposon-trap vecotrs. 2) Develop SB transposon traps with greater
sensitivities for detection of genes expressing low levels of transcripts. 3)
Establish lines of zebrafish that express SB transposase to improve efficiency
of transposon tagging. 4) Measure the efficiencies of local transposition in
lines of zebratish that already contain transposon-trap vectors. 5) Develop
lines of zebrafish that have genes containing the transposon-traps for further
mapping of genetic pathways in zebrafish.
StatusFinished
Effective start/end date5/1/016/30/14

ASJC

  • Medicine(all)