Discipline: Ecology Environmental and Earth Sciences
Subcategory: Cell and Molecular Biology
Viveca M. Vélez-Negrón - University of Puerto Rico-Rio Piedras Campus, PR
Co-Author(s): Luis R. Col?n-Cruz, University of Puerto Rico-Medical Sciences Campus, PR; Roberto E. Rodr?guez-Morales, University of Puerto Rico-Medical Sciences Campus, PR and Martine L. Behra, University of Puerto Rico-Medical Sciences Campus, PR
Rapid and cost-effective screens for waterborne pollutants that affect the flora and fauna are needed. We propose to use the larval zebrafish lateral line (LL), which is a superficial sensory system made of stereotypically distributed sensory patches named neuromasts (NMs) which are mainly composed of highly sensitive mechanoreceptors called the hair cells (HCs). LL-HCs, were previously presented as a toxicological endpoint, but we proposed to assess how their regeneration is affected. Our laboratory has generated loss-of-function mutant alleles for several genes susceptible to interfere with proper HC regeneration using CRISPR-Cas9 genome technology. To identify alleles of interest and grow stable mutant lines we have to genotype the potential heterozygote or homozygote carriers. To do so, we prepare genomic DNA (gDNA) from fin clips of juvenile and adult fish or whole embryos and larvae. We designed PCR primers to amplify the targeted region in each gene and sequence the amplicons looking for insertions or deletions (INDELS) creating a translational frame shift and eventually introducing an early stop codon. So far, we have selected several alleles for 4 different genes. Three out of 4 lines are homozygote viable, meaning that unchallenged animals which are completely lacking the given gene products, are developing into fertile adults. Both, adult fish and larvae from those 3 lines will be ideal for testing water quality.
Funder Acknowledgement(s): This work was supported by a grant from PRCEN-CREST, #534034.
Faculty Advisor: Martine L. Behra, email@example.com
Role: Through the time I have been working at the laboratory, I have participated mainly in the extraction of genomic DNA by doing fin clipping and performing PCR protocol. Also, maintaining our mutant lines healthy and growing.