Retroviral-mediated Insertional Mutagenesis in Zebrafish

Since the initial publication of this chapter in 2004, additional methodologies have been developed which could improve and/or complement the original retroviral-mediated insertional mutagenesis. Retroviral vectors have also been shown to be useful for goals other than mutagenesis. In addition, retroviral-mediated insertional mutagenesis has been applied to zebrafish for use in reverse genetics as well as forward screening. Finally, the insertional mutant collection described herein has been screened by a number of labs to find a host of mutants (with genes already identified) with developmental and/or growth defects affecting the eye, liver, skin, craniofacial skeleton, kidney, myeloid cells, hematopoietic stem cells, and axon pathfinding, as well as mutants with defects in the cell cycle or DNA damage response, altered aging properties, and modulated cardiac repolarization. The major complementary approaches and new uses of this technique include: Pseudotyped retroviruses have been used to deliver enhancer trap vectors, which allows selection of insertions in or near genes with particular expression patterns. Although the mutagenicity of these vectors has yet to be determined, they are useful purely because they generate a large number of transgenic lines with visible reporters (e.g., Green Fluorescent Protein GFP) expressed in interesting patterns and they provide information regarding gene regulation in the context of genomic organization. Gain-of-function vectors have been designed to allow for dominant genetic screens. Thus, genes whose overexpression results in phenotypes of interest can be efficiently identified. Retroviruses can be used to make a library of insertions in which hundreds of thousands of mapped insertions can be recovered from frozen sperm samples. Such libraries could serve as on-the-shelf reverse genetic resources, whereby one can obtain a mutation in nearly any gene by simply recovering an insertion in that gene from the frozen sperm, similar to the use of gene-trap insertions and genome-wide gene targeting in ES cells in the mouse. Transposons have been shown to be nearly as effective transgenesis vectors as retroviruses and thus may be used in similar screens - both for mutagenicity and gene traps and enhancer traps. It is possible that retroviruses and transposons could have different insertion site biases, making them important complementary technologies for genome-wide screening.