Reprogramming of X-linked genes by nuclear transfer

通过核移植重编程 X 连锁基因

• 批准号: 6612983
• 负责人: Xiuchun TIAN
• 金额: $ 7.15万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612983
• 关键词: alleles; animal tissue; biopsy; biotechnology; cow; developmental genetics; egg /ovum; embryogenesis; embryogenic cleavage; fibroblasts; gene expression; genetic manipulation; nuclear transfer; sex chromosomes; tissue /cell culture

DESCRIPTION (provided by applicant): Placental abnormalities have been propose as a major cause for developmental defects of animals clone from differentiated somatic cells. In mice and cattle, X chromosome inactivation (XCI) is established at blastocyst stage with the paternal X chromosome (XP) preferentially inactivated (XiP) in the trophectoderm. Abnormal XCI has been found to have deleterious effect on placental growth and causes fetal death. The abnormal XCI may be caused by the impaired ability of cloned embryos to erase epigenetic marks of XCI in the donor cells, which interfere with the re-establishment of new imprinting marks. Here the investigators propose to test the hypothesis that the avoidance of both the reversal of epigenetic marks of XCI during nuclear reprogramming as well as the conflict between epigenetic marks for inactivation (Xi) and maternal origin (Xm) should improve the reprogramming of the donor nuclei and result in normal expression of X-linked genes in cloned embryos. Conversely, embryos in which Xi becomes Xa or where Xim persists in the trophectoderm will have more abnormal gene expression and poorer embryo development. Therefore, embryos in which the epigenetic marks in trophectoderm are reprogrammed to those mimicking the placenta in natural reproduction, i.e., XiP, should have the best embryo development and normal expression of X-linked genes. The Specific Aims of this application are 1, to compare blastocyst development of cloned embryos which "maintained" versus reversed the XCI patterns of the somatic donor cells. Fibroblast cells from a female donor cow with known XCI patterns will be individually cultured, clonally expanded and categorized into two groups: XipXam or XaPXim. Nuclear transfer will be conducted using these two groups of cells and the blastocyst development of the resulting embryos in which reversal and "maintenance" of the XCI pattern in the donor cells will be compared. 2, to compare the patterns and levels of expression of l0 X-linked genes in the two groups of embryos generated from Specific Aim 1. These genes include MAOA, XIST. ELF4, FACL4; PLP1, PPEF1, SYP, SYNl, BGN, and TBLl. In vivo produced XX embryos will be used as controls for normal gene expression. Aberrant expression of all of these genes (except for TBLl which escapes XCI) was found in the investigators' died bovine clones. Findings from this pilot project should generate sufficient initial data on the mechanisms of epigenetic reprogramming in cloned embryos during early embryogenesis which could lead to a full-blown investigation on the reprogramming of X-linked genes and other epigenetic marks during peri- and post-implantation development.

描述（由申请人提供）：胎盘异常被认为是由分化的体细胞克隆的动物发育缺陷的主要原因。 在小鼠和牛中，X染色体失活（XCI）在胚泡阶段建立，其中父方X染色体（XP）在滋养外胚层中优先失活（XiP）。 已发现异常XCI对胎盘生长具有有害影响并导致胎儿死亡。 异常XCI可能是由于克隆胚胎清除供体细胞中XCI表观遗传标记的能力受损，干扰了新印迹标记的重建。 在这里，研究人员提出测试的假设，即避免在核重编程过程中XCI的表观遗传标记的逆转以及表观遗传标记失活（Xi）和母体来源（Xm）之间的冲突，应该改善供体核的重编程，并导致克隆胚胎中X连锁基因的正常表达。 相反，Xi变成Xa或Xim持续存在于滋养外胚层的胚胎将具有更多的异常基因表达和更差的胚胎发育。 因此，其中滋养外胚层中的表观遗传标记被重编程为模仿自然生殖中的胎盘的那些的胚胎，即，XiP，应该具有最佳的胚胎发育和正常表达的X连锁基因。 本申请的具体目的是1，比较“维持”与逆转体细胞供体细胞的XCI模式的克隆胚胎的胚泡发育。 将来自具有已知XCI模式的雌性供体奶牛的成纤维细胞单独培养、克隆扩增并分为两组：XipXam或XaPXim。 将使用这两组细胞进行核移植，并比较所得胚胎的胚泡发育，其中将比较供体细胞中XCI模式的逆转和“维持”。 2，比较10个X连锁基因在由特异性目的1产生的两组胚胎中的表达模式和水平。 这些基因包括MAOA、XIST。ELF 4、FACL 4; PLP 1、PPEF 1、SYP、SYN 1、BGN和TBL 1。 体内产生的XX胚胎将用作正常基因表达的对照。 在研究者的死牛克隆中发现所有这些基因的异常表达（除了逃避XCI的TBL 1）。 该试点项目的研究结果应产生足够的初步数据，克隆胚胎在早期胚胎发育过程中的表观遗传重编程机制，这可能导致对X连锁基因和其他表观遗传标记在胚胎发育和植入后发育过程中的重编程进行全面调查。