title: From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.
abstract: Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.
authors.affiliation: Laboratory of Molecular Pharmacology, National Cancer Institute (NCI, NIH), Bldg 37 Room 4 E28, 37 Covent Drive, Bethesda, MD, 20892-4255, USA, kouprinn@mail.nih.gov.
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
authors.forename: Nicholas C O
authors.initials: NC
authors.lastname: Lee
authors.forename: Artem V
authors.initials: AV
authors.lastname: Kononenko
authors.forename: Alexander
authors.initials: A
authors.lastname: Samoshkin
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
mesh_terms: D000818:Animals; D016466:CHO Cells; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry / Q000378:metabolism; D022222:Chromosomes, Artificial, Human / Q000737:chemistry / Q000378:metabolism; D018244:Chromosomes, Artificial, Yeast / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003412:Cricetulus; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013104:Spheroplasts / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D004271:DNA, Fungal