Howard Hughes Medical Institute, Boston, MA 02115, USA.
Stem Cell Program, Children's Hospital Boston, Boston, MA 02115, USA.
Dev Dyn. 2013 Aug;242(8):949-963. doi: 10.1002/dvdy.23989. Epub 2013 Jul 3.
Linear DNA-based and Tol2-mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long-term transgene stability unpredictable with variable expression. Targeted, site-directed transgene integration into pre-determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site-directed transgenesis.
We report the implementation of the phiC31 integrase-mediated attP/attB recombination for site-directed zebrafish transgenics of attB-containing transgene vectors into single genomic attP landing sites. We generated Tol2-based single-insertion attP transgenic lines and established their performance in phiC31 integrase-catalyzed integration of an attB-containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue-specific transgene applications as well as long-term stability of phiC31-mediated transgenes.
Our results establish phiC31 integrase-controlled site-directed transgenesis into single, genomic attP sites as space-, time-, and labor-efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community.
基于线性 DNA 的转染和 Tol2 介导的转染是生成转基因斑马鱼的强大工具。然而，多个拷贝或随机整合在基因组位置的转基因会使比较转基因分析复杂化，并使长期转基因稳定性变得不可预测，其表达情况也会有所不同。靶向、定点转基因整合到预定的基因组位置可以避免这些问题。phiC31 整合酶催化异源 attP 和 attB 位点之间的单向重组反应，是定点转基因的有效平台。
我们报告了 phiC31 整合酶介导的 attP/attB 重组在定向斑马鱼转基因中的应用，将含有 attB 的转基因载体靶向整合到单个基因组 attP 着陆位点。我们生成了基于 Tol2 的单个插入 attP 转基因系，并建立了它们在 phiC31 整合酶催化的 attB 含有转基因载体整合中的性能。我们发现，在所有测试的动物中，有 34%的动物能够稳定地将 attB 报告基因传递给下一代。我们进一步对两个功能 attP 着陆位点系进行了特征描述，并确定了它们的基因组位置。我们的实验还证明了组织特异性转基因的应用以及 phiC31 介导的转基因的长期稳定性。
我们的结果建立了 phiC31 整合酶控制的靶向转基因到单个基因组 attP 位点的空间、时间和劳动力高效的斑马鱼转基因技术。所描述的试剂可用于分配给斑马鱼社区。
