Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 285-288 |
| Number of pages | 4 |
| Journal | Genomics |
| Volume | 62 |
| Issue number | 2 |
| DOIs | |
| State | Published - Dec 1 1999 |
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All Science Journal Classification (ASJC) codes
- Genetics
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Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper. / Bhargava, Jaya; Shashikant, Cooduvalli S.; Carr, Janet L.; Juan, Hsin; Bentley, Kevin L.; Ruddle, Frank H.
In: Genomics, Vol. 62, No. 2, 01.12.1999, p. 285-288.Research output: Contribution to journal › Article
TY - JOUR
T1 - Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper
AU - Bhargava, Jaya
AU - Shashikant, Cooduvalli S.
AU - Carr, Janet L.
AU - Juan, Hsin
AU - Bentley, Kevin L.
AU - Ruddle, Frank H.
PY - 1999/12/1
Y1 - 1999/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0033369150&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033369150&partnerID=8YFLogxK
U2 - 10.1006/geno.1999.6000
DO - 10.1006/geno.1999.6000
M3 - Article
C2 - 10610723
AN - SCOPUS:0033369150
VL - 62
SP - 285
EP - 288
JO - Genomics
JF - Genomics
SN - 0888-7543
IS - 2
ER -