LoxP Site Design
Since 1998, ingenious targeting laboratory has designed and implemented LoxP sites in over 1,500 conditional alleles, establishing precise frameworks for Cre mediated recombination that enable spatial and temporal control of gene expression.
LoxP site design determines the success of conditional knockout and knockin strategies. Proper positioning ensures normal gene function prior to exposure to Cre.
LoxP Site Structure
The LoxP sequence is a 34 base pair DNA element recognized by Cre recombinase:
ATAACTTCGTATA GCATACAT TATACGAAGTTATInverted Repeats
Size: 13 bp each
Function: Cre binding sites
Spacer Region
Size: 8 bp
Function: Determines orientation and outcome
Positioning Guidelines
Distance from Splice Sites
At least 100 to 200 bp from exon boundaries to avoid disrupting splicing
Avoid Regulatory Elements
Introns contain enhancers, silencers, and branch points that must be avoided
Minimum Floxed Region
500 bp to 5 kb optimal; regions under 200 bp reduce efficiency
Maximum Floxed Region
Regions over 10 kb can recombine but with decreased efficiency
Common Design Patterns
Single Exon Floxing
Simplest approach when one exon contains catalytic domain, is in all isoforms, and deletion causes frameshift
Multi Exon Floxing
Required when gene has multiple functional domains, single exon maintains frame, or isoform specific exons exist
First Coding Exon
Can eliminate all downstream sequence and possibility of truncated functional proteins.
Common Design Pitfalls
Hypomorphic Alleles
Cause: LoxP sites or cassette disrupt regulatory elements before Cre exposure
Prevention: Careful intronic placement. Test floxed allele homozygotes for normal phenotype.
Incomplete Null After Recombination
Cause: Floxed exon not essential, alternative splicing bypasses region, or in frame deletion preserves function
Prevention: Thorough gene structure analysis. Target functionally essential domains.
Inefficient Recombination
Cause: LoxP sites too close together, chromatin inaccessibility, or low Cre expression
Prevention: Optimal spacing (500 bp to 5 kb). Use robust Cre drivers.
What Researchers Say
“I've been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, but the entire process was transparent with the opportunity at any time along the way to discuss my questions or concerns with scientists who had significant insight into the process. The mice were delivered on time, as billed!”
— Raghu Mirmira, MD, PhD
University of Chicago
Start Your LoxP Design Project
Our scientific team evaluates each gene for LoxP placement, including gene structure review, exon essentiality analysis, and frameshift verification.