Zebrafish Functional Genomics Development at UW-Stout

Since the completion of the human genome sequencing project, morpholino phosphorodiamidate oligonucleotide (MO) knockdown in zebrafish has been increasingly used to elucidate human gene function. As part of the effort to expand the functional genomics screening capacity at the University of Wisconsin-Stout, this project investigated MO microinjection techniques, embryo production, and brine shrimp survival. Oneto two-cell embryos injected with the pigmentinhibiting tyrosinase MO were observed at 48 hours post fertilization for pigmentation. Injection efficiency was calculated by dividing the number of zebrafish lacking pigment by the total number of injected zebrafish. To obtain sufficient embryos for MO experiments, the effects of a dry food diet and live (brine shrimp) food diet on embryo production were analyzed. Effects of a yeast diet on brine shrimp survival were also studied to provide zebrafish with healthy brine shrimp. Results included a 92% injection efficiency, greater embryo production with the dry food diet, and increased brine shrimp survival with a yeast diet. This work provided an important foundation in the development of a reverse-genetic screen for future students. Introduction The sequencing of the human genome has yielded thousands of potential genes with unknown function (HGMIS, 2005). Due to the similarities in genetic composition across species and technological advancements, the functions of these genes can be determined relatively quickly by screening them in model organisms, such as the zebrafish (Pickart et al., 2004; Dahm & Geisler, 2006). Common screening tools for deciphering gene function include mutagenesis and forwardand reverse-genetic screens. Mutagenesis screens mutate genes and determine gene function based on what is observed (the phenotype). Forward-genetic screens generally consist of exposing the organism to a chemical, observing the phenotype, and ascertaining the gene affected. Reverse-genetic screens utilize agents such as morpholino phosphorodiamidate oligonucleotides (MOs) to alter specific gene expression and phenotype and provide insight to gene function. All three of these methods have been used in zebrafish. With the genome of the zebrafish approximately 70% sequenced (TDRSP, 2006), MO technology is becoming a popular antisense tool (targets the matching nucleic acid sense strand). MOs are synthetically-made, neutral, nucleic acid analogs that can be ordered online from Gene Tools, LCC (Philomath, OR) (Pickart et al., 2006; Deiters & Yoder, 2006). Structurally, MOs have a morpholine group in place of the ribose sugar and a phosphorodiamidate (not phosphodiester) backbone. As a result of these properties, MOs are soluble, bind to specific messenger ribonucleic acid (mRNA) sequences via Watson-Crick base pairing, and are not degraded as easily as some antisense mechanisms. Functionally, MOs work by binding to and Zebrafish Functional Genomics Development preventing mRNA from translating into protein, thereby inhibiting gene expression. The ~25 nucleotide-long sequence of a MO is designed to bind to the 5' end of the mRNA (to block cellular translational machinery) and, thus, generally contains the translational start codon AUG. Alternatively, MOs can be designed to bind to splice sites to alter or prevent intron splicing, which inhibits the production of normal protein. Gene sequence information for MO design can be obtained from such websites as The Zebrafish Information Network (www.zfin.org), Ensembl Zebrafish (www.ensembl.org/Danio_rerio), and others at Zebrafish Genome Resources (www.ncbi.nlm.nih.gov/genome/guide/zebrafish). Once designed, MOs can be microinjected into an embryo (typically at the one to two-cell stage) and the effects analyzed hours to days afterward. The ability to quickly assess gene function is one of the many advantages of using zebrafish. A native to the Ganges River in India, the ~2-inch black-striped zebrafish is an emerging model organism (Badman et al., n. d.). Named Danio rerio in 1822 by Francis HamiltonBuchanan, the zebrafish’s potential in genetic research was not recognized until the 1970s by the viral geneticist and fish hobbyist George Streisinger, who highlighted the advantages of zebrafish in the journal Nature (Dahm, 2006). Indeed, the zebrafish has many favorable attributes (Badman et al.). They are easy to maintain at low labor and cost. Each female can produce hundreds of eggs per week, which allows for plentiful data collection. The embryos (eggs fertilized by sperm) develop outside the females in transparent sac-like chorions, so their development can be readily observed. Easily manipulated embryos allow such technologies as MO knockdown to determine gene function. Compared to a human gestation of nine months, zebrafish’s development time of 48 hours equates to quick analysis. Importantly, the conservation of genes and biological processes between humans and zebrafish increases the relevancy of data collected. Research fields utilizing the zebrafish are very diverse, from the study of organ development (organogenesis), nerves, blood vessels (angiogenesis and vasculargenesis), and bone (osteogenesis) to cancer research, toxicological assays, and therapeutic drug screening (Pickart et al., 2004; Badman et al.). Today, over 500 laboratories utilize the zebrafish (Dahm), including the $10-million, 5000-square foot facility in Bethesda, Maryland that has over a half million zebrafish capacity (Agres, 2003). In spring 2005, the University of Wisconsin-Stout’s Zebrafish Laboratory was started by Assistant Professor Michael A. Pickart as part of the Genomics Technology Access Core (GTAC) facility to enable students to conduct hands-on, cutting-edge biological research. Methods for chemical screening were established that fall (Hoage, 2005). The following spring, techniques for using MO microinjection technology were developed and tested (this article). Initiated in support of UW-Stout’s growing Biotechnology curriculum and the GTAC, this project was the first student-lead effort to develop the functional genomics capacity of the Zebrafish Laboratory. As diagramed in Figure 1, the project consisted of three parts: MO microinjection techniques, embryo production, and brine shrimp (Aretemia) survival. Microinjection techniques referred to the manipulation of and efficient MO injection into zebrafish embryos. To establish microinjection techniques and measure injection efficiency, the tyrosinase MO was injected into 1to 2-cell embryos. Similar to the human disease condition oculocutaneous albinism (OCA1) characterized by a lack of pigment in the eyes, hair, and skin, the tyrosinase MO prevented the formation of the tyrosinase protein and the production of black pigment (melanin) in zebrafish (Pickart et al., 2004). Injection efficiency was easily measured by dividing the number of zebrafish lacking pigment by the total number of injected zebrafish at 48 hours post fertilization (hpf). The effects of a dry food diet and live (brine shrimp) food diet on Zebrafish Functional Genomics Development embryo production were analyzed to obtain sufficient embryos for injection experiments. To provide adequate amounts of healthy brine shrimp, the effects of a yeast diet on brine shrimp survival were studied. Ideas for the live food diet and yeast diet came from Westerfield (2000) and Cleveland et al. (1998), respectfully.