SELECTION AND ISOLATION OF RADIATION INDUCIBLE GENES

辐射诱导基因的选择和分离

基本信息

批准号：
2438943
负责人：
JEAN-NUMA N LAPEYRE
金额：
$ 24.32万
依托单位：
SALEM INTERNATIONAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-02-10 至 1998-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2438943
关键词：
Retroviridae X ray complementary DNA gamma radiation gene induction /repression genetic promoter element genetic regulation human genetic material tag ionizing radiation nucleic acid sequence polymerase chain reaction radiobiology recombinase transfection

项目摘要

The goal of this application is to identify and isolate by a drug selection-mediated approach genes whose activity is induced by ionizing radiation (IR) with the purpose of generating probes for these genes and defining the regulation of their promoters. This is a first step in determining the molecular and biochemical functions carried out by uncharacterized radiation modulated proteins (RMPS) that are induced by radiation and will help elucidate many poorly understood radiologic end- points in mammalian cells. The human genome will be saturated by retroviral vector-mediated random integration of a promoter less 2 micron yeast recombinase gene (FLPase) containing a splice acceptor and IVS leader for enhanced stability in a large population of HT1080 cells. This marker also permits positive dominant selection of ionizing radiation induced (IRI) genes induced by radiation doses employed in radiotherapy in cells that have been transfected with a neomycin/hygromycin flip-flop dominant selection gene. This approach to isolating IRI genes is a spin- off of a previously described gene tagging approach that employed a FACS protocol to sort radiation induced expressors of B-galactosidase after random integration of pROS-Bgal-pgkneo, a retroviral vector that encodes the promoterless lacZ indicator gene, a neor marker to select for integrants with G418, and a supF marker for rescue of the genetic region. The drug selection approach eliminates the problem of background and null cells creeping through multiple FACS screens that hampers the screening of complementary deoxyribonucleic acid (cDNA) libraries for actual IRI gene isolation. The applicant's preliminary experiments using this gene tagging approach suggest the existence of about 100-200 IRI genes. So far, other approaches have only defined or isolated about a dozen genes induced or involved in ionizing radiation response. The goal of Aim I is to identify, then isolate, IRI cDNAs, which will be sequenced and characterized. The goal of Aim II is to use the IRI cDNA probes to isolate IRI genes from a genomic library, including the upstream regulator regions for characterization of the response elements transducing the inductive effect.

该应用的目的是通过药物识别和隔离选择介导的方法基因通过电离引起活性辐射（IR）的目的是为这些基因生成探针和定义其发起人的调节。这是第一步确定由未经表征的辐射调制蛋白（RMP）辐射，将有助于阐明许多鲜为人知的放射学端哺乳动物细胞中的点。人类基因组将被逆转录病毒媒介介导的随机整合启动子较少2微米包含剪接受体和IVS的酵母重组酶基因（FLPase）在大量HT1080细胞中增强稳定性的领导者。这标记还允许积极的电离辐射选择诱导的（IRI）基因是由放疗中采用的放射剂诱导的（IRI）基因已转染新霉素/湿霉素触发器转染的细胞主要选择基因。这种分离IRI基因的方法是一种自旋 - 使用先前描述的基因标记方法，该方法采用FACS 在B-galactosidase的辐射诱导表达的方案之后 Pros-Bgal-Pgkneo的随机集成，这是一种编码的逆转录病毒矢量无启动子LACZ指示基因，一种选择的神经标记物具有G418的整合体，以及用于营救遗传区域的SUPF标记。药物选择方法消除了背景和无效的问题细胞蔓延到多个FACS筛选，从而阻碍了筛查的筛查实际IRI基因的互补脱氧核糖核酸（cDNA）文库隔离。使用此基因标记的申请人的初步实验方法表明存在约100-200个IRI基因。到目前为止，其他方法仅定义或隔离大约十几个诱导的基因或参与电离辐射响应。目标的目标是识别，然后分离的IRI cDNA，将进行测序，并特征。 AIM II的目标是将IRI cDNA探针用于来自基因组文库的分离IRI基因，包括上游调节剂表征响应元件的区域转导的区域电感效应。