Full Length Research Paper

Construction and characterization of a cDNA library from human brain glioma cell line U251 with overexpressed exogenous p53 gene

Kun-xian Shu¹, Yan-zhe Li¹, Hong-bo Shao^2,3,5*, Ming-hua Zhang⁴ and Li-xiang Wu⁴

¹College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing 400065, China.

²Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources , Yangling 712100, China.

³Yantai Institute of Costal Zone Research, Chinese Academy of Sciences, Yantai 264003, China.

⁴Xiangya School of Medicine, Central South University, Changsha 410008, China.

⁵Institute for Life Sciences, Qingdao University of Science and Technology, Qingdao 266042, China.

*Corresponding author. E-mail: shaohongbochu@126.com.  

Tel: +86 532 84023984.

Abbreviations: LDPCR, Long distance polymerase chain reaction; MCK, muscle creatine kinase; MDM2, murine double minute 2; PCNA, proliferating cell nuclear antigen; PGM, phosphoglycerate mutase; TIGAR, TP53-induced glycolysis and apoptosis regulator; SCO2, synthesis of cytochrome c oxidase 2; TRE, tetracycline-response element; Dox, doxycycline; FBS, fetal bovine serum; PBS, phosphate buffered saline; DSN, duplex-specific nuclease; ds, double stranded; PCR, polymerase chain reaction.

Accepted 28 July, 2010

   Abstract

The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain glioma cell line U251 while exogenous p53 gene is overexpressed. The constructed cDNA library contained 1.3×10⁶ directional recombinants, and its insert size ranged from 0.5 to 2.0 kb. Screening the cDNA library, we obtained two novel p53 downstream genes, PAP1 and PAP2. Polymerase chain reaction (PCR) analyses of the library for specific genes revealed the presence of cDNAs for p53 downstream genes such as p21, gadd45, and PCNA. These results demonstrate the sequence complexity and relatively low redundancy of our cDNA library. It is a valuable and unique resource for studying p53 gene expression, regulatory mechanisms and screening p53 downstream genes.

Key words: p53 Gene, p53 downstream gene, cDNA library, normalization.