NIF LinkOut Portal

Options
Only Pubmed Central
Include Pubmed Central
Sections
Title
Abstract
Introduction
Methods
Results
Supplement
Appendix
Contributions
Background
Commentary
Funding
Limitations
Caption
FILTERS

Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs.

Authors:
te Riele H, Maandag ER, Berns A
Affiliation:
Journal:
Proceedings of the National Academy of Sciences of the United States of America

Abstract

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 divergences 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.

  1. Welcome

    Welcome to NIF. Explore available research resources: data, tools and materials, from across the web

  2. Community Resources

    Search for resources specially selected for NIF community

  3. More Resources

    Search across hundreds of additional biomedical databases

  4. Literature

    Search Pub Med abstracts and full text from PubMed Central

  5. Insert your Query

    Enter your search terms here and hit return. Search results for the selected tab will be returned.

  6. Join the Community

    Click here to login or register and join this community.

  7. Categories

    Narrow your search by selecting a category. For additional help in searching, view our tutorials.

  8. Query Info

    Displays the total number of search results. Provides additional information on search terms, e.g., automated query expansions, and any included categories or facets. Expansions, filters and facets can be removed by clicking on the X. Clicking on the + restores them.

  9. Search Results

    Displays individual records and a brief description. Click on the icons below each record to explore additional display options.

X