This thesis describes my work to develop methods and assays to study transgenerational inheritance in the widely used genetic model organism Caenorhabditis elegans (C. elegans). In the first chapter, I describe a novel method that uses an exogenous histamine-selective chloride channel (HisCl1) for negative selection in transgenesis. C. elegans transgenesis is a core technique used by most laboratories and often requires distinguishing between rare animals with a single-copy transgene inserted into the genome from more frequent animals that carry multiple copies of the transgene in extra-chromosomal arrays. I demonstrate that histamine-selection induces rapid and irreversible paralysis in only array animals thus allowing quick identification of the desired transgenic animals.
In the second chapter, I develop a high-throughput assay for quantifying transgenerational epigenetic inheritance of endogenous gene silencing. Small RNA -mediated gene silencing leads to an increased incidence of males in the population which can be inherited for four to six generations. I identify a fluorescent marker that specifically fluoresces in males and show that I can use a large-particle particle sorter to quantify the frequency of males in a population. This automated system will allow me to follow
inheritance patterns over at least ten generations in various mutant backgrounds in parallel to determine the genetic basis and the rules of epigenetic inheritance.
|Date of Award
- Biological, Environmental Sciences and Engineering
|Christian Froekjaer Jensen (Supervisor)
- Transgenerational Inheritance
- C. elegans
- Synthetic Biology