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A major strength of zebrafish as a model organism is their rapid, in vitro development. The easy access to embryos compared to mammals, allows larval molecular and cellular composition to be manipulated by microinjection, providing a powerful avenue for biological and translational studies. Here, we describe the essential steps and different applications of microinjection in zebrafish for genome editing and functional studies, along with some experimental tips that are critical for microinjection success.

Binding of transcription factors to promoters is a necessary step to initiate transcription. From an evolutionary standpoint, the regulatory proteins and their binding sites are considered to have molecularly coevolved. We developed an efficient yeast strategy, an "inverse one-hybrid system", to identify binding targets of transcription factors globally in a genome of interest. The technique consists of a yeast strain expressing a -transcription factor of interest mated to yeast containing a library of random genomic fragments cloned upstream of a reporter gene (URA3). Positive growth on media without uracil denotes a fragment being bound by the transcription factor, e.g., zebrafish FoxI1. The bound fragments in hundreds of positive clones are sequenced and retested for their binding activities using a colony PCR and sequencing strategy. The resulting tools allow for rapid and genomic-wide identification of transcriptional binding targets.

This chapter describes a method for generation of the high-titer pseudotyped Moloney murine leukemia virus (MLV) that efficiently infects zebrafish embryos (i.e., more than 25 retroviral copies per cell). Injection techniques are also described for production of the retrovirus-infected mosaic "founder" fish. We describe a quantitative PCR (qPCR)-based assay as a quick way to assess the infectivity after each round of viral production and injection. Most of the required equipment is commercially available and commonly present in most research laboratories.