Splinkerette PCR for mapping transposable elements in Drosophila.
Splinkerette PCR for mapping transposable elements in Drosophila.
复制标题
DOI：
10.1371/journal.pone.0010168
复制
发表时间：
2010-04-13
期刊：
影响因子：
3.7
通讯作者：
Luo L
中科院分区：
文献类型：
--
作者：
Potter CJ;Luo L
转座因子（如P-因子和piggyBac）已被用于将数千个转基因构建体导入果蝇基因组。这些转基因构建体具有许多作用，从测定基因/细胞功能到控制染色体臂重排。通常需要知道转座因子的精确基因组插入位点。这使得能够鉴定由增强子陷阱捕获的基因组增强子区域，鉴定由转座子插入突变的基因，或简化重组实验。最常用的转基因定位方法是反向PCR（iPCR）。虽然iPCR通常是有效的，但其局限性阻碍了其分离复杂基因组基因座（如含有天然转座子的基因座）中侧翼基因组DNA的能力。在这里，我们报告了splinkerette PCR（spPCR）方法的适应性，用于分离任何P元件或piggyBac的侧翼基因组DNA。我们报告一个简单而详细的协议spPCR。我们使用spPCR来1）定位位于天然转座子内的GAL 4增强子陷阱，精确定位大脑中嗅觉神经元表达的主调控区;和2）定位所有常用的着丝粒FRT插入位点。spPCR的简便、高效和有效性使其成为果蝇转座因子定位的有利选择。
Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.
登录
查看更多内容
影响因子：
56.9
作者：
COOLEY, L;KELLEY, R;SPRADLING, A
通讯作者：
SPRADLING, A
影响因子：
25
作者：
Liu, Xu;Davis, Ronald L.
通讯作者：
Davis, Ronald L.
影响因子：
4
作者：
Bharathi, V;Pallavi, SK;Shashidhara, LS
通讯作者：
Shashidhara, LS
影响因子：
14.8
作者：
O'Malley, Ronan C.;Alonso, Jose M.;Ecker, Joseph R.
通讯作者：
Ecker, Joseph R.
影响因子：
30.8
作者：
Markstein, Michele;Pitsouli, Chrysoula;Perrimon, Norbert
通讯作者：
Perrimon, Norbert