When to Use Conditional Knockout
Floxed mouse models provide maximum experimental flexibility. A single conditional allele can generate tissue specific knockouts across dozens of organ systems simply by crossing to different Cre driver lines.
Essential Genes
Many genes are essential for embryonic development. Global knockout of these genes results in embryonic lethality. Conditional knockout allows bypass of developmental requirements by restricting gene deletion to specific tissues or adult stages.
Pleiotropic Genes
Genes with functions in multiple organ systems can produce complex phenotypes when deleted globally. Tissue specific conditional knockout isolates gene function in individual tissues, clarifying which phenotypes are cell autonomous.
Cell Type Specific Questions
When the research question focuses on gene function in a specific cell type, conditional knockout provides direct answers without confounding effects from other tissues.
How Conditional Knockout Works
The Cre Lox System
Conditional knockout relies on the Cre lox recombination system. LoxP sites are 34 base pair DNA sequences recognized by Cre recombinase. When two LoxP sites flank a DNA segment in the same orientation, Cre recombinase excises the intervening sequence, leaving a single LoxP site.
In a conditional knockout allele, LoxP sites flank one or more critical exons of the target gene. The floxed allele functions normally in the absence of Cre. When crossed to a Cre driver line, cells expressing Cre recombinase excise the floxed exons, creating a null allele in those specific cells while gene function is preserved elsewhere.
Learn more about Cre Lox SystemTissue Specific Gene Deletion
Tissue specificity is achieved through Cre driver lines that express Cre recombinase under tissue specific promoters. Hundreds of Cre driver lines are available, targeting virtually every organ system and cell type.
Albumin Cre
Hepatocyte specific deletion
Nestin Cre
Neural progenitor and CNS deletion
LysM Cre
Myeloid cell specific deletion (macrophages, granulocytes)
CD4 Cre
T cell specific deletion
Myh6 Cre
Cardiomyocyte specific deletion
Villin Cre
Intestinal epithelium specific deletion
Inducible Gene Deletion
Temporal control is achieved using inducible Cre systems. The most common approach uses CreERT2, a fusion of Cre recombinase with a modified estrogen receptor that is inactive until tamoxifen administration.
- Trigger gene deletion at any developmental stage or in adult animals
- Study gene function after normal development is complete
- Investigate acute versus chronic phenotypes
- Model disease onset in mature animals
Allele Design and Configuration
Critical Exon Selection
Successful conditional knockout requires careful selection of exons to flank with LoxP sites. The targeted exon or exons must be essential for gene function.
- Exons present in all transcript variants
- Exons encoding essential functional domains
- Exon deletion that causes frameshift in downstream sequence
- Avoidance of alternative splicing that could bypass the deletion
Conditional Knockout with Reporter
Adding a reporter gene to your conditional knockout model enables visualization of when and where gene inactivation occurs, providing confirmation at the single cell level.
- Inverted Reporter Strategy: Reporter placed in opposite orientation, activated when Cre inverts the region
- Post Exon Reporter: Reporter placed after the last exon, activated after Cre mediated deletion
- Dual Reporter Systems: One reporter expressed with the gene, a different reporter expressed after knockout
Derivative Allele System
A standard stop cassette configuration enables generation of four derivative alleles from a single targeted mouse line:
Knockout First
Original targeted allele with full cassette. Gene is disrupted by stop cassette insertion.
Conditional Ready
Flp excision removes the cassette while leaving LoxP sites flanking the critical exon.
Conditional Null
Cre excision removes the critical exon, creating the tissue specific null allele.
TruView Conditional Knockout™
ingenious targeting laboratory's proprietary TruView technology provides strong reporter expression after knockout and can be used with genes of almost any size.
- Visualization of recombined cells regardless of target gene expression levels
- All from one target gene locus
- Demonstration of successful knockout
- Labeling of affected cells and tissues
Selected Publications
Conditional knockout models generated by ingenious targeting laboratory:
Wang L, Noyer L, Jishage M, Wang YH, Tao AY, McDermott M, et al. (2025).
CLNS1A regulates genome stability and cell cycle progression to control CD4 T cell function and autoimmunity. ↗Sci Immunol 10(108): eadq8860
Clausen BE et al. (1999).
Conditional gene targeting in macrophages and granulocytes using LysMcre mice. ↗Transgenic Research 8(4): 265-277
What Researchers Say
“ingenious Targeting Laboratory is highly recommended for generating custom animal models. Past 2 years, we have made 2 conditional knockout mouse lines. All processes of each project were scientifically and professionally handled. Their scientific consulting to initiate the project was superb compared to other companies, and transparency of the project progress reported by project managers was excellent. Their excellency and dedication to meet our needs in a timely manner are invaluable to continuation of our research progress.”
— Hyekyung Plumley, PhD
Warren Center for Neuroscience Drug Discovery
Start Your Conditional Knockout Project
Our scientific consultants are ready to discuss your research requirements and recommend the optimal conditional allele design for your experimental goals. Initial consultation is provided at no charge and includes exon analysis, configuration recommendations, and Cre driver line guidance.
Key Terms
Understanding the terminology used in conditional knockout mouse model generation helps you communicate effectively with our scientific team and interpret project documentation.
Conditional Knockout
A gene targeting strategy where gene inactivation is controlled by a recombinase system, allowing tissue-specific or temporally-controlled gene deletion.
Floxed Allele
A gene with LoxP sites flanking a critical region. The allele functions normally until Cre recombinase excises the flanked region.
Cre Recombinase
A site-specific recombinase enzyme that catalyzes recombination between LoxP sites, used in conditional knockout systems.
LoxP Site
A 34 base pair DNA sequence recognized by Cre recombinase. Two LoxP sites in the same orientation enable excision of the flanked sequence.
Conditional Knockout Resources
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Frequently Asked Questions
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