Long-Range Transcriptional Regulation of Placental and O

我们使用大插入克隆获得酵母和细菌人工染色体克隆（YAC和BAC），其中包含两个在人类生殖中具有重要意义的基因：PLAC1，仅在胎盘的三层之一中表达，FOXL2，仅在发育中的眼睑和卵泡的滤泡细胞中表达。[FOXL2的缺乏会导致一些女性的卵巢早衰（POF）。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。与其他情况如肝特异性基因相反，组织特异性不被PLAC1或FOXL2 5'端高达10 Kb的推定启动子DNA片段复制。这表明，较长期的监管是有效的。与这一观点相一致的是，破坏FOXL2转录的易位距离基因序列非常远（约168 Kb）。 在这些研究的下一阶段，我们将重点关注人类和小鼠的Plac1基因。在细菌中使用基于重组的方法，我们已经将Flag标签引入到PLAC1的C末端，用哺乳动物选择性药物标记杀稻瘟菌素改造BAC，并从外显子1上游的65Kb序列中的最5'处开始进行10Kb的连续块的缺失。这些构建体现在被转染到细胞系中以研究它们的表达，然后用抗FLAG抗体在蛋白质水平上进行探测。 用PCR方法回收了含有Plac1外显子3和侧翼序列的8.3Kb片段。外显子3被哺乳动物选择性PGKNeo标记物取代，并且敲除构建体目前被转染到小鼠ES细胞中以获得Plac1敲除小鼠，以分析其与其特异性表达相关的功能。 在一种互补的方法中，用兔抗体进行的针对来自人和小鼠蛋白质的选定表位的实验表明，PLAC1蛋白在体内以二聚体形式存在，经历大量蛋白质修饰，并且定位于细胞质中。实验正在进行中，以追踪具有FLAG表位的人PLAC1蛋白，并通过与抗FLAG抗体的免疫沉淀来寻找相互作用的伴侣。FLAG表位将BAC编码的PLACl与内源性表达的蛋白质区分开。

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: PLAC1, expressed uniquely in one of the three layers of the placenta, and FOXL2, expressed only in developing eyelids and in follicular cells of the ovarian follicles. [Deficiency in FOXL2 leads to Premature Ovarian Failure (POF) in some women.] The goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. In contrast to other instances such as liver-specific genes, the tissue specificity is not reproduced by segments of putative promoter DNA up to 10 Kb 5' of PLAC1 or FOXL2. This indicates that longer-range regulation is operative. Consistent with that notion, a translocation that disrupts transcription of FOXL2 lies very far (about 168 Kb away) from the gene sequence. In the next phase of these studies we are focusing on both human and mouse Plac1 genes. Using recombination-based methods in bacteria, we have introduced a Flag tag into the C-terminal end of the PLAC1, retrofitted the BAC with mammalian selectable drug marker, blasticidin, and made deletions of successive blocks of 10Kb, starting at the 5' most in the 65Kb sequence upstream of Exon 1. These constructs are now being transfected into cell-lines to study their expression, followed by probing at the protein level with anti-FLAG antibody. Recombineering methods were also used to recover 8.3Kb fragment containing Plac1 exon3 and flanking sequences. Exon3 was replaced with mammalian selectable PGKNeo marker and the knockout construct is currently being transfected into mouse ES cells to derive a Plac1 knockout mouse, in order to analyze its function in relation to its specific expression. In a complementary approach, experiments with rabbit antibodies raised against selected epitopes from both human and mouse proteins suggest that the PLAC1 protein exists in vivo as a dimer, undergoes substantial protein modification, and is localized in the cytoplasm. Experiments are in progress to track the human PLAC1 protein with the FLAG epitope, and to look for interacting partners by immunoprecipitation with anti-FLAG antibodies. The FLAG epitope distinguishes the BAC-encoded PLAC1 from endogenously expressed protein.