Larsen Camilla, Franch-Marro Xavier, Hartenstein Volker, Alexandre Cyrille, Vincent Jean-Paul
University of California, Life Sciences Building 4214, Los Angeles, CA 90025, USA.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17813-7. doi: 10.1073/pnas.0607652103. Epub 2006 Nov 8.
Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain- and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit.
转座元件已被用于果蝇中以检测基因表达、使基因功能失活,并诱导异位表达或过表达。我们已将所有这些特性整合到一个单一构建体中。当该构建体插入转录单元内时,无启动子的GAL4 cDNA会表达,并且GAL4会激活存在于同一转座子中的编码GFP的基因。在初次筛选中,在不育雄性的活体后代中,可将有模式的基因表达检测为GFP荧光。只需相对较少的努力就能获得许多以不同模式表达GFP的动物。正如预期的那样,许多插入会导致功能丧失。在基因组位置插入后,转座子的特定部分可被FLP重组酶切除,从而使其能够诱导标记基因的条件性错误表达。因此,通过在根据其表达模式鉴定出的基因中的单次插入,就可以进行功能获得和功能丧失研究。使用这种启动子陷阱方法,我们鉴定出了一组支配蘑菇体萼的细胞,因此可能定义了一个以前未被识别的记忆回路。