Effect of nuclear localization and hydrodynamic delivery-induced cell division on phiC31 integrase activity.
Effect of nuclear localization and hydrodynamic delivery-induced cell division on phiC31 integrase activity.
复制标题
核定位和流体动力学递送诱导的细胞分裂对 phiC31 整合酶活性的影响。
DOI：
10.1038/gt.2009.136
复制
发表时间：
2010
期刊：
影响因子：
5.1
通讯作者：
Calos,MP
中科院分区：
文献类型：
--
作者：
Woodard,LE;Hillman,RT;Keravala,A;Lee,S;Calos,MP
噬菌体φC31整合酶是一种重组酶，可以在哺乳动物细胞中表达，以将携带attB序列的质粒整合到基因组中特定的假attP位置。我们通过免疫荧光显示，野生型φC31整合酶位于细胞质中，而加入SV40核定位信号（NLS）后，φC31整合酶定位于细胞核。出乎意料的是，NLS降低了HeLa细胞中的整合效率，并且当用于将人因子IX（hFIX）基因整合到小鼠肝脏中时没有提供益处。由于有丝分裂期间核膜的破裂可以允许细胞质整合酶进入染色体，我们分析了体内整合到肝细胞中是否需要细胞分裂。用碘脱氧尿苷标记肝细胞，以标记在流体动力学注射后一周内经历DNA复制的细胞。流体动力学递送导致三分之一的肝细胞中的DNA复制。大约四分之三的具有φC31整合酶介导的稳定hFIX表达的细胞没有进行复制，表明细胞分裂不是肝脏中整合酶功能所必需的。因此，尽管大部分φC31整合酶蛋白在哺乳动物细胞中似乎位于细胞质中，但即使没有细胞分裂，整合仍然可以发生在细胞核中。
Phage φC31 integrase is a recombinase that can be expressed in mammalian cells to integrate plasmids carrying an attB sequence into the genome at specific pseudo attP locations. We show by immunofluoresence that wild-type φC31 integrase is cytoplasmic and that addition of the SV40 nuclear localization signal (NLS) localized φC31 integrase to the nucleus. Unexpectedly, the NLS depressed integration efficiency in HeLa cells and provided no benefit when used to integrate the human Factor IX (hFIX) gene into mouse liver. As breakdown of the nuclear membrane during mitosis could allow cytoplasmic integrase access to the chromosomes, we analyzed whether cell division was required for integration into liver cells in vivo. Hepatocytes were labeled with iododeoxyuridine to mark cells that underwent DNA replication during the week after hydrodynamic injection. Hydrodynamic delivery led to DNA replication in one-third of hepatocytes. Approximately three out of four cells having φC31 integrase-mediated stable hFIX expression did not undergo replication, indicating that cell division was not required for integrase function in liver. Therefore, although the bulk of φC31 integrase protein seems to be cytoplasmic in mammalian cells, integration can still occur in the nucleus, even without cell division.