使用非放射性原位雜交術於人鼠共同高度保留性基因觀察其mRNA於胚胎及組織切片之表現

已知許多與遺傳疾病相關之基因具人鼠共同基因高度保留性,此類基因多 為常駐性基因; 觀察其mRNA於組織中隨時間與空間之分布變化是探討該種 基因功能及其異常病變之機轉的重要基礎研究之一. 非放射性標誌法進 行原位雜交其優點在於其避免了放射性同位素之使用且利於長期儲放,利 用單股長度僅45-mer 之寡核甘酸鏈可達到實驗之專一性;同時應用各組寡 核甘酸鏈之GC比例,僅改變雜交緩衝液中formamide的濃度可適用於多種不 同探針之雜交,其結果具相當的特異性. 應用本實驗法研究FMR-1, PCM 基因產物於不同天數之胚胎與成鼠組織切片,其結果fmr-1 mRNA 主要表現 於發育早期,同時於高度分化性組織有高度表現,而於不同的序列組合之探 針進進行之雜交中,我們發現其mRNA 的表現強度與序列之互補性組合有正 相關性,同時其分布區域除廣泛性存在外,另於alternative splicing form mRNA isoforms 發現其表現區域不同,意味著不同的fmr-1 mRNA其具 有不同之組織專一性. pcm mRNA 則著重於較高鼠齡時期腦部,及各項分化 活力高之組織如肌肉骨骼系統中表現. 應用本實驗法於此二項基因產物, 由其表現形態之特異性研究證實,此一非放射性探針標誌原位雜交法之穩 定性及應用性將可應用至其他基因產物之研究,堪稱其為一便利而有效之 研究工具. 同時也有助於對於此二項基因之功能表現有更進一步瞭解.

In this study the expression of FMR-1 and PCMT, two genes that are highlyconserved in human and mouse, was localized using nonradioactive in situhybridization. Mouse tissue and embryo sections of different develpomentalstages were formalin-fixed and paraffin-embeded. Oligonucleotides of thelenght of 45-mer were synthesized and labeled nonradioactively with digoxigenin. By changing the formamide concentrations in the hybridization buffer, insitu hybridization with probes of different GC content can be performed at the same temperature, which greatly improves the application of the technique. The expression of FMR-1 was mainly in the early develpomental stages, especiallyin highly differentiated tissues, such as brain. We designed three olgonucleotide probes that can distinquish different alternative splicing productsof FMR-1 . The strength and subtle localization differences of the mRNA expressioncan be observed by using three probes. The expression of PCMT, although can bedetected as early as in 10-day embryos, are strongest in certain areas of brain in aged mices. Different expression pattern of the experimental results are genuine tissue specific. Since oligonucleotides can be easily synthesized and the nonradioactive label is stable, the reproducibility of our experiments suggests that in situ hybridization with non-radioactive oligonucleotide probes has the potential to be applied generally for studying the expression of genes.