Major: Medical Sciences
Faculty Advisor: Diana Mitchell
Identification and sequencing of zebrafish CRISPR mutants carrying Cas9-induced mutations in the apoc1 gene
From previous research we found the apoc1 gene to be one of the most highly expressed genes in microglia during retinal regeneration in zebrafish. Microglia are specialized macrophages resident in the central nervous system of vertebrates. Despite high expression by microglia, the function of apoc1 in the central nervous system is currently not well understood. Given this, and the genetic association of APOC1 and Alzheimer’s disease in humans, we decided to use CRISPR/Cas9 to generate zebrafish with mutations within this gene. The goal of this project was to identify zebrafish carrying apoc1 mutations, and to sequence the mutant alleles, from possible crispant fish previously generated by the lab. To this end, we began by extracting genomic DNA (gDNA) from possible zebrafish mutants from fin clip samples. The gDNA was then used as a template for a polymerase chain reaction with primers specific to the apoc1 gene. The PCR products were ligated into a plasmid vector, then transformed into bacteria. We selected clones that carried correct size inserts. Once plasmids were purified, they were sent out for DNA sequencing of the PCR product inserts. We used bioinformatic software to compare the returned sequences to the apoc1 wild-type gene sequence. Out of the 11 fish analyzed, we found 9 fish carried a mutant allele containing an insertion or deletion. Within these 9 fish, the mutations varied dramatically, ranging from 4 to 182 base pair deletions and 2 to 18 base pair insertions at the Cas9 target site. We are now outcrossing selected mutant carriers to propagate mutant lines and will develop genotyping assays. In the future we plan to analyze phenotypes of homozygous apoc1 mutants to gain a better understanding of the function of the apoc1 gene in the central nervous system, using zebrafish as our model.
Funding: NIH R01 EY030467, NIH Idaho INBRE program Developmental Research Project Award P20 GM103408