Tags

Type your tag names separated by a space and hit enter

Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange.
J Mol Biol. 1999 Oct 01; 292(4):779-85.JM

Abstract

Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.

Authors+Show Affiliations

Department of Medicine/Division of Hematology, Albert Einstein College of Medicine, Bronx, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10525404

Citation

Feng, Y Q., et al. "Site-specific Chromosomal Integration in Mammalian Cells: Highly Efficient CRE Recombinase-mediated Cassette Exchange." Journal of Molecular Biology, vol. 292, no. 4, 1999, pp. 779-85.
Feng YQ, Seibler J, Alami R, et al. Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J Mol Biol. 1999;292(4):779-85.
Feng, Y. Q., Seibler, J., Alami, R., Eisen, A., Westerman, K. A., Leboulch, P., Fiering, S., & Bouhassira, E. E. (1999). Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. Journal of Molecular Biology, 292(4), 779-85.
Feng YQ, et al. Site-specific Chromosomal Integration in Mammalian Cells: Highly Efficient CRE Recombinase-mediated Cassette Exchange. J Mol Biol. 1999 Oct 1;292(4):779-85. PubMed PMID: 10525404.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. AU - Feng,Y Q, AU - Seibler,J, AU - Alami,R, AU - Eisen,A, AU - Westerman,K A, AU - Leboulch,P, AU - Fiering,S, AU - Bouhassira,E E, PY - 1999/10/20/pubmed PY - 1999/10/20/medline PY - 1999/10/20/entrez SP - 779 EP - 85 JF - Journal of molecular biology JO - J Mol Biol VL - 292 IS - 4 N2 - Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/10525404/Site_specific_chromosomal_integration_in_mammalian_cells:_highly_efficient_CRE_recombinase_mediated_cassette_exchange_ DB - PRIME DP - Unbound Medicine ER -