利用Minos转座子和镶嵌克隆技术筛选调节果蝇幼虫脂肪储存代谢的基因

Misregulation of fat storage is associated with obesity, diabetes II and angiocardiopathy. Therefore it is necessary to perform research on lipid metabolism. Although some genes have been reported through RNAi screen, P-element insertional mutagenesis and other technologies, screening genes for fat homeostasis is severely hindered by their disadvantages. The Minos transposon almost has no preference in Drosophila genome, so a modified Minos insertional mutagenesis ( Mi{MIC} ) library will be created under the background of fly with P-element containing FRT element. These lethal lines will be selected to identify their insertional sites in order to find out the genes mutated by inverse PCR. Furthermore, genes related to fat storage will be screened by FRT/Flp mediated mosaic clone system and Nile Red staining will be performed in the larva fat body. Lastly, the expression pattern of screened genes in different tissues will be observed due to the gene trap cassette of Mi{MIC}. I am performing various fly experiments and crosses in five years and have generated the tools used in the project. In conclusion, we have no concerns with the off-target effects derived from RNAi technology and the advantage of our screen is that mosaic clone analysis of lethal genes is directly made without wasting time and energy to recombination of lethal gene with FRT element.

脂肪储存代谢异常会导致肥胖症、2型糖尿病和心血管疾病发生，进行脂肪储存代谢机制研究是必要的。利用RNAi技术和P因子插入突变等方法已经筛选出一些与脂肪代谢有关的基因，但是由于这些技术自身的缺陷也限制了筛选。我们利用果蝇的Minos转座子几乎没有偏好性的特点，以含有FRT元件的P因子果蝇为背景，通过遗传杂交，构建Minos被改造的Mi{MIC}插入突变库；筛选出插入突变致死的果蝇，利用反向PCR方法确定插入突变影响的基因；利用FRT/Flp技术在果蝇幼虫脂肪体里产生镶嵌克隆，通过尼罗红染色筛选影响脂肪储存代谢的基因；Mi{MIC}可以标记插入基因组织表达情况，利用荧光显微镜观察插入基因在果蝇各组织里表达情况。申请人从事五年的果蝇研究，精通果蝇实验方法手段，并且已经构建了筛选所需要的工具果蝇。我们的筛选避免了RNAi筛选脱靶现象的影响,最重要的是可以直接进行致死突变体的镶嵌克隆分析。