ER intrabody mediated protein knockdown in zebrafish

Abstract

Gene knockout and knockdown approaches have been extensively used in zebrafish to study genes playing a pivotal role during the development, or to create animal models of human diseases. In the past ten years, evidence has accumulated that using different loss-of-function methods, acting at DNA or RNA level to interfere with the expression of a specific gene may often result in the generation of completely independent phenotypes. Genetic compensation on one side and off-targeting effects on the other have been identified as main contributors to this phenomenon. The ER intrabody knockdown approach, acting at protein level with high specificity, is intrinsically less prone to such drawbacks, and additionally offers a higher level of protein isoform discrimination. In this study, we describe the process of antibody validation for highly specific protein identification in different assays and for targeted protein knockdown in vitro and in vivo of zebrafish Cadherin-2 (Cdh2). We have generated 11 novel sequence-defined human scFv antibodies against zebrafish Cdh2 via phage display. Cdh2 parachute mutant and Cdh2-GFP transgenic lines were used to compare antibody specificity in immunofluorescent staining and flow cytometry analysis, as well as whole mount immunohistochemistry. Four of these recombinant monoclonal antibodies showed drastically improved specificity against zCdh2 compared to the only commercial available reagent, which was found highly cross-reactive towards zCdh2-- cells, independently from the type of sample preparation. Further, we generated ER intrabodies to zCdh2 via genetically fusing the newly generated scFv antibody genes to the DNA encoding for the ER localization KDEL peptide. We evaluated ER intrabodies‘ capability to mediate zCdh2 knockdown from the surface of PAC2 cells and to induce target antigen degradation. In vivo, the ER intrabody SH1352-D7-KDEL expressed in atoh1a+ progenitors of zebrafish embryos was shown to interfere with tegmental hindbrain nuclei primordia formation. Our validated protein knockdown approach constitutes an expansion of the available zebrafish tool box for gene function studies and for modeling human diseases in this organism model. We indirectly proved that protein based approaches can have a beneficial impact on the zebrafish community, provided that the process of generation of these tools is subjected to an extensive and thoughtful characterization processes.