Fuentes Fernando, Reynolds Eric, Lewellis Stephen W, Venkiteswaran Gayatri, Knaut Holger
Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016.
Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016 Kimmel Center for Stem Cell Biology, New York University Langone Medical Center, New York 10016
G3 (Bethesda). 2016 Apr 7;6(4):829-34. doi: 10.1534/g3.115.026344.
Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.
大型DNA构建体的转基因对于基因功能分析至关重要。最近，Tol2转座酶介导的转基因已成为将细菌人工染色体（BAC）DNA构建体插入斑马鱼基因组的强大工具。为了实现高效转基因，BAC构建体中的基因组DNA片段需要侧翼有Tol2转座子位点，并且构建体应包含一个转基因标记，以便于识别转基因动物。我们报告了一组质粒，其包含靶向盒，可将Tol2位点和不同的转基因标记插入BAC中。使用含有这些靶向盒的BAC，我们表明转基因效率与iTol2相同，预先选择转基因标记的表达可提高转基因率，并且BAC转基因忠实地重现内源性基因表达模式，并允许估计内源性基因表达水平。
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