Highly Efficient Gene Targeting in Embryonic Stem Cells Through Homologous Recombination with Isogenic DNA Constructs

A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors i...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1992-06, Vol.89 (11), p.5128-5132
Main Authors: Riele, Hein te, Maandag, Els Robanus, Berns, Anton
Format: Article
Language:English
Subjects:
Alleles
Animals
Biological and medical sciences
Biotechnology
Cell lines
Cellular biology
Deoxyribonucleic acid
DNA
DNA probes
Exons
Fundamental and applied biological sciences. Psychology
Gene targeting
Genes
Genes, Retinoblastoma
Genetic engineering
Genetic Engineering - methods
Genetic loci
Genetic technics
Genetics
Homologous recombination
Methods. Procedures. Technologies
Mice
Mice, Inbred Strains
Miscellaneous
Polymorphism, Genetic
Recombination, Genetic
Restriction Mapping
Stem Cells
Synthetic digonucleotides and genes. Sequencing
Transfection
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Summary:A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors in efficient homologous recombination is the use of isogenic DNA to prepare the targeting vectors. Homologous recombination at the retinoblastoma susceptibility gene (Rb) in embryonic stem cells derived from mouse strain 129 was 20-fold more efficient with a 129-derived targeting construct than with a BALB/c-derived construct. The two constructs were identical, except for a number of base sequence divergencies between 129 and BALB/c DNA, including base-pair substitutions, small deletions/insertions, and a polymorphic CA repeat. Transfection with an isogenic DNA construct, containing 17 kilobases of homology, yielded a targeting frequency of 78% (of a total of 20,000 drug-resistant colonies), without the use of an enrichment protocol for homologous recombination. This result indicates that, also in mammalian cells, homologous recombination rather than random integration can be the predominant event.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.89.11.5128