352— Investigating the function of MHC Class I in Xenopus laevis tadpoles

Xenopus laevis, is an excellent model organism for transgenesis and immunological research due to their large eggs and the similarities between mammalian and amphibian immune systems. MHC Class I is found on nearly all cells and educates T cells to recognize between self and non-self which is an essential immune system function. However, tadpoles don’t have detectable MHC Class I expression while adult frogs do. Despite this tadpoles are still immunocompetent; thus the role of MHC Class I in tadpoles is unknown. To investigate MHC Class I function we inactivated the corresponding gene in Xenopus laevis using CRISPR/Cas9. The CRISPR/Cas9 gene editing system has two components; a guide RNA that targets a specific DNA sequence and the Cas9 protein that cuts the DNA. After the DNA is cut, the cell repairs the DNA break, which introduces mutations that theoretically inactivate this gene. Previously, we generated transgenic tadpoles with potentially inactivated MHC Class I genes, and we are in the process of determining the success of our transgenesis. To detect MHC Class I inactivation we will use PCR to amplify the MHC class I gene from genomic DNA of the transgenic tadpoles which will then be sent for sequencing.