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: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3809754
• 关键词: Retroviridae; cell differentiation; chronic granulomatous disease; gene expression; gene therapy; genetic manipulation; human tissue; laboratory mouse; molecular pathology; tissue /cell culture; transfection

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.

这个非临床 IIDEA 项目的目的是研究可行性 使用逆转录病毒载体转移异常基因 人类常染色体隐性慢性肉芽肿病（p47-phox） 造血细胞。开发具有逆转录病毒生产能力的细胞系 感染人类血细胞将提供研究机会 该蛋白在前体和分化造血细胞中的表达 细胞。含有蛋白质编码序列的 p47-phox 的 cDNA 已被 克隆到称为 pLXSN 的逆转录病毒载体中。两种小鼠生产细胞 已经建立了一些品系，其中一种能够产生亲嗜性逆转录病毒 感染啮齿动物细胞和其他产生双嗜性逆转录病毒的细胞 能够感染多种哺乳动物和鸟类细胞。这 正常（有义）和 使用反向（反义）方向感染两种白血病细胞系， HL-60 和 U937。 RNA 和蛋白质分析证明是逆转录病毒 将 p47-phox 基因转移到这些细胞中。此外，爱泼斯坦·巴尔病毒 来自正常个体和 AR-CGD 患者的转化 B 淋巴细胞 也被感染了。这些线似乎也已被转导 基于蛋白质和 RNA 分析的逆转录病毒。确定是否 由转移基因制成的蛋白质仍具有功能性 进步。源自 AR-CGD 的 EBV 转化 B 淋巴细胞系 患者细胞可能有助于修复缺陷的研究，因为 正常情况下这些细胞会产生少量的超氧化物，并且可以 在功能和分子水平上进行研究。表达 小鼠骨髓中转移的 p47-phox 和造血重建 在灵长类动物和人类研究之前，需要对受辐射的小鼠进行研究 可以继续。初步实验表明可以 将该基因转移到小鼠骨髓中。