Patterns /Expression Levels of Imprinted Genes in Clones

遗传印迹基因只能通过母系或父系等位基因表达。这是由于配子体形成过程中精子和卵母细胞DNA上建立的不同“标记”，这些标记在胚胎发生和随后的发育过程中保持不变。然而，在核移植的体细胞克隆中，不涉及具有不同标记基因的单独配子，两组染色体来自同一个供体细胞。因此，研究印迹基因在体细胞克隆中的表达是如何受到调控的是一个很有意义的问题。这个问题很有趣，因为无论供体细胞类型或供体年龄如何，在克隆牛中都观察到印记破坏的迹象。发育缺陷，如高胎儿和新生儿死亡率和大犊牛，让人想起小鼠实验产生的印迹中断（印迹基因的双等位基因表达）和人类自然发生的印迹疾病。因此，我们假设在体细胞克隆中一致观察到的一些缺陷可能与克隆后印迹基因的异常表达有关。我们的长期目标是了解印记基因是否以及如何在克隆中被改变。我们这一建议的具体目的是：1)确定来自成年体细胞的克隆动物的印迹模式是否被破坏；2)确定体细胞克隆中印迹基因的mRNA水平是否被改变。这些特定的目标是基于我们的初步结果：1)我们收集了11个活的或死的体细胞牛克隆的样本，这些克隆显示出印迹中断的迹象；2)我们建立了牛IGF2和IGF2R的多态性分析，我们的供体在两个位点上都是杂合的。这些探索性研究的发现不仅有助于我们了解体细胞克隆的低效率，而且有助于我们进一步了解哺乳动物发育过程中遗传印迹的建立和调控。

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.