Edholm Eva-Stina, Robert Jacques
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14620
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14620.
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):pdb.prot101519. doi: 10.1101/pdb.prot101519.
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.
通过将外源DNA稳定整合到基因组中来生成转基因青蛙的方法在[具体物种]中已得到充分确立。本方案描述了转基因技术与稳定RNA干扰相结合的方法，作为一种在[具体物种]中研究基因功能的有效反向遗传学方法。这种转基因方法最初是在青鳉鱼中开发的，后来适用于[具体物种]，它使用I-SceI巨核酸酶，一种具有18bp识别序列的“稀有切割”内切核酸酶。在本方案中，使用双启动子表达盒来生成特定基因表达被敲低的转基因[具体物种]。该表达盒两侧是I-SceI识别位点，包含在人U6启动子控制下的所选shRNA和在人EF-1α启动子控制下的绿色荧光蛋白（GFP）报告基因。在显微注射前，用I-SceI消化使质粒线性化，然后将整个反应显微注射到单细胞期的卵中。I-SceI高度严格的识别序列被认为能使线性化质粒保持非串联状态，这有利于质粒转基因在基因组中的随机整合。注射后的胚胎饲养至幼虫56期，然后通过荧光显微镜筛选GFP表达，并使用尾部活检通过定量RT-PCR评估有效敲低情况。通常，I-SceI巨核酸酶转基因技术的转基因效率为35%-50%，存活率高（>35%）且GFP表达明亮且无镶嵌现象。该技术的一个关键优势是，高效率和非镶嵌转基因表达允许直接使用F代动物。
