人载脂蛋白E7转基因小鼠肾脏cDNA削减文库与cDNA文库

The cDNA Subtractive Library and cDNA Library of the Kidney of Human Apolipoprotein E7 Transgenic Mice

目的 以人apoE7基因制备的高脂血症转基因小鼠模型已建成 ,要用于探查这一突变基因致病的分子机制 ,首先揭示人apoE7在小鼠体内诱发了那些基因的表达异常 ,是重要的环节之一。cDNA削减文库是寻找异常高表达的已知和未知基因有效的手段 ,而cDNA文库更是获取全长基因所必须。方法 自正常小鼠与转基因小鼠肾脏同时提取总RNA ,再分离mRNA ,逆转录制备cDNA后 ,用两次分子杂交削除转基因鼠cDNA中与正常小鼠相同的部分 ,再用PCR与择需PCR法扩增转基因鼠特异的基因 ,克隆入pGEM T载体后 ,制备削减文库。未经削减的转基因小鼠cDNA克隆入λgt11载体 ,制备基因表达文库。结果和结论 自高脂血症转基因小鼠肾脏构建的削减文库得到约 4 0 0个削减克隆 ,测定了部分克隆的序列 ,并进行同源性比较。λgt11 cDNA文库滴度 1 7× 10 7pfu ml,随机测定的克隆其插入片段长约 5 0 0bp以上。

Objective A hyperlipidemia transpenic mouse model has been established with human apoE7 gene. It provides a useful means for discovery of the pathogenetic mechanisms of human apoE mutant. In connection with this regard to find the over expression of some other genes, known or unknown, induced by apoE mutants within the cells would be one of the important links at first. Establishment of the subtractive cDNA library is the most effective way toward this aim and of the cDNA library is necessary for obtaining the whole gene. Method The total RNAs were extrated from the kidney of normal mice and transgenic mice. Then the mRNAs were purified from the total RNAs. The cDNA were synthesized according to protoccal of reverse transcription. The transgenic cDNAs were subjected to hybridization twice with normal cDNA to remove the common genes and then the specific transgenic genes amplified with PCR and suppression PCR. Following cloning the specific cNDA into the pGEM T vector the subtractive library was constructed finally. The cDNAs of the apoE7 transgenic mouse were cloned into λgt11 to prepare the cDNA library. Results and Conclusion About 400 colonies were found in the subtractive cDNA library. Several colonies were sequenced and the sequence homology was compared. The titer of the cDNA library is 1 7×10 7pfu/ml. The size of cDNA segments insertion determined randomly is longer than 0 5kb.