RNAi-Mediated Loss of Function of Immune Genes by Transgenesis.

Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Initially developed in the fish medaka and later adapted to , this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic with knocked down expression of a specific gene are generated using a double promoter expression cassette. This cassette, which is flanked by I-SceI recognition sites, contains the shRNA of choice under the control of the human U6 promoter and a green fluorescent protein (GFP) reporter gene under the control of the human EF-1α promoter. Prior to microinjection the plasmid is linearized by digestion with I-SceI and the entire reaction is then microinjected into one-cell stage eggs. The highly stringent recognition sequence of I-SceI is thought to maintain the linearized plasmid in a nonconcatamerized state, which promotes random integration of the plasmid transgene in the genome. The injected embryos are reared until larval stage 56 and then screened for GFP expression by fluorescence microscopy and assessed for effective knockdown by quantitative RT-PCR using a tail biopsy. Typically, the I-SceI meganuclease transgenesis technique results in 35%-50% transgenesis efficiency, a high survival rate (>35%) and bright nonmosaic GFP expression. A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F animals.