Functional Analysis of Mouse Antisense RNA and Evolution of X Chromosome

我有2500对有义反义基因的初步数据。这些基因对是使用cDNA数据鉴定的，但尚未获得其表达的直接证据。为了确认这些基因的实际表达，构建了使我们能够区分正义和反义基因表达和定量的微阵列（60聚体寡核苷酸）。从上述2500对中选择2000对，并在成纤维细胞、脑、心脏和睾丸中进行表达分析。结果表明，超过90%的基因实际上是转录的，并且正义和反义基因之间的表达价在组织中是不同的。在印迹上检测到不同大小的转录物，无论基因的编码或非编码状态如何。多为poly（A）阴性，多位于细胞核内。这些发现表明，很难敲除或敲除的反义基因，只有通过常规的方法来继续功能分析。我分析了7个正义或反义基因在X染色体上是否逃脱X染色体失活。它们的表达是在X染色体失活的情况下进行的。为了进一步分析这些转录物的功能，有必要精确鉴定每个位点中的转录单位。

I had preliminary data of 2500 pairs of sense-antisense genes identified in silico. These pairs of the genes were identified using cDNA data, but direct evidence of their expression had not been obtained. In order to confirm the actual expression of these genes, microarrays (60mer oligo nucleotides) that enabled us to distinguish sense and antisense gene expression and quantitation were constructed. Out of 2500 pairs above,2000 pairs were chosen and subject to expression analysis in fibroblast cells, brain, heart, and testis. It turned out that more than 90% of the genes were actually transcribed, and that the expression valance between the sense and antisense genes varied among tissues.Seven gene pairs were subject to Northern analysis. Various-sized transcripts were detected as smear on the blots, regardless of coding or non-coding status of the genes. They were mostly poly(A)-negative and tended to be localized in nucleus. These finding suggested that it was difficult to knockout or knockdown the antisense genes only to continue functional analysis by conventional methods.I analyzed seven sense or antisense genes on X chromosome whether or not they escaped from X chromosome inactivation. Their expression was under X chromosome inactivation.We found out unusual characters of the RNA transcribed from the sense-antisense gene loci. In order to further analyze function of these transcripts, it is necessary to precisely identify the transcription unit in each locus.