Two vectors for the insertion of mammalian selectable genes into yeast artificial chromosome cloned DNA.
The introduction of cloned DNA into mammalian cells allows functional testing of genes contained on the fragments. In many cases, the exogenous DNA introduced into mammalian cells requires selectable genes that mark the presence of input DNA. Two new vectors, carrying mammalian selectable markers encoding for either neomycin-resistance (neo) or histidinol-resistance (hol), have been constructed for targeted integration to specific single-copy sites within yeast artificial chromosome (YAC) insert DNA. The integration cassettes comprise a single selectable yeast gene adjacent to a mammalian selectable gene, either LEU2 with neo or HIS3 with hol. Modification of the YAC occurs in yeast by transfection with linear DNA containing YAC-specific, unique, recombinogenic ends, thereby ensuring co-integration of the markers. Analysis of modified YACs confirms that both vectors correctly integrate into the targeted unique sites. The precise localization of selectable marker genes in the cloned DNA ensures the integrity of the genomic fragments during functional testing. Placement of mammalian selectable markers within the YAC insert DNA should allow for YAC-based gene targeting experiments in a variety of mammalian cell lines.
Tucker
R M
RM
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109, USA.
Burke
D T
DT
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular; D018390:Gene Targeting / Q000379:methods*; D005796:Genes / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006836:Hydro-Lyases / Q000235:genetics; D019868:Kanamycin Kinase / Q000235:genetics; D008322:Mammals; D051379:Mice; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001426:Bacterial Proteins; D005819:Genetic Markers; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D050539:3-Isopropylmalate Dehydrogenase; D019868:Kanamycin Kinase; D006836:Hydro-Lyases; C021434:imidazoleglycerolphosphate dehydratase