Figure 1

Figure 1

Approaches for Genetic Manipulation of Zebrafish. (A) Injection of constructs and chemicals in zebrafish eggs. Transient depletion can be performed by injection of morpholino oligonucleotides, RNA-binding oligomers that block translation/maturation of a specific (pre)-mRNA. Morpholinos can sometimes elicit off-target effects, therefore, it is important to validate phenotypes using alternative strategies and/or rescue experiments before conclusions can be fully drawn. Transient expression of genes can be obtained by injection of synthesized mRNA or plasmid DNA bearing an expression construct. Injected mRNAs will be expressed ubiquitously, while injection of plasmids enables cell- or tissue-specific expression. Zebrafish eggs can stably integrate DNA, which can be used to obtain stable transgenic lines or insertional mutants. The frequency of transgenesis is low when injecting DNA alone, but can be increased using transposases (i.e., Tol2) or meganucleases (i.e., I-SceI meganuclease). Zebrafish stable mutants can be efficiently generated with ZFNs, TALENs, or CRISPR/Cas9. These systems are based on induction of a site-specific double-stranded break, which is repaired via an error-prone non-homologous end joining mechanism. The CRISPR/Cas9 system has recently become the most common method to generate zebrafish mutants. Additionally, the CRISPR/Cas9 system has also been adapted to generate conditional/tissue-specific knockouts. Mutants are obtained by injecting mRNA or protein for the nuclease (together with guide RNA in the case of CRISPR/Cas9) in zebrafish eggs. Conditional/tissue-specific mutants are obtained by integration of a construct where Cas9 expression is controlled by an inducible or tissue-specific promoter. DNA constructs for stable integration can be designed with flanking homology recombination arms, which drive integration into a precise locus, and allow generation of knock-in lines. Generation of precise knock-in zebrafish is still challenging but can be facilitated by introducing double strand breaks at the site of interest (i.e., using TALENs or CRISPR/Cas9) [6]. Embryos manipulated using these techniques can be used for downstream functional studies, or, in the case of stable modifications, be raised to adulthood to establish a novel line. (B) Selection of stable transgenic or mutant lines. Carriers from (A) can be outcrossed to obtain heterozygote carriers. These offspring can be used for experiments, or raised to adulthood and inbred (i.e., to obtain homozygotes). (C) Systematic mutagenesis. Large libraries of random mutations can be obtained by exposure of sperm to chemical or physical mutagens (i.e., ENU or γ-radiation) prior to in vitro fertilization. Abbreviations: CRISPR/Cas9, clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9; ENU, N-ethyl N-nitrosurea; TALEN, transcription activator-like effector nuclease; ZFN, zinc-finger nuclease.