2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38739The long-range objective is to understand the genetic program residing in the zygote that enables it to become a complete mammal. The homeobox genes play important roles in the regulating gene expression during development. Our research was focus on screening novel homeobox genes and identifying their functions. The specific goal of this proposed research is to determine the developmental functions of a gonad-expressed gene cluster containing Cphx, mDux and Tgcs genes in mouse chromosome 14A3. We found that Cphx gene is a maternal homeobox gene specifically expressed in oocytes. In this research, we will characterize the oocyte maturation and early embryo development in Cphx-RNAi transgenic mouse. On the other hand, to determine the developmental functions of a novel double homeobox gene, mDux, preferentially expressed in ovary and Tgcs genes, which is testis germ cell expressed gene. Specific experimental objectives are outlined below: 1. Determination of the full length and alternative splicing forms of mDux, Tgcs transcripts. The full length and alternative splicing forms of mDux and Tgcs transcripts will be analyzed by 5’ and 3’ RACE reactions, respectively. Cphx, mDux and Tgcs orthologs will be examined by database search and degenerated PCRs, respectively. 2. Gene structure analyses. Full-length cDNAs for Cphx, mDux and Tgcs will be isolated and sequences of cDNAs and corresponding genomic DNAs will be determined, respectively. The intron-exon junctions will be indicated, and the amino acid sequences of encoded protein will be predicted. These works will provide the framework for later analyses of cis-acting regulatory elements and physiological functions of these genes. 3. Developmental expression patterns. A combination of techniques including RT-PCR, Northern blotting, in situ hybridization and immuno-staining will be used to examine the expression patterns of Cphx, mDux and Tgcs genes, respectively. The data of their transcript and protein distributions will indicate the expression profiles of Cphx, mDux and Tgcs genes during development. 4. Functional studies of Eso-1 by transgenic RNAi. The expressions of Cphx, mDux and Tgcs genes will be down-regulated to examine their functions by transgenic RNAi techniques. Cells or mice carrying precise changes in these gene expressions can be generated, and the resulting perturbations in fertility, development, proliferation, and differentiation can be directly observed. 5. Characterization of cis-acting regulatory element. To localize the cis-acting sequences controlling Cphx, mDux and Tgcs gene expressions, respectively, we will generate transgenic cells or mice carrying putative regulatory elements of these genes connected with a reporter gene. After analyzing reporter gene products in transgenic animals or cells, expression levels of reporter gene will be obtained. Related regulatory elements of each gene under different differentiation conditions or mouse developmental stages will be identified.此計畫的最終目的是想了解在哺乳動物中，胚胎發育成個體的整個遺傳機制。由於同源箱基因在胚胎發育的過程扮演上游調控的角色，因此研究早期胚胎表現的新的同源箱基因並研究這些新基因的功能及對胚胎發育的影響，一直是我們的研究目標。本計畫之主要目標為研究14A3區已知母源性基因Cphx基因的功能，延續過去的研究，分析已建立之Cphx-RNAi基因轉殖鼠，其Cphx基因表現減少對卵細胞及胚胎發育的影響，同時研究位於Cphx基因下游的雙同源箱基因mDux及精細胞專一性表達的Tgcs基因在卵細胞、精細胞成熟或早期胚胎發育所扮演的角色，計畫之特定實驗項目與目標如下： 1. 找出全長的cDNA序列，並分析在不同物種上是否存在對應基因 利用5’及3’RACE的結果，獲得全長的mDux, Tgcs完整cDNA序列，並觀察是否有不同編譯的cDNA序列存在。再進一步找出其他物種是否存在此Cphx, mDux, Tgcs基因之orthologs，它們在不同的物種發育上是否扮演相同的角色。 2 基因結構分析： 根據mDux, Tgcs基因的完整cDNA序列，找出相對應之genomic DNA序列，並推算出蛋白質的氨基酸序列。這些資料可提供將來分析基因之調控因子及詳細結構功能特徵的骨架。 3. 基因表現的模式分析： 利用RT-PCR, Northern blot及in situ hybridization甚至immunostaining等各種技術的結合，分析小鼠發育過程中Cphx,mDux, Tgcs基因表現的分佈趨勢。由初步的結果顯示Cphx基因專一性的表現於卵細胞，mDux表現於卵巢組織及睪丸組織，Tgcs則為精細胞專一性表達的基因，因此進一步分析卵巢組織或睪丸組織中之體細胞，及不同減數分裂時期的生殖細胞中Cphx,mDux, Tgcs基因的表現，確認基因群表現的時間與部位。 4. 基因功能的分析： 分析已產製之Cphx-RNAi基因轉殖鼠對卵細胞及胚胎發育之影響，同時未來將利用RNAi基因轉殖技術，改變mDux或Tgcs基因在卵巢組織或睪丸組織或細胞的表現量，直接觀察對小鼠卵細胞或精細胞成熟過程及早期胚胎發育過程之影響。 5. cis-acting調節序列的探討： Cphx,mDux, Tgcs基因可能是調控哺乳動物卵細胞、精細胞或早期胚胎發育的重要成員，我們將以Cphx,mDux, Tgcs基因啟動子上可能的調節序列接上EGFP 表達基因，送 入細胞中或產製基因轉殖鼠，以螢光來觀察轉殖細胞中EGFP 表達基因之表現，比較不同的調節因子片段接上表達基因的表現情形，可得知調控Cphx,mDux, Tgcs基因在不同情況下表現的最小片段之cis-acting element。Project