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RETROVIRAL TRANSFER OF P47-PHOX INTO MURINE CELLS AND HUMAN MYELOID CELL LINES

RETROVIRAL TRANSFER OF P47-PHOX INTO MURINE CELLS AND HUMAN MYELOID CELL LINES
P47-PHOX 逆转录病毒转移至鼠细胞和人骨髓细胞系
批准号:
3809754
负责人:
K LOMAX
金额:
$0.0万
依托单位国家:
美国
项目类别:
财政年份:
--
资助国家:
美国
项目状态:
未结题
起止时间:

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中文摘要
翻译
这个非临床的IIDEA项目的目的是研究其可行性 利用逆转录病毒载体转移人类不正常的基因 常染色体隐性遗传性慢性肉芽肿性疾病(P47-Phox) 造血细胞。能够产生逆转录病毒的细胞系的建立 感染人类血细胞将为研究提供机会 该蛋白在前体和分化造血细胞中的表达 细胞。含有该蛋白编码序列的p47-Phox的cDNA已被克隆。 克隆到名为pLXSN的逆转录病毒载体上。两个小鼠生殖细胞 已经建立了品系,其中一株产生了一种能够产生生态逆转录病毒的 感染啮齿动物细胞和产生两性逆转录病毒的另一种 能够感染多种哺乳动物和禽类细胞。这个 在正常(正义)和正常(正义)中都含有P47-Phox的两性病毒 反向(反义)定向被用于感染两个白血病细胞系, HL-60和U937。RNA和蛋白质分析证实了逆转录病毒 在这些细胞中转移P47-Phox基因。此外,爱泼斯坦-巴尔病毒 正常人和AR-CGD患者转化的B淋巴细胞 也被感染了。这些线似乎也是由 基于蛋白质和RNA分析的逆转录病毒。检测以确定 由转移的基因产生的蛋白质仍然具有功能。 进步。AR-CGD来源的EB病毒转化B淋巴细胞系 患者细胞可能对纠正缺陷的研究有帮助,因为 正常人的这些细胞会产生少量的超氧化物,并可以 在功能和分子水平上进行了研究。的表达 P47-Phox在小鼠骨髓中的转移与造血重建 在灵长类动物和人类研究之前,有必要对受辐射的小鼠进行检查 就可以继续了。初步实验表明,有可能 将这种基因转移到小鼠骨髓中。
英文摘要
The purpose of this non-clinical, IIDEA project is to study the feasibility of using retroviral vectors to transfer the gene which is abnormal in autosomal recessive Chronic Granulomatous Disease(p47-phox) into human hematopoietic cells. Development of retrovirus producer cell lines capable of infecting human blood cells will provide the opportunity to study expression of this protein in precursor and differentiated hematopoietic cells. A cDNA for p47-phox containing the protein coding-sequence has been cloned into a retroviral vector known as pLXSN. Two murine producer cell lines have been established, one producing an ecotropic retrovirus capable of infecting rodent cells and the other producing amphotropic retrovirus capable of infecting a wide range of mammalian and avian cells. The amphotropic virus containing p47-phox in both normal(sense) and reverse(antisense) orientations was used to infect two leukemia cell lines, HL-60 and U937. RNA and protein analysis demonstrated retrovirally transferred p47-phox genes in these cells. In addition, Epstein Barr Virus transformed B lymphocytes from a normal individual and an AR-CGD patient were also infected. These lines also appear to have been transduced by the retroviruses based on protein and RNA analysis.Assays to determine whether the protein made from the transferred gene is functional are still in progress. EBV transformed B lymphocyte cell lines derived from AR-CGD patient cells may be helpful in studies of correction of the defect since these cells in normals produce a small amount of superoxide and can be studied at the functional as well as molecular level. Expression of transferred p47-phox in murine bone marrow and hematopoietic reconstitution of an irradiated mouse will be necessary before primate and human studies can proceed. Preliminary experiments indicate that it is possible to transfer this gene into murine bone marrow.
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CHARACTERIZATION OF THE MURINE HOMOLOG FOR THE HUMAN P47-PHOX GENE
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