同源箱基因群（ homeobox gene superfamily ）在生物的發育過程中，扮演著重要的調控角 色。同源區蛋白中的同源區 ( homeodomain ) 包含 60 個高度保留性的氨基酸。同源區 蛋白即藉由此同源區去辨認下游的目標基因的調控區，並與之結合，以調節該基因的表 現。近年來發現某些同源箱基因在動物出生後，對器官的成熟與在胚胎發育過程中亦具 有調控的作用。本實驗即針對此方面的基因作研究，利用已知的同源區蛋白最保守的序 列，設計退化性引子 (degenerate primer) ，以 PCR 方式對小鼠 testis cDNA 基因庫進行 新同源箱基因的分子選殖，再將此方式所得的新同源箱基因片段作為探針，去篩選小鼠 的 testis cDNA library 中更長片段的 cDNA clone ，結果我們得到一段長度為 839 bp 的 新同源箱基因 Tse1 片段。由小鼠各不同器官之總 RNA 作多組織北方墨點法 (northern blot) 分析，結果顯示 Tse1 的 RNA 主要表現在睪丸，全長約為 1.2Kb 。進一步再取各 時期小鼠睪丸的總 RNA 作北方墨點法，由結果得知 Tse1 在 20 天的睪丸表現量最高。
由另一個小鼠 testis cDNA library 之篩選，得到 Tse1 的 full length cDNA 總長為 1117 bp 。 經由原位雜交 (in situ hybridization) 實驗顯示 Tse1 表現在小鼠睪丸的精細胞產生過程 以及萊氏細胞 (Leydig cells) ，由此推測 Tse1 可能參與精子成熟的過程並可能參與雄性 賀爾蒙的分泌調節。在 Genomic DNA 方面由 Genomic DNA library 篩選及南方墨點法 (southern blot) 得到兩個包含 Tse1 cDNA 片段的 Genomic DNA 菌株， 經過核酸定序分 析並配合由美國國家衛生院所建立的 NCBI 網站搜尋 genomic 的資訊結果，目前已得 到部分的 3’ 端延伸片段，未來將進一步釐清 Tse1 Genomic DNA 的詳細序列，以便進 行 Tse1 基因剔除鼠的建立。日後可藉由基因剔除鼠，以了解 Tse1 之基因功能並深入探 討此基因與賀爾蒙之間的關係。
小鼠睪丸之同源箱基因 Tse1 之分子選殖與分析
The homeobox genes play a central role in animal development. These genes encode homeo domain proteins that act as transcription factors which regulate expression of gene cascades.
The homeodomain of these homeobox genes, consisting of a conserved 60 amino-acid region , recognizes and binds to regulatory sites of their target genes, and then initiates gene cascade s and cell type specificities. Tse1 is a novel mouse homeobox gene identified by degenerate d-oligonucleotide PCR screening from a mouse testis cDNA library. A 839 bp cDNA fragme nt of Tse1 was used as a probe to screen testis cDNA library and obtained a 1117 bp full—le ngth cDNA clone which encoded 293 a.a. with a Prd-like homeodomain. Analysis of tissue a nd stage expression patterns of Tse1 gene by Northern hybridization, we found that Tse1 tran script was about 1.2kb and mainly expressed in testis with the highest level at postnatal day 2 0. Tse1 transcript was also detected in c-kit mutant (w/ wv and wv/ wv) testes in which germ cells were depleted. These results suggest that Tse1 gene is possible expressed in somatic cell s of testes. In situ hybridization further confirmed that Tse1 expressions were in spermatids a nd leydig cells and implied that Tse1 may play a role during spermatogenesis and may be inv olved in hormonal reguation during the puberty. Gene structure of Tse1 was identified by seq uence comparison between cDNA and genomic DNA. Information obtained from these geno mic DNA sequences will be used to construct the Tse1 knock-out construct and establish the Tse1 knock-out mice to elucidat the in vivo function of Tse1 in spermatogenesis.
Molecular Cloning and Analysis of a Testis — Spe cific Expression Homeobox Gene , Tse1
