DNA METHYLATION IDENTIFIES AND REGULATES IMPRINTS

印记是哺乳动物正常发育所必需的，其定义为 配子发生过程中染色体的表观遗传修饰， 将印记基因的表达限制在母方或父方 等位基因。父亲特有的印记丢失(LOI)和 杂合性缺失(LOH)在几种类型的儿童中很常见 癌症(如肾母细胞瘤、视网膜母细胞瘤、横纹肌肉瘤、 神经母细胞瘤和急性髓系白血病)和几种基因 疾病(如Prader-Willi和Beckwith-Wiedermann综合征)。十六, 在估计的100个印记基因中，已经在老鼠和 父母特有的DNA甲基化区域已经在许多地方被发现 这些基因座的。DNA甲基转移酶(DNMT)失活会中断 印迹基因调控及DNMT基因的重新导入 ES细胞(被挽救的细胞)无法恢复父母特有的甲基化。 这意味着通过以下方式寻找新的印迹基因座是一个很好的机会 筛查正常ES与挽救ES的DNA甲基化差异 细胞。一种更有针对性的小鼠亲本特异性甲基化筛查 已被确认为印迹区域的12号染色体也将 使用流动分选的小鼠中期染色体t(4：12)的DNA来完成， 无论是父系遗传还是母系遗传。这些DNA将被消化并 在甲基DNA结合域柱(MBD)上分离得到 高度甲基化的片段随后将用于基于PCR-SELECT 减影方案以产生印记相关片段。的克隆 系统地检查人们对序列同源性的兴趣， 表达和印记。最终，将对已识别的基因座进行测试 同源重组基因敲除的发育功能及其获得 转基因小鼠的功能插入。此外，一个可逆的 将制造DNMT的可抑制等位基因，并用于研究DNMT的作用 在配子发生过程中的基因印记。

Imprinting is necessary for proper mammalian development and is defined as the epigenetic modification of chromosomes during gametogenesis, which limits expression of imprinted loci to either the maternal or paternal allele. Paternal-specific loss of imprinting (LOI) and loss of heterozygosity (LOH) are common occurrences in several types of childhood cancer (e.g. Wilms' tumor, retinoblastoma, rhabdomyosarcoma, neuroblastoma, and acute myelogenous leukemia) and in several genetic disorders (e.g. Prader-Willi and Beckwith-Wiedermann syndromes). Sixteen, of an estimated 100, imprinted genes have been identified in mice and regions of parental-specific DNA methylation have been identified at many of these loci. Inactivation of the DNA methyltransferase (Dnmt) disrupts imprinted gene regulations and re-introduction of the Dnmt cDNA into these ES cells (rescued cells) fails to restore parental-specific methylation. This suggests a great opportunity to search for new imprinted loci by screening DNA for methylation differences between normal and rescued ES cells. A more focused screen for parental-specific methylation on mouse chromosome 12, which has been identified as an imprinted region, will also be done using DNA from flow-sorted metaphase mouse chromosomes t(4:12), either paternally or maternally inherited. These DNAs will be digested and fractionated on a methyl-DNA binding domain column (MBD) to isolated highly methylated fragments which will then use used in PCR-select based subtraction protocols to yield imprint-associated fragments. Clones of interest will be systematically examined for sequence homologies, expression, and imprinting. Ultimately, identified loci will be tested for developmental function by homologous recombinant knockouts and gain of function insertions in transgenic mice. In addition, a reversibly repressible allele of Dnmt will be made and used to study the role of Dnmt in gene imprinting during gametogenesis.