Isolation and characterization of cDNA clones of mRNAs expressed selectively in the immature cells of developing brain.

我们首先建立了几个改进的程序来分离在发育中的脑的未成熟细胞中选择性表达的mRNA的cDNA克隆。利用两种载体（pT 7 T3 D和pBluescript）系统的改进的差异筛选程序显示出具有低背景水平的清晰信号。扩增的cDNA文库DNA的Southern印迹分析显示能够取代北方印迹分析。这些程序是可靠的，特别是用于分析的cDNA克隆，其相应的mRNA相对丰富的表达。一种改进的消减cDNA克隆方法，命名为定向标签PCR消减，被证明是有效的分离相对很少表达的mRNA的差异表达克隆。这三个程序不需要额外的RNA筛选后的cDNA文库的建设，使微小的大脑区域的分析特别interests.By使用上述程序，我们然后分离出大鼠胎脑富集（FBE）克隆，其相应的mRNA表达优先在产前阶段的大脑发育。我们成功地鉴定了22个不同的FBE克隆，其mRNA在胎儿脑中的表达至少是成人脑中的5倍。22个FBE克隆的核苷酸序列分析显示，其中13个与数据库中报道的序列没有显著匹配，而其中9个与先前报道的序列匹配（α微管蛋白M α 1、β微管蛋白M β 5、胸腺素β 10、stathmin、β微管蛋白M β 2、α-internexin、铁蛋白Lg亚基、neuronatin和aperterin）。原位杂交分析表明，FBE克隆对应的mRNA在脑发育过程中表达减少，并呈现不同的表达模式。新分离的FBE克隆的mRNA在小脑外胚层和发育中的脑室下区的未成熟细胞中选择性表达。

We first established several improved procedures to isolate cDNA clones of mRNAs expressed selectively in the immature cells of developing brain. A modified differential screening procedure, which utilized two vector(pT7T3D and pBluescript) -system, showed clear signals with low background levels. Southern blot analysis of amplified cDNA library DNAs were shown to be able to replace the Northern blot analysis. These procedures were reliable especially for the analysis of cDNA clones whose corresponding mRNAs were relatively abundantly expressed. An improved subtractive cDNA cloning procedure, named Directional tag PCR subtraction, were demonstrated to be efficient to isolate differentially expressed clones of relatively rarely expressed mRNAs. These three procedures require no additional RNA for the screening after construction of the cDNA libraries, enabling the analysis of tiny brain regions of particular interest.By use of the procedures described above, we then isolated rat fetal brain-enriched (FBE) clones whose corresponding mRNAs were expressed preferentially in the prenatal stages of brain development. We successfully identified 22 distinct FBE clones whose mRNAswere expressed at least 5-fold more in the fetal brain than in the adult brain. The nucleotide sequence analysis of the 22FBE clones revealed that 13 of them had no significant matches to the sequences reported in the databases, whereas 9 of them matched previously reported sequences (alpha tubulin M alpha 1, beta tubulin M beta 5, thymosin beta 10, stathmin, beta tubulin M beta 2, alpha-internexin, ferritin Lg subunit, neuronatin, and amphoterin). In situ hybridization analysis showed that mRNAs corresponding tothe FBE clones decreased during braindevelopment with various expression patterns. The mRNA of a newly isolated FBE clone was shown to be expressed selectively in the immature cells in theexternal germinal layr of the cerebellum and the subventricular zone of developing brain.