Human nerve growth factor receptor and cytosine deaminase fusion genes.
Human nerve growth factor receptor and cytosine deaminase fusion genes.
复制标题
人神经生长因子受体和胞嘧啶脱氨酶融合基因。
作者：
V. Lewis;L. Basso;N. Blake;J. Salo;T. Lund;R. McIvor;P. Orchard
胞嘧啶脱氨酶（CD）将5-氟胞嘧啶（5-FC）转化为毒性代谢物5-氟尿嘧啶（5-FU），并已被广泛研究作为选择性细胞根除的潜在工具。在本文中，遗传构建体的设计表达的CD酶融合的跨膜和细胞外结构域的人神经生长因子受体（NGFR），从而允许阳性鉴定的转导细胞的流式细胞术和阳性选择的磁珠技术。构建体被设计为在NGFR和CD基因的核酸编码序列之间编码[Gly（4）Ser]（2）柔性接头。用野生型CD和NG/CD融合基因构建的逆转录病毒载体用于转染3 T3成纤维细胞和人T细胞系CEM。CD融合基因的功能与细胞毒性试验中测定的野生型基因的功能相当。通过流式细胞术，NGFR抗原的表达后，来自大肠杆菌（NG/CDe）或酿酒酵母（NG/CD）的融合基因可检测到，但最大的抗原密度被观察到在细胞与NG/CD载体转导。同样，NG/CD融合基因在鼠成纤维细胞和人T细胞中均观察到上级5-FC敏感性。此外，表达NG/CD的CEM细胞在体内被更有效地消除。利用嵌合NG/CD基因的细胞工程化提供了一种新的基因治疗模式，允许使用单个蛋白质编码序列进行阳性和阴性选择。
Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.
影响因子：
11.2
作者：
E. Kievit;M. Nyati;E. Ng;L. Stegman;J. Parsels;B. Ross;A. Rehemtulla;T. Lawrence
通讯作者：
E. Kievit;M. Nyati;E. Ng;L. Stegman;J. Parsels;B. Ross;A. Rehemtulla;T. Lawrence
影响因子：
2.7
作者：
Miller Ad;Rosman Gj
通讯作者：
Miller Ad;Rosman Gj