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An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.
One powerful approach for gene function studies is to microinject in vitro transcribed capped RNA in zebrafish embryos. Capped RNA behaves similarly to eukaryotic mRNAs found in vivo due to the presence of the CAP analog. Zebrafish researchers routinely utilize this method to overexpress or misexpress their gene of interest. In this demonstration, we will microinject an EGFP-tagged transcript and use live whole-embryo GFP-expression as a visible readout for a successful injection.
Morpholino antisense oligonucleotides are widely used to modify gene expression by blocking translation of a targeted protein or by modifying pre-mRNA splicing 2,3. Morpholinos in the zebrafish serve as a powerful reverse genetics tool by knocking down gene function. In this article, we will microinject a morpholino oligo targeted to the translational initiation site of pkd2 (5′-AGGACGAACGCGACTGGAGCTCATC-3′). Based on previously established work, we expect the injected embryos to phenotypically mimic pkd2 mutant fish4.
EGFP mRNA overexpression: To verify the success of the microinjection, we will monitor the expression of GFP in the developing embryos beginning at shield stage (~6 hpf) by in vivo whole-embryo fluorescence microscopy (Leica Microsystems GmBh, MZFLIII). Expression of the construct is most strong during the early events of embryonic development and will often diminish during development as the injected capped RNA is gradually degraded and depends on the stability of the expressed protein.
pkd2 translational-blocking morpholino: Based on previously published results, we expect the pkd2 morphants to mimic the dorsal body axis curvature as found in pkd2 mutant zebrafish and present kidney cysts at approximately 2 - 3 dpf4.
Figure 1: Representative results from microinjection of EGFP mRNA and pkd2 AUG morpholino. (a) TAB embryos were microinjected at the 1-cell stage with 0.17 ng of EGFP mRNA and visualized at 1 dpf for in vivo GFP expression. (b,c) TAB embryos were microinjected at the 1-cell stage with ~1.7 nl of a pkd2 translational blocking morpholino at 0.50 mM and scored for dorsal body curvature at 4 dpf.
Microinjection into zebrafish embryos is a well-established and robust technique for exploring the role of a particular gene in development. Applications include overexpression, misexpression, and knockdown assays of your gene of interest as well as epistatic analysis between multiple genes. Microinjection in zebrafish has been widely used for generating transgenic animals, and mapping cell fate in early blastula embryos5,6,7,8,9. In addition, the application of this technique serves as a key step in generating germ-line chimeras by cell transplantation methods10.
One alternative method for exploring a gene’s ‘gain-of-function’ phenotype is to microinject constitutively active forms of that gene. In addition, a gene’s pseudo ‘loss-of-function’ phenotype may be explored by microinjection of dominant negative forms of that gene. Microinjection into zebrafish embryos may also include DNA and small molecules11,12.
A critical step in this microinjection technique is the quality of the micropipette needle. We make our micropipette from capillary tubes shaped by a pipette puller device. It is essential that the pipette puller is properly calibrated to yield optimal needle shape and size. Micropipettes that are too long and narrow often lack rigidity, break easily and struggle to penetrate the chorion and yolk. Short micropipettes are often more prone to damaging the embryo during microinjection.
This work was supported by the NIH and PKD foundation to ZS. All animal experiments were conducted according to Yale Animal Resources Center (YARC) and Institutional Animal Care and Use Committee (IACUC) guidelines.