Long-Range Transcriptional Regulation of Placental and Ovary Specific Genes

胎盘和卵巢特异性基因的长程转录调控

• 批准号: 7592024
• 负责人: Ramaiah Nagaraja
• 金额: $ 23.29万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592024
• 关键词: Alternative Splicing; Attention; Bacterial Artificial Chromosomes; Cells; Cloning; Code; Databases; Development; Elements; Embryo; Exons; Expressed Sequence Tags; Eyelid structure; Functional RNA; Genes; Genetic Transcription; Genome; Goals; Human; Knock-out; Link; Luciferases; Maps; Methods; Mus; Mutate; Mutation; Neomycin; Outcome Study; Ovarian Follicle; Ovary; Personal Satisfaction; Placenta; Pregnancy; Pregnancy Outcome; Premature Ovarian Failure; Process; Protein Isoforms; Range; Regulation; Reporter; Reporter Genes; Reproduction; Resistance; Structure; Surveys; Techniques; Tissue-Specific Gene Expression; Tissues; Transcriptional Regulation; Up-Regulation; Woman; YAC Clone; Yeasts; base; embryonic stem cell; fetal; gene function; insight; mouse model; promoter; vector

We have used large-insert cloning to obtain yeast and bacterial artificial chromosome clones (YACs and BACs) containing two genes of importance in human reproduction: One, PLAC1, is expressed uniquely in placenta, where its expression is restricted to specific placental regions facing the maternal tissue; and it has been implicated both in cases of placental problems in inter-specific crosses of mice, and in fetal well being and successful outcome of pregnancy in humans. The other gene, FOXL2 is expressed only in developing eyelids and in follicular cells of the ovarian follicles and deficiency in FOXL2 leads to Premature Ovarian Failure (POF) in some women (see AG000647-05). The goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. We have recently focused our attention on the regulation and function of PLAC1. Recent EST database survey suggested that the human PLAC1 might have additional exons 5 to the 3 exons previously defined. We have now shown that the gene has 3 additional non-coding exons, resulting in a total of 6 exons spanning 200 Kb, with at least five isoforms made by alternative splicing. The distance between the putative start of transcription of the 1st exon and the end of the next gene in the genome is only 5300 bp, suggesting that most promoter elements are located within this region. We cloned and fused this 5.3 Kb fragment, including the 1st exon, to a Luciferase reporter gene, and find that it gives up to 60 fold higher luciferase activity in placental derived cells, when compared to a promoter-less vector. Promoter activity thus resides in this fragment. Deletion constructs removing 700 bp unique sequence immediately neighbouring the 1st exon abolishes the reporter activity. We are now doing fine structure mapping of the promoter sequences necessary for specific transcription. To study the function of the gene in placenta, recombineering methods were also used to recover a fragment containing Plac1 coding and flanking sequences from a mouse BAC. The cloned fragment was modified to ablate the Plac1 sequence and replace it with a selectable neomycin resistance marker. This construct is in the process of being transfected into C57/BL6 ES cells to generate a knockout mouse model.

我们已经使用大插入克隆获得酵母和细菌人工染色体克隆（YAC和BAC），其含有在人类生殖中重要的两个基因：一个是PLAC 1，其在胎盘中独特地表达，其中其表达限于面向母体组织的特定胎盘区域;并且它与小鼠种间杂交中的胎盘问题以及人类的胎儿健康和妊娠成功结果有关。另一种基因FOXL 2仅在发育中的眼睑和卵泡的滤泡细胞中表达，FOXL 2的缺乏导致一些女性的卵巢早衰（POF）（参见AG 000647 -05）。我们的目标是确定这些基因的非常选择性的组织特异性表达的基础。 我们最近将注意力集中在PLAC 1的调节和功能上。 最近的EST数据库调查表明，人类PLAC 1除了之前定义的3个外显子之外，可能还有额外的第5个外显子。 我们现在已经表明，该基因有3个额外的非编码外显子，导致总共6个外显子跨越200 Kb，至少有五个异构体通过选择性剪接。 基因组中第一个外显子的假定转录起始点和下一个基因的末端之间的距离仅为5300 bp，这表明大多数启动子元件位于该区域内。 我们克隆并融合了这个5.3 Kb的片段，包括第一个外显子，荧光素酶报告基因，并发现它给出了高达60倍高的荧光素酶活性在胎盘来源的细胞相比，启动子少载体。 因此，启动子活性存在于该片段中。 缺失构建体去除紧邻第1外显子的700 bp独特序列，消除了报告活性。 我们现在正在对特异性转录所必需的启动子序列进行精细结构作图。 为了研究该基因在胎盘中的功能，还使用重组工程方法从小鼠BAC中回收含有Plac 1编码和侧翼序列的片段。对克隆的片段进行修饰，以消除Plac 1序列，并用可选择的新霉素抗性标记替换它。该构建体正在被转染到C57/BL 6 ES细胞中以产生敲除小鼠模型。