Microfluidic devices for early (less than 48 hpf), non-destructive zebrafish genotyping

Project Summary 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 hpf hpf 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 design and fabricate a device for collection of DNA samples from the chorionic fluid of zebrafish embryos and design and fabricate an integrated device that extracts DNA from zebrafish chorions and directly analyzes the DNA for mutations and 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 embryos are kept alive after DNA collection and analysis genotyping is performed early in the life cycle of the embryo saving time and costs and the processes are semi automated and are efficient for high throughput studies 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