Generation and Profiling of a Mosiac Zebrafish (Danio Rerio) Neurofibromatosis Type IIb Knockout Line.
Location
Village Square
Start Date
6-5-2022 12:00 PM
Department
Biology
Abstract
Neurofibromatosis Type 2 (NF2) is a genetic disorder that presents as benign peripheral nerve sheath tumors known as vestibular schwannomas (VS). Neurofibromatosis manifests as three disorders: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis. The gene encoding for NF2 is located on human chromosome 22q12.2, contains 17 exons, two splicing isoforms, and encodes for the 595 amino acid protein Merlin. Merlin is a member of the ERM protein family and has two isoforms that act as a scaffolding protein to indirectly link F-actin, transmembrane receptors, and other intracellular effectors that control cell proliferation and adhesion. The model organism zebrafish (Danio rerio) has been used to study early vertebrate development and various cancer types and investigate NF2 pathogenesis. Zebrafish lack a fully developed immune system during early larval development stages, which allow for the transplantation of tumor cells free of host rejection. Compared to human NF2, the zebrafish-encoded orthologs NF2a and NF2b have retained 79% and 72% amino acid identity, respectively, including high conservation of the signaling “blue box” motif. Previous studies used NF2a and NF2b CRISPR-mediated knockdown during embryonic development to determine zebrafish NF2 gene activity. NF2a and NF2b oligonucleotides were designed to synthesize guide RNA (gRNA) microinjected with dCas9 into 2-cell stage embryos. NF2a and NF2b were shown to have different temporal expression patterns, with NF2b expression seen at cleavage 2-cell stage through 5 days post fertilization (dpf). In addition, NF2 knockdown animals showed increased cell proliferation in the posterior blood island notochord curvature. This project aims to establish a stable mosaic NF2b knockdown line to assess the effects of long-term NF2b gene loss in zebrafish. The first step was to design NF2b CRISPR-Cas9 gRNAs to truncate the gene at either exon three or seven. These constructs were then microinjected into 2-cell stage animals to be grown into adulthood and crossed to generate stable lines. Another project aim is to analyze zebrafish NF2b developmental expression to pinpoint exact NF2a and NF2b expression timepoints. Using semi-quantitative RT-PCR, we profiled Profile NF2b and NF2a temporal expression in the dome, shield, 1-4 somites, and Prim-15 stages of development.
Faculty Sponsor
Jorge Cantu, Northeastern Illinois University
Generation and Profiling of a Mosiac Zebrafish (Danio Rerio) Neurofibromatosis Type IIb Knockout Line.
Village Square
Neurofibromatosis Type 2 (NF2) is a genetic disorder that presents as benign peripheral nerve sheath tumors known as vestibular schwannomas (VS). Neurofibromatosis manifests as three disorders: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis. The gene encoding for NF2 is located on human chromosome 22q12.2, contains 17 exons, two splicing isoforms, and encodes for the 595 amino acid protein Merlin. Merlin is a member of the ERM protein family and has two isoforms that act as a scaffolding protein to indirectly link F-actin, transmembrane receptors, and other intracellular effectors that control cell proliferation and adhesion. The model organism zebrafish (Danio rerio) has been used to study early vertebrate development and various cancer types and investigate NF2 pathogenesis. Zebrafish lack a fully developed immune system during early larval development stages, which allow for the transplantation of tumor cells free of host rejection. Compared to human NF2, the zebrafish-encoded orthologs NF2a and NF2b have retained 79% and 72% amino acid identity, respectively, including high conservation of the signaling “blue box” motif. Previous studies used NF2a and NF2b CRISPR-mediated knockdown during embryonic development to determine zebrafish NF2 gene activity. NF2a and NF2b oligonucleotides were designed to synthesize guide RNA (gRNA) microinjected with dCas9 into 2-cell stage embryos. NF2a and NF2b were shown to have different temporal expression patterns, with NF2b expression seen at cleavage 2-cell stage through 5 days post fertilization (dpf). In addition, NF2 knockdown animals showed increased cell proliferation in the posterior blood island notochord curvature. This project aims to establish a stable mosaic NF2b knockdown line to assess the effects of long-term NF2b gene loss in zebrafish. The first step was to design NF2b CRISPR-Cas9 gRNAs to truncate the gene at either exon three or seven. These constructs were then microinjected into 2-cell stage animals to be grown into adulthood and crossed to generate stable lines. Another project aim is to analyze zebrafish NF2b developmental expression to pinpoint exact NF2a and NF2b expression timepoints. Using semi-quantitative RT-PCR, we profiled Profile NF2b and NF2a temporal expression in the dome, shield, 1-4 somites, and Prim-15 stages of development.