*Please note that this project can be completed as a PhD or MPhil*
Normal sperm production and function is critical for reproduction and thus species survival. An important aspect of reproduction is the performance of sperm once they enter the female reproductive tract - they are exposed to a hostile environment (females make spermicides), and have to compete with sperm from other males to achieve fertilisation of the egg. In many species this has resulted in the males making two or more different types of sperm. One type is capable of fertilisation, while the other(s) act as helpers, by counteracting the spermicide and by dislodging sperm from other males. This is an important evolutionary adaptation, but leaves us as molecular biologists wondering "how do the males manage to make these different sperm types?".
Drosophila pseudoobscura males make three types of sperm (two short, one long), of which just one (long) is capable of fertilising eggs. We have identified a set of genes that are expressed more highly in precursors of either long or short sperm, as well as some regulators that probably activate and/or repress these genes in the appropriate cells.
We are exploiting the CRISPR-CAS9 system for genome editing in D. pseudoobscura, to insert tags onto proteins of interest and thus investigate protein localisation and function, or to make knock out lines. We are currently testing this system, and also using conventional transgeneis to generate lines expressing tagged proteins. You will investigate the function of these factors – what target sites do they bind in the genome (ChIP-seq with tagged proteins); what is the effect of mis-expressing them in the “wrong” cells (transgenics, single cell analysis); what is the effect of mutating them (CRISPR knock outs followed by RNAseq)?
Drosophila genetics, including injection and generation of transgenics.
Molecular biology – PCR, Q-PCR, RT-PCR, cloning, CRISPR construct designs and application.
Cell biology – in situ hybridisation, immunostaining, cytology.
RNAseq and ChIP-seq, including bioinformatics analysis.