Direct Cloning of Genomic DNA by Recombinogenic Targeting Method Using a Yeast–Bacterial Shuttle Vector, pClasper

We have developed a method to clone genomic DNA selectively into a yeast–bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region t...

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Bibliographic Details
Published in:Genomics (San Diego, Calif.) Calif.), 1999-12, Vol.62 (2), p.285-288
Main Authors: Bhargava, Jaya, Shashikant, Cooduvalli S., Carr, Janet L., Juan, Hsin, Bentley, Kevin L., Ruddle, Frank H.
Format: Article
Language:English
Subjects:
Animals
Antigens, CD - genetics
Biological and medical sciences
Cell Line
Chromosomes, Bacterial - genetics
Cloning, Molecular - methods
Diverse techniques
DNA, Circular - genetics
Fundamental and applied biological sciences. Psychology
Gene Targeting - methods
Genetic Vectors - chemistry
Genetic Vectors - genetics
Humans
Mice
Molecular and cellular biology
Receptors, Adrenergic, beta-2 - genetics
Receptors, Tumor Necrosis Factor - genetics
Receptors, Tumor Necrosis Factor, Type I
Recombination, Genetic
Saccharomyces cerevisiae - genetics
Transfection
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Summary:We have developed a method to clone genomic DNA selectively into a yeast–bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region to be cloned. Linearized, recombinogenic pClasper targeting vector and native genomic DNA were cotransformed into yeast. The gene of interest is selectively cloned by recombination between the recombinogenic ends in the targeting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene construct from a mouse embryo fibroblast cell line and single-copy genes from total human genomic DNA. The frequency of capture of the recombinant insert was 0.05–3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous replicating sequences. This approach provides a powerful method with which to clone and modify genes precisely for functional analysis.
ISSN:0888-7543
1089-8646
DOI:10.1006/geno.1999.6000