Transforming Mouse Genetics
This technology has transformed mouse genetics by enabling:
Study of essential genes that cause embryonic lethality when deleted globally
Dissection of gene function in specific tissues without confounding systemic effects
Temporal control over gene inactivation to model disease onset or acute loss of function
How Cre Lox Works
The Components
The Cre lox system consists of two components that work together:
Cre Recombinase
A 38 kDa enzyme from bacteriophage P1 that catalyzes recombination between specific DNA sequences. Cre recognizes and binds to LoxP sites, bringing two sites together and catalyzing strand exchange to excise or invert the intervening DNA.
LoxP Sites
34 base pair DNA sequences consisting of two 13 bp inverted repeats flanking an 8 bp asymmetric spacer. The spacer sequence determines directionality. When two LoxP sites are oriented in the same direction, Cre mediated recombination excises the intervening sequence, leaving a single LoxP site.
Recombination Outcomes
The relative orientation of LoxP sites determines the recombination outcome:
| LoxP Orientation | Recombination Outcome |
|---|---|
| Same orientation (direct repeats) | Excision of intervening sequence, leaving single LoxP site |
| Opposite orientation (inverted repeats) | Inversion of intervening sequence |
| Different chromosomes or distant sites | Translocation or large deletion (less efficient) |
Conditional Knockout Mechanism
In a conditional knockout (floxed) allele:
This design enables gene inactivation restricted to specific cell types, tissues, or developmental stages based on the Cre driver used.
LoxP Site Design
Positioning Considerations
Proper LoxP site placement is essential for conditional allele function:
Exon Selection Criteria
The exon or exons flanked by LoxP sites must meet specific criteria:
- Present in all known transcript variants
- Encode essential functional domains
- Deletion causes frameshift in downstream sequence (for single exon targeting)
- No alternative splicing that could bypass the deletion
ingenious targeting laboratory analyzes gene structure, transcript architecture, and protein domains to identify optimal exon targets for each project.
Cre Driver Lines
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.
Neural System
| Cre Driver | Target Population | Applications |
|---|---|---|
| Nestin Cre | Neural progenitors | Pan neural deletion |
| CamKII Cre | Forebrain excitatory neurons | Learning, memory, behavior |
| DAT Cre | Dopaminergic neurons | Parkinson disease models |
| GFAP Cre | Astrocytes | Glial function |
| Olig2 Cre | Oligodendrocytes | Myelination studies |
Immune System
| Cre Driver | Target Population | Applications |
|---|---|---|
| CD4 Cre | T cells | T cell development and function |
| CD19 Cre | B cells | B cell biology |
| LysM Cre | Macrophages, granulocytes | Innate immunity |
| CD11c Cre | Dendritic cells | Antigen presentation |
| Foxp3 Cre | Regulatory T cells | Immune tolerance |
Metabolic Tissues
| Cre Driver | Target Population | Applications |
|---|---|---|
| Albumin Cre | Hepatocytes | Liver metabolism |
| Adiponectin Cre | Adipocytes | Adipose biology |
| Insulin Cre | Pancreatic beta cells | Insulin secretion |
| Villin Cre | Intestinal epithelium | Gut function |
| MyoD Cre | Skeletal muscle | Muscle metabolism |
Other Systems
| Cre Driver | Target Population | Applications |
|---|---|---|
| Myh6 Cre | Cardiomyocytes | Cardiac function |
| Col2a1 Cre | Chondrocytes | Cartilage development |
| Krt14 Cre | Keratinocytes | Skin biology |
| Tie2 Cre | Endothelial cells | Vascular biology |
Inducible Cre Systems
Tamoxifen Inducible (CreERT2)
CreERT2 is a fusion of Cre recombinase with a modified estrogen receptor ligand binding domain. The fusion protein is sequestered in the cytoplasm until tamoxifen administration, which triggers nuclear translocation and Cre activity.
- Gene deletion triggered by tamoxifen injection
- Recombination occurs within days of treatment
- Enables adult onset gene deletion after normal development
- Allows study of acute versus chronic loss of function
Doxycycline Inducible (Tet Systems)
Tetracycline regulated systems use doxycycline to control Cre expression:
- Tet On: Cre expressed in presence of doxycycline
- Tet Off: Cre expressed in absence of doxycycline
- Reversible control (unlike CreERT2 which causes permanent deletion)
- Useful when reversible gene regulation is needed
Combining Tissue and Temporal Control
Tissue specific CreERT2 lines combine spatial and temporal control. For example, Albumin CreERT2 enables tamoxifen inducible deletion specifically in hepatocytes, allowing study of gene function in adult liver without developmental effects.
Applications
Essential Genes
Many genes cause embryonic lethality when deleted globally. Conditional knockout bypasses developmental requirements by restricting gene deletion to specific tissues or to adult stages after development is complete.
Pleiotropic Genes
Genes with functions in multiple organ systems produce complex phenotypes when deleted globally. Tissue specific conditional knockout isolates gene function in individual tissues, clarifying cell autonomous versus secondary effects.
Cell Type Specific Questions
When research focuses on gene function in a specific cell type, conditional knockout provides direct answers without confounding effects from other tissues.
Temporal Studies
Inducible Cre systems enable study of gene function at specific times: during development, in young adults, or in aged animals. This temporal control is valuable for modeling disease onset and studying acute versus chronic phenotypes.
Selected Publications
Cre lox 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
“iTL produced four conditional knockout mouse models on our behalf. They have been extremely helpful and informative at all stages of the project; all the way from construct design to breeding strategies and genotyping the new mouse models. I know where to turn when the needs comes up again for another mouse project; it is certainly faster and cheaper than doing this by ourselves.”
— William A. Coetzee, DSc
NYU School of Medicine
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