This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hanson, K. D.
Right arrow Articles by Sedivy, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hanson, K. D.
Right arrow Articles by Sedivy, J. M.

 Previous Article  |  Next Article 

Mol. Cell. Biol., 01 1995, 45-51, Vol 15, No. 1
Copyright © 1995, American Society for Microbiology

Analysis of biological selections for high-efficiency gene targeting

KD Hanson and JM Sedivy
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.

A two-marker selection system that allows the efficient isolation of diploid gene knockouts by two sequential rounds of targeted homologous recombination has been developed. A systematic evaluation of the biological parameters that govern the selection process showed that a successful strategy must match the expression level of the target gene, the efficacy of the marker, and the selection stringency. An enrichment ratio of 5,000- to 10,000-fold, which resulted in a 30% targeting efficiency of the c-myc gene in a fibroblast cell line, has been achieved. Such efficiency brings the difficulty of gene targeting effectively down to the level of simple transfections, since only 10 to 20 drug-resistant clones need to be screened to recover several homologous hits. The general utility of the targeting strategy is of interest to investigators studying gene function in a large variety of mammalian tissue culture systems.


This article has been cited by other articles:

  • Endo, M., Osakabe, K., Ichikawa, H., Toki, S. (2006). Molecular Characterization of True and Ectopic Gene Targeting Events at the Acetolactate Synthase Gene in Arabidopsis. Plant Cell Physiol 47: 372-379 [Abstract] [Full Text]  
  • Topaloglu, O., Hurley, P. J., Yildirim, O., Civin, C. I., Bunz, F. (2005). Improved methods for the generation of human gene knockout and knockin cell lines. Nucleic Acids Res 33: e158-e158 [Abstract] [Full Text]  
  • Piedrahita, J. A., Mir, B., Dindot, S., Walker, S. (2004). Somatic Cell Cloning: The Ultimate Form of Nuclear Reprogramming?. J. Am. Soc. Nephrol. 15: 1140-1144 [Abstract] [Full Text]  
  • Mir, B., Piedrahita, J. A. (2004). Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts. Nucleic Acids Res 32: e25-e25 [Abstract] [Full Text]  
  • Kohli, M., Rago, C., Lengauer, C., Kinzler, K. W., Vogelstein, B. (2004). Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res 32: e3-e3 [Abstract] [Full Text]  
  • Hostager, B. S., Haxhinasto, S. A., Rowland, S. L., Bishop, G. A. (2003). Tumor Necrosis Factor Receptor-associated Factor 2 (TRAF2)-deficient B Lymphocytes Reveal Novel Roles for TRAF2 in CD40 Signaling. J. Biol. Chem. 278: 45382-45390 [Abstract] [Full Text]  
  • Vasquez, K. M., Marburger, K., Intody, Z., Wilson, J. H. (2001). Manipulating the mammalian genome by homologous recombination. Proc. Natl. Acad. Sci. USA 98: 8403-8410 [Abstract] [Full Text]  
  • Luo, Z., Macris, M. A., Faruqi, A. F., Glazer, P. M. (2000). High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells. Proc. Natl. Acad. Sci. USA 10.1073/pnas.160004997v1 [Abstract] [Full Text]  
  • Faruqi, A. F., Datta, H. J., Carroll, D., Seidman, M. M., Glazer, P. M. (2000). Triple-Helix Formation Induces Recombination in Mammalian Cells via a Nucleotide Excision Repair-Dependent Pathway. Mol. Cell. Biol. 20: 990-1000 [Abstract] [Full Text]  
  • Sedivy, J. M., Vogelstein, B., Liber, H. L., Hendrickson, E. A., Rosmarin, A. (1999). Gene Targeting in Human Cells Without Isogenic DNA. Science 283: 9a-9 [Abstract] [Full Text]  
  • Brown, J. P., Wei, W., Sedivy, J. M. (1997). Bypass of Senescence After Disruption of p21CIP1/WAF1 Gene in Normal Diploid Human Fibroblasts. Science 277: 831-834 [Abstract] [Full Text]  
  • Datta, H. J., Chan, P. P., Vasquez, K. M., Gupta, R. C., Glazer, P. M. (2001). Triplex-induced Recombination in Human Cell-free Extracts. DEPENDENCE ON XPA AND HsRad51. J. Biol. Chem. 276: 18018-18023 [Abstract] [Full Text]  
  • Luo, Z., Macris, M. A., Faruqi, A. F., Glazer, P. M. (2000). High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells. Proc. Natl. Acad. Sci. USA 97: 9003-9008 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»