A novel expression system for genomic DNA loci using a human artificial chromosome vector with transformation-associated recombination cloning.
Following the recent completion of the human genome sequence, genomics research has shifted its focus to understanding gene complexity, expression, and regulation. However, in order to investigate such issues, there is a need to develop a practical system for genomic DNA expression. Transformation-associated recombination (TAR) cloning has proven to be a convenient tool for selective isolation of a genetic locus from a complex genome as a circular YAC using recombination in yeast. The human artificial chromosome (HAC) vector containing an acceptor loxP site has served as a platform for the reproducible expression of transgenes. In this study, we describe a system that efficiently expresses a genetic locus in mammalian cells by retrofitting a TAR-YAC with the donor loxP site and loading it onto the HAC vector by the Cre/loxP system. In order to demonstrate functional expression of genomic loci, the entire human hypoxanthine phosphoribosyl transferase (HPRT) locus contained in a 100 kb YAC was loaded onto the HAC vector and was shown to complement the genetic defect in Hprt-deficient CHO cells. Thus, the combination of TAR cloning and the HAC vector may serve as a powerful tool for functional genomic studies.
Ayabe
Fumiaki
F
Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.
Katoh
Motonobu
M
Department of Human Genome Science, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.
Inoue
Toshiaki
T
Department of Human Genome Science, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.
Kouprina
Natalay
N
Laboratory of Biosystems and Cancer, National Cancer Institute, NIH, Bldg. 37, Room 5032, 90000 Rockville Pike, Bethesda, MD, 20892, USA.
Larionov
Vladimir
V
Laboratory of Biosystems and Cancer, National Cancer Institute, NIH, Bldg. 37, Room 5032, 90000 Rockville Pike, Bethesda, MD, 20892, USA.
Oshimura
Mitsuo
M
Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan. oshimura@grape.med.tottori-u.ac.jp.|Department of Human Genome Science, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan. oshimura@grape.med.tottori-u.ac.jp.
D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D016521:Electrophoresis, Gel, Pulsed-Field; D020869:Gene Expression Profiling / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics*; D015183:Restriction Mapping; D014170:Transformation, Genetic / Q000235:genetics*
D017931:DNA Primers; D007041:Hypoxanthine Phosphoribosyltransferase