Patterns /Expression Levels of Imprinted Genes in Clones

基因印记的基因只能在母亲或父亲的等位基因中表达。这是由于在配子发生过程中，精子和卵母细胞的DNA上建立了不同的“标记”，这些标记在整个胚胎发生和随后的发育过程中一直保持不变。然而，在核移植的体细胞克隆中，不涉及带有差异标记基因的单独配子，两组染色体都来自同一供体细胞。因此，了解印记基因在体细胞克隆中的表达是很有意义的。这个问题很耐人寻味，因为无论供体细胞类型或供体年龄，克隆牛都观察到了印迹破坏的迹象。发育缺陷，如胎儿和新生儿死亡率高，以及大牛犊，让人想起实验中在小鼠身上造成的印记中断(印记基因的双等位表达)，以及在人类中自然发生的印记疾病。因此，我们假设，在体细胞克隆中持续观察到的一些缺陷可能与克隆后印记基因的异常表达有关。我们的长期目标是了解是否以及如何在克隆中改变印记基因。我们这个建议的具体目的是1)确定来自成年体细胞的克隆动物的印记模式是否被破坏，以及2)确定体细胞克隆中印记基因的mRNA水平是否改变。这些特定的目的是基于我们的初步结果：1)我们收集了11个活的或死亡的体细胞克隆的样本，这些克隆显示出印迹中断的迹象；2)我们已经建立了牛的IGF2和IGF2R的多态分析，我们的供体在这两个基因座上都是杂合子。从这些探索性研究中发现，不仅将为体细胞克隆的低效率提供深入的见解，而且将极大地加深我们对哺乳动物发育过程中遗传印记的建立和调控的理解。

Genetically imprinted genes are only expressed from either the maternal or paternal allele. This is due to differential "marks" established on the DNA of sperm and oocytes during gametogenesis, which are maintained throughout embryogenesis and subsequent development. In somatic cloning by nuclear transfer, however, no separate gametes with differentially marked genes are involved, both sets of chromosomes are derived from the same donor cell. Therefore, it is of interest to find out how the expression of imprinted genes is regulated in somatic clones. This question is intriguing because signs of imprinting disruption have been observed in cloned cattle regardless of donor cell type or donor age. Developmental defects such as high fetal and neonatal deaths and large calves are reminiscent of experimentally created imprinting disruptions (biallelic expression of imprinted genes) in mice and naturally occurring imprinting diseases in humans. Therefore, we hypothesize that some of the defects consistently observed in somatic clones are likely associated with aberrant expressions of imprinted genes after cloning. Our long-term objective is to understand whether and how imprinted genes are altered in clones. Our specific aims of this proposal are 1) to determine whether imprinting patterns are disrupted in cloned animals derived from adult somatic cells, and 2) to determine if the levels of mRNA for imprinted genes are altered in somatic clones. These specific aims are capitalized on our preliminary results that 1) we have collected samples from eleven live or dead somatic cattle clones which showed signs of imprinting disruptions, and 2) we have established polymorphic assays for IGF2 and IGF2R in bovine and our donor is heterozygous in both loci. Finding fromthese explorative studies will not only provide insights into the low efficiency of somatic cloning but also significantly further our understanding of the establishment and regulation of genetic imprinting during mammalian development.