Genetics, Development and Cell Biology Ph.D. candidate — Jordan M. Welker

Major professor — Jeff Essner

Major — Interdepartmental Genetics and Genomics

Title — Reverse genetics using precise exogenous DNA integration In zebrafish

Abstract — Directional nodal flow initiated by motile cilia in the left-right (L-R) organizer of developing embryos is required for proper establishment of L-R asymmetry. Previous work in Zebrafish has shown that morpholino knockout of the gap junction (GJ) protein, connexin43.4 (cx43.4), causes disruption of the L-R organizer, Kupffer’s Vesicle (KV), and correlated randomization of L-R asymmetry (Hatler et al., 2009). Here we present phenotype analysis of an indel (10 bp deletion) mutant of cx43.4 showing that cx43.4 is required for KV inflation but is not solely responsible for proper L-R designation. We also present an application for the GeneWeld precise exogenous DNA integration strategy (Wierson et al., 2018) using the pPRISM plasmid series (Welker et al., in prep) to generate visually traceable alleles including a 2A-RFP knock-in, and cDNA knock-in alleles. We show that a cx43.4 2A-RFP knock-in allele phenocopies the indel allele, and that a hemichannel cDNA knock-in allele (cDNA containing a SNP to disable the ability of the GJ to dock with other GJs) is sufficient for partial inflation of KV.