Characterizing the Role of the AATF Gene in Apoptosis Using the Model Organism Crithidia fasciculata

Jackqueline Ardo and Dr. Swati Agrawal, Department of Biological Sciences, University of Mary Washington, 1301 College Ave, Fredericksburg, VA 22401

Kinetoplastids are unicellular eukaryotic parasites that include many species that infect humans like African trypanosomes the causative agent of African sleeping sickness and Leishmania the causative agent of leishmaniasis. Crithidia fasciculata is a kinetoplastid parasite which does not infect humans however its biology and genome is very similar to the parhogenic parasites, therefore making it an excellent model organism. Using the C. fasciculata, applicable findings can be made to other kinetoplastids including how they cause disease and identification of drug targets. It is speculated that apoptosis, or programmed cell death, helps regulate the parasite infection through “selfish altruism”, the favoring of the fittest parasite for survival and transmission, in order to successfully continue infection in the host and vector. In mammalian apoptosis, the gene AATF has been shown to be a regulator of p53 activating cell death. We have identified an AATF homolog that is conserved in all kinetoplastids. The purpose of this project is to understand the role of AATF in apoptosis using C. Fasciculata as a model system for Kinetoplastids. We are working on characterizing the gene function using tagging and knock out studies that utilize CRISPR-Cas9 tools to modify the gene locus. We are currently generating sgRNA and the drug repair cassette. The drug repair cassette is created using Fusion PCR that combines a 500 bp homology arms on either side of the gene and a drug resistance marker (Puromycin or Blasticidin). We have successfully amplified the three segments for tagging constructs and are currently working on combining them in a Fusion PCR reaction. Future work will focus on transfection of these constructs into C. fasciculata followed by drug selection and screening of successful transgenic parasites.

Additional Abstract Information

Presenter: Jackqueline Ardo

Institution: University of Mary Washington

Type: Poster

Subject: Biology

Status: Approved

Time and Location

Session: Poster 3
Date/Time: Mon 4:30pm-5:30pm
Session Number: 3156