TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques
dc.contributor.author | Friis-Hansen, Lennart | en_US |
dc.contributor.author | Gillespie, Patrick J. | en_US |
dc.contributor.author | Samuelson, Linda C. | en_US |
dc.contributor.author | Lay, Jean M. | en_US |
dc.date.accessioned | 2006-09-11T13:56:25Z | |
dc.date.available | 2006-09-11T13:56:25Z | |
dc.date.issued | 1998-03 | en_US |
dc.identifier.citation | Lay, Jean M.; Friis-Hansen, Lennart; Gillespie, Patrick J.; Samuelson, Linda C.; (1998). "TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques." Transgenic Research 7(2): 135-140. <http://hdl.handle.net/2027.42/43859> | en_US |
dc.identifier.issn | 1573-9368 | en_US |
dc.identifier.issn | 0962-8819 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/43859 | |
dc.identifier.uri | http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=9608741&dopt=citation | en_US |
dc.description.abstract | Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells | en_US |
dc.format.extent | 467881 bytes | |
dc.format.extent | 3115 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.language.iso | en_US | |
dc.publisher | Kluwer Academic Publishers; Chapman and Hall ; Springer Science+Business Media | en_US |
dc.subject.other | Biomedicine | en_US |
dc.subject.other | Human Genetics | en_US |
dc.subject.other | Animal Anatomy / Morphology / Histology | en_US |
dc.subject.other | Polymerase Chain Reaction | en_US |
dc.subject.other | Stem Cells | en_US |
dc.subject.other | Gene Targeting | en_US |
dc.subject.other | Gastrin | en_US |
dc.subject.other | Cholecystokinin | en_US |
dc.subject.other | Plant Sciences | en_US |
dc.title | TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques | en_US |
dc.type | Article | en_US |
dc.subject.hlbsecondlevel | Biological Chemistry | en_US |
dc.subject.hlbsecondlevel | Genetics | en_US |
dc.subject.hlbsecondlevel | Molecular, Cellular and Developmental Biology | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.subject.hlbtoplevel | Health Sciences | en_US |
dc.description.peerreviewed | Peer Reviewed | en_US |
dc.contributor.affiliationum | Department of Physiology, The University of Michigan, Ann Arbor, USA | en_US |
dc.contributor.affiliationum | Cellular Molecular Biology Program, The University of Michigan, Ann Arbor, USA | en_US |
dc.contributor.affiliationum | Cellular Molecular Biology Program, The University of Michigan, Ann Arbor, USA | en_US |
dc.contributor.affiliationum | Department of Physiology, The University of Michigan, Ann Arbor, USA | en_US |
dc.contributor.affiliationumcampus | Ann Arbor | en_US |
dc.identifier.pmid | 9608741 | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/43859/1/11248_2004_Article_172516.pdf | en_US |
dc.identifier.doi | http://dx.doi.org/10.1023/A:1008876526826 | en_US |
dc.identifier.source | Transgenic Research | en_US |
dc.owningcollname | Interdisciplinary and Peer-Reviewed |
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