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Updated: Oct 27, 2025
Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction
Published on: November 20, 2018
Sites of Cre-recombinase activity in mouse lines targeting skeletal cells
Greig Couasnay1, Maria-Bernadette Madel1, Joohyun Lim2
1Department of Orthopedic Surgery, Baylor College of Medicine, Houston, TX, USA.
Abstract:
The Cre/Lox system is a powerful tool in the biologist's toolbox, allowing loss-of-function and gain-of-function studies, as well as lineage tracing, through gene recombination in a tissue-specific and inducible manner. Evidence indicates, however, that Cre transgenic lines have a far more nuanced and broader pattern of Cre activity than initially thought, exhibiting "off-target" activity in tissues/cells other than the ones they were originally designed to target. With the goal of facilitating the comparison and selection of optimal Cre lines to be used for the study of gene function, we have summarized in a single manuscript the major sites and timing of Cre activity of the main Cre lines available to target bone mesenchymal stem cells, chondrocytes, osteoblasts, osteocytes, tenocytes, and osteoclasts, along with their reported sites of "off-target" Cre activity. We also discuss characteristics, advantages, and limitations of these Cre lines for users to avoid common risks related to overinterpretation or misinterpretation based on the assumption of strict cell-type specificity or unaccounted effect of the Cre transgene or Cre inducers. © 2021 American Society for Bone and Mineral Research (ASBMR).
Insights
The Cre/Lox system offers gene manipulation but Cre transgenic lines show unexpected activity in unintended cells. This review details Cre activity in bone and connective tissue cells to guide researchers in selecting appropriate Cre lines for accurate gene function studies.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
- Skeletal Biology
Background:
- The Cre/Lox recombination system is widely used for genetic manipulation in biological research.
- Cre transgenic lines are essential for loss-of-function, gain-of-function, and lineage tracing studies.
- Concerns exist regarding the specificity of Cre activity, with evidence of "off-target" recombination in unintended cell types.
Purpose of the Study:
- To consolidate information on the activity patterns of major Cre lines targeting bone and connective tissue cells.
- To document both intended and "off-target" Cre activity sites and timing for key skeletal cell types.
- To aid researchers in selecting appropriate Cre lines for precise gene function studies in skeletal biology.
Main Methods:
- Literature review and synthesis of published data on Cre/Lox system activity.
- Compilation of major Cre lines used for targeting bone mesenchymal stem cells, chondrocytes, osteoblasts, osteocytes, tenocytes, and osteoclasts.
- Documentation of reported sites and timing of both on-target and off-target Cre activity for selected Cre lines.
Main Results:
- Cre transgenic lines exhibit broader and more nuanced activity patterns than initially assumed.
- Significant "off-target" Cre activity is reported in various tissues and cell types beyond the intended targets.
- Detailed summaries of Cre activity are provided for specific bone and connective tissue cell lineages.
Conclusions:
- Researchers must be aware of the potential for "off-target" Cre activity when using Cre/Lox systems.
- Careful selection of Cre lines based on comprehensive activity profiles is crucial to avoid misinterpretation of experimental results.
- This review provides essential information for the judicious use of Cre lines in skeletal research.
