Radial transformation-associated recombination cloning from the mouse genome: Isolation of Tg.AC transgene with flanking DNAs

被引:13
作者
Humble, MC
Kouprina, N
Noskov, VN
Graves, J
Garner, E
Tennant, RW
Resnick, MA
Larionov, V
Cannon, RE
机构
[1] NIEHS, Lab Environm Carcinogenesis & Mutagenesis, Res Triangle Pk, NC 27709 USA
[2] Univ N Carolina, Curriculum Toxicol, Chapel Hill, NC 27514 USA
[3] NIEHS, Mol Genet Lab, Res Triangle Pk, NC 27709 USA
关键词
D O I
10.1006/geno.2000.6384
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Transformation-associated recombination (TAR) cloning allows entire genes and large chromosomal regions to be specifically, accurately, and quickly isolated from total genomic DNA. We report the first example of radial TAR cloning from the mouse genome. Tg.AC mice carry a zeta-globin promoter/v-Ha-ras transgene. Fluorescence in situ hybridization localized the transgene integrant as a single site proximal to the centromere of chromosome 11. Radial TAR cloning in yeast was utilized to create orientation-specific yeast artificial chromosomes (YACs) to explore the possibility that cis-flanking regions were involved in transgene expression, YACs containing variable lengths of 5' or 3' banking chromosome 11 DNA and the Tg.AC transgene were specifically chosen, converted to bacterial artificial chromosomes (BACs), and assayed for their ability to promote transcription of the transgene following transfection into an FVB/N carcinoma cell line. A transgene-specific reverse transcription-polymerase chain reaction assay was utilized to examine RNA transcripts from stably transfected clones. All Tg.AC BACs expressed the transgene in this in vitro system. This report describes the cloning of the v-Ha-ras transgene and suggests that transcriptional activity may not require cis elements flanking the transgene's integration site. (C) 2000 Academic Press.
引用
收藏
页码:292 / 299
页数:8
相关论文
共 29 条
[1]   CENTROMERE ANALYSIS OF MICRONUCLEI INDUCED BY 2-AMINOANTHRAQUINONE IN CULTURED MOUSE SPLENOCYTES USING BOTH A GAMMA-SATELLITE DNA-PROBE AND ANTI-KINETOCHORE ANTIBODY [J].
AFSHARI, AJ ;
MCGREGOR, PW ;
ALLEN, JW ;
FUSCOE, JC .
ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, 1994, 24 (02) :96-102
[2]  
Bates G, 1996, Methods Mol Biol, V54, P139
[3]  
BIRNBOIM HC, 1979, NUCLEIC ACIDS RES, V7, P1513
[4]  
Cannon RE, 1997, MOL CARCINOGEN, V20, P108, DOI 10.1002/(SICI)1098-2744(199709)20:1<108::AID-MC12>3.0.CO
[5]  
2-5
[6]  
Cannon RE, 1998, MOL CARCINOGEN, V21, P244, DOI 10.1002/(SICI)1098-2744(199804)21:4<244::AID-MC3>3.0.CO
[7]  
2-K
[8]  
CARDIFF RD, 1993, AM J PATHOL, V142, P1199
[9]   DNAzol(R): A reagent for the rapid isolation of genomic DNA [J].
Chomczynski, P ;
Mackey, K ;
Drews, R ;
Wilfinger, W .
BIOTECHNIQUES, 1997, 22 (03) :550-553
[10]  
CHOMCZYNSKI P, 1993, BIOTECHNIQUES, V15, P532