In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish
miRNA constructs and their effects in zebrafish embryos. (a) Schematic of the miRNA and the eGFP sensor vectors showing the miRNA target sites as well as the qRT-PCR primer locations. The miRNA vector contains a CMV promoter for ubiquitous expression in all tissues, and a DsRed Express reporter gene and the pre-miRNAs. The pre-miRNAs can be single (only one miRNA) or chained (containing more than one miRNA under the same promoter). The eGPF sensor miRNA also contains a CMV promoter, an eGFP reporter gene, and the mature miRNA target at the 3′ UTR of the eGFP gene. The miRNA target sites for zerg-2 are located in exons 9 (miRNA 1), 14 no. 1 (miRNA 2), 14 no. 2 (miRNA 3), and 3′ UTR (miRNA 4). The miRNA targets for zerg-3 are located in exon 8 (miRNA 1), exon 15 (miRNA 2), and two targets located on the 3′ UTR. (b) The injection of eGFP sensor mRNA alone produces embryos that exhibit green fluorescence only, and embryos injected with both eGFP sensor and miRNA mRNA exhibit both green and red fluorescence. (c) An example of embryos injected with either miRNA with eGFP sensor or scrambled miRNA (negative control) with eGFP sensor. The * indicates the presence of the midbrain hindbrain boundary.