Radial transformation-associated recombination cloning from the mouse genome: isolation of Tg.AC transgene with flanking DNAs.
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' flanking 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.
Humble
M C
MC
Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514, USA.
Kouprina
N
N
Noskov
V N
VN
Graves
J
J
Garner
E
E
Tennant
R W
RW
Resnick
M A
MA
Larionov
V
V
Cannon
R E
RE
D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D016678:Genome; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D019076:Transgenes
D017931:DNA Primers; D004247:DNA