Efficient disruption of Zebrafish genes using a Gal4-containing gene trap

Area of Science:

• Genetics
• Developmental Biology
• Molecular Biology

Background:

• Zebrafish embryos are ideal for in vivo insertional mutagenesis due to external development and optical transparency.
• Fluorescent proteins aid in visualizing gene expression patterns in mutated genes.
• Gene Breaking Transposon (GBT) vectors enhance the fidelity and mutagenicity of transposon-based gene trapping.

Purpose of the Study:

• To construct and evaluate a bipartite GBT vector utilizing Gal4-VP16 as a reporter.
• To enable direct detection of gene trap events using a UAS:eGFP cassette.
• To assess the vector's efficiency in generating stable gene trap lines and identifying novel mutations.

Main Methods:

• Construction of a bipartite GBT vector with Gal4-VP16 and UAS:eGFP.
• Generation of a UAS:mRFP tester line for confirming gene trap events.
• Screening of 270 potential founders to establish gene trap lines.
• Utilizing Cre and Flp recombinases for allele reversion and cassette removal.

Main Results:

• Successfully established 41 gene trap lines from 270 screened founders.
• Identified three homozygous lethal alleles: nsf(tpl6), atp1a3a(tpl10), and flr(tpl19).
• Demonstrated successful reversion of gene trap alleles using Cre mRNA.
• Showcased the utility of the vector for detecting gene expression (e.g., nsf in pancreas) and creating specific Gal4 drivers.

Conclusions:

• The developed Gal4-containing bipartite GBT vector offers high specificity and mutagenicity.
• Gene trap alleles are revertible by Cre, enhancing experimental flexibility.
• The vector's ability to generate specific Gal4 drivers is valuable for diverse research applications.