This proposal describes a project to ascertain the potential for using zinc finger nuclease-mediated gene targeting in the unicellular green alga Chlamydomonas. The process of intraflagellar transport (IFT) was first described in Chlamydomonas, and much of our knowledge of the proteins required for IFT comes from studies of the assembly of Chlamydomonas flagella. Defects in IFT have been directly associated with processes required for kidney development, for establishment of laterality and the vertebrate body plan, for the assembly of rod cells in the retina, and many other key processes in mammalian development and cell function. The continued dissection of the machinery of IFT in a simple model system like Chlamydomonas will help inform our understanding of IFT processes in higher eukaryotes. This project combines two new and very promising technical approaches: iTRAQ, a mass spectrometry-based technique to identify the relative abundance of individual proteins in complex mixtures;and gene targeting using zinc finger nucleases (ZFNs) to generate mutations in genes of interest. We have identified a series of novel gene products in Chlamydomonas flagella that are likely to be involved in IFT, but they have not been identified as IFT components by standard biochemical approaches. We seek to examine a potential role for these proteins by creating directed gene knockouts using ZFNs. In this procedure, artificial zinc finger proteins designed to bind to a sequence in a target gene are attached to a Fok1 nuclease domain, and when this fusion protein is expressed in cells, cleavage of the target sequence in the genome leads to gene disruption. We propose to first, demonstrate the feasibility of using this technique in Chlamydomonas by disrupting and making modifications in the nitrate reductase gene;and second, to determine whether three newly identified potential IFT proteins are required for flagellar assembly.
The research described in this proposal will make the unicellular green algae Chlamydomonas an even more useful system for uncovering fundamental components of the intraflagellar transport system. The discovery of IFT in Chlamydomonas has led to a greatly enhanced understanding of basic biological process that underlies human diseases such as polycystic kidney disease, retinal degeneration, Bardet-Biedl syndrome, and many other inherited disorders. The research in this project will likely uncover more basic knowledge about IFT that can be applied to human diseases.
|Tam, Lai-Wa; Ranum, Paul T; Lefebvre, Paul A (2013) CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas. Mol Biol Cell 24:588-600|