多形性腺瘤基因1高表达转基因小鼠动物模型的建立  

Development of salivary gland tumors in pleomorphic adenoma gene 1 transgenic mice

作　　者：赵旭东[1] 杨雯珺[2] 王龙 孔辉 任维华[1] 张梅[4] 费俭 张陈平[2] 王铸钢[1] 

机构地区：[1]上海第二医科大学医学遗传学教研室,200025 [2]上海第二医科大学附属第九人民医院口腔颌面外科 [3]上海南方模式生物研究中心 [4]上海第二医科大学医学病理学教研室,200025

出　　处：《中华医学遗传学杂志》2003年第5期390-395,共6页Chinese Journal of Medical Genetics

基　　金：由国家杰出青年科学基金( 3992 50 2 3);教育部"长江学者奖励计划"( OOTPJS1 1 1 );国家自然科学基金( 30 2 71 42 0 );国家 86 3计划 ( 2 0 0 1 AA2 1 6 0 81 );上海市科学技术发展基金 ( 99JC1 40 2 9;99XD1 40 0 5)

摘　　要：目的　克隆人多形性腺瘤基因 1(pleomorphic adenoma gene1,PL AG1)基因 ,构建 PL AG1组织非特异性和特异性表达载体 ,并建立 PL AG1转基因小鼠模型 ,阐明 PL AG1基因的高表达与唾液腺肿瘤发生的关系。方法　取 PL AG1高表达的瘤组织或正常胎盘组织 ,提取总 RNA,逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)扩增获得 PL AG1c DNA全部编码框序列。将PL AG1分别克隆至 p CMV-EGFP和 p MMTV-EGFR-stop载体获得 p CMV-EGFP/PL AG1和 p MMTV-PL AG1表达载体。用 p CMV-EGFP/PL AG1瞬时转染 NIH3 T3细胞以观察融合基因的表达及 PL AG1的细胞内定位。经显微注射分别获得 p CMV-EGFP/PL AG1和 p MMTV-PL AG1转基因小鼠。结果　从不同组织来源的 RNA克隆的 PL AG1c DNA,经测序后发现与 Gen Bank中的 PL AG1基因 (Gen Bank No.U650 0 2 )比较有一个碱基差 ,相应氨基酸由苏氨酸变为脯氨酸。构建的组织非特异性表达载体 p CMV-EGFP/PL AG1和组织特异性表达载体 p MMTV-PL AG1经酶切和测序鉴定 ,证实 PL AG1序列及插入方向正确。用 p CMV-EGFP/PL AG1转染 NIH3 T3细胞 ,在细胞水平证明 PL AG1的表达及其在细胞核的定位。两种表达载体分别经显微注射 ,获得了多个转基因阳性小鼠Objective: Activation and overexpression of pleomorphic adenoma (PLAG1) gene due to t (3;8)(p21;q12) translocation are associated with the development of human pleomorphic adenomas of the salivary glands. This study was conducted to generate ubiquitously-expressed or tissue-specific expressed PLAG1 transgenic mice and to elucidate the role of PLAG1 gene in tumorigenesis in vivo. Methods: Human PLAG1 cDNA was cloned from salivary gland tumor or placenta tissues by RT-PCR. Ubiquitous expression vector pCMV-EGFP/PLAG1 driven by CMV promoter and tissue-specific expression vector pMMTV-PLAG1 driven by MMTV LTR were constructed. NIH3T3 cells transiently transfected with pCMV-EGFP/PLAG1 showed high expression of PLAG1 in nucleus. Transgenes were microinjected into pronucleus of zygotes to generate transgenic mice. Results: It was found that the human PLAG1 cDNA cloned from several salivary gland tumor and normal placenta tissues consistently showed a variation of a single nucleotide at the same position when compared with the human PLAG1 cDNA sequence in GenBank (Accession No. U65002), which led to T458P at protein level. It might be a single nucleotide polymorphism (SNP)locus. Fused EGFP/PLAG1 protein was found to be localized in the nucleus of NIH3T3 cells transiently transfected with pCMV-EGFP/PLAG1. Several pCMV-EGFP/PLAG1 and pMMTV-PLAG1 transgenic mouse lines were obtained respectively. As might be expected, pMMTV-PLAG1 transgenic mice spontaneously developed salivary gland tumors in three independent lines, among which, line 42 showed tumorigenic phenotype in 100% of transgenic mice within three months after birth. Conclusion: Overexpression of PLAG1 gene plays a crucial role in tumorigenesis of salivary gland tumors.

关 键 词：多形性腺瘤基因1 转基因小鼠 基因表达 动物模型 肿瘤 基因突变 

分 类 号：R-332[医药卫生]