Zebrafish is a powerful vertebrate model organism for biomedical research. However, the toolbox for zebrafish research has not kept pace with its potential for large-scale screens. In particular, the capability for genotyping currently is a time, labor, and training intensive process. Currently, embryos must either be raised to adulthood or embryos must be sacrificed to determine genotypes; mutants are difficult to genotype; and screens or drug/therapeutics trials cannot be performed on animals of known genotype until an older age. Finally, high- throughput technologies based on advances in genomic editing technology such as transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are limited by a requirement for manual screening. We propose to address this bottleneck in zebrafish research using microfluidic technology. Microfluidics, the science and technology of manipulating fluids at the microscale, is ideally geared for processing zebrafish embryos. We have designed and tested a prototype microfluidic system to rapidly genotype and sort live zebrafish embryos. The device has three sub-components: the Embryo Processor extracts chorionic fluid from zebrafish embryos aged 24hpf-48hpf; the DNA Extractor isolates DNA from the chorionic fluid; and the third sub-system performs PCR and high-resolution melt analysis (HRMA). Our specific aims for this project are to: (1) design and fabricate a device for collection of DNA samples from the chorionic fluid of zebrafish embryos; and, (2) design and fabricate an integrated device that extracts DNA from zebrafish chorions and directly analyzes the DNA for mutations; and (3) validate the integrated device by demonstrating its ability to screen a novel zebrafish mutant generated using CRISPRs. Our work meets a crucial gap in zebrafish research, and provides an essential framework for enhanced utilization of zebrafish genetics and screening potential. In addition, this is an important resource for the next- generation sequencing era, with the abundance of untested gene variants for which zebrafish mutants can provide a critical means to assay biological relevance. Our integrated system provides three advantages: (1) embryos are kept alive after DNA collection and analysis, (2) genotyping is performed early in the life cycle of the embryo, saving time and costs; and (3) the processes are semi-automated and are efficient for high-throughput studies.
Public Health Relevance Statement: Project Narrative This project will develop methods and tools for rapid processing of zebrafish embryos. Zebrafish serve as animal models for a variety of medical research projects including projects related to drug discovery for a vast array of medical applications, including specifically neurological and blood-related disorders.
Project Terms: Address; Adult; Age; aged; Alleles; Animal Model; Animals; base; behavior test; Biological Assay; Biomedical Research; Blood; Caenorhabditis elegans; Chorion; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Computer software; cost; design; Detection; Development; Devices; Disease; DNA; DNA analysis; DNA Sequence Alteration; drug discovery; drug testing; Embryo; Enzymes; Fertilization; genetic analysis; Genetic Screening; genetic variant; genome editing; Genotype; Health; high throughput screening; high throughput technology; Hour; Human Development; human disease; instrument; interest; Life; Life Cycle Stages; Liquid substance; Maintenance; Manuals; Mechanics; Medical; Medical Research; meetings; melting; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molecular; mouse development; mutant; Mutation; Neurologic; next generation sequencing; novel; novel therapeutics; Organ; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Problem Solving; Process; Protocols documentation; prototype; Research; Research Methodology; Research Personnel; Research Project Grants; Resolution; Resources; Sampling; Savings; Science; screening; Sorting - Cell Movement; Staging; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Trials; Time; tool; Training; transcription activator-like effector nucleases; Vertebrates; White Matter Disease; Work; Zebrafish; zebrafish development
