The target genotype
For a standard conditional knockout study you need two groups from the same crosses:
Experimental
homozygous floxed, Cre positive
Littermate control
homozygous floxed, Cre negative
Both carry the floxed allele. Only one carries the recombinase. That is what helps isolate the effect of gene deletion from the effect of the floxed allele itself.
The cross path
Genotype notation used below: Flox slash plus means one floxed allele and one wild type allele. Flox slash Flox means two floxed alleles, that is homozygous floxed. Cre slash plus means one copy of the Cre transgene. The letter x between two genotypes means crossed to.
Generation 1
Cross a Cre driver line to the floxed line. Flox/+ crossed to Cre/+ produces compound heterozygotes carrying one floxed allele and the Cre transgene at an expected 50 percent.
Generation 2
Cross the compound heterozygote to a homozygous floxed animal. Flox/+ Cre/+ crossed to Flox/Flox produces the experimental genotype, Flox/Flox Cre/+, at an expected 25 percent, and the matched control genotype, Flox/Flox Cre negative, at an expected 25 percent. Half of the offspring are expected to be homozygous floxed.
Generation 3
Expand from confirmed Flox/Flox Cre/+ and Flox/Flox breeders to produce the cohort at scale, age matched to your study window.
Standard C57BL/6 reproductive parameters put gestation at 19 to 21 days, weaning at 21 days, and breeding age at 6 to 8 weeks, so one generation runs about 10 to 12 weeks. Three generations plus growth to study age can span several months, depending on the starting genotypes and breeding scheme.
Expected yields
| Cross | Experimental yield, Flox/Flox Cre positive | Control yield, Flox/Flox Cre negative | Notes |
|---|---|---|---|
| Flox/+ x Cre/+ | 0 percent | 0 percent | Generation 1 only, produces the compound heterozygote at 50 percent |
| Flox/+ Cre/+ x Flox/Flox | 25 percent | 25 percent | Recommended route, half of each litter usable |
| Flox/+ Cre/+ x Flox/+ Cre/+ | 12.5 percent | 12.5 percent | Fewer lines to maintain, lower yield |
| Flox/Flox Cre/+ x Flox/Flox | 50 percent | 50 percent | Production cross once breeders are confirmed |
| Any cross with linked loci | Frequency depends on parental haplotypes and recombination rate | Frequency depends on parental haplotypes and recombination rate | Independent assortment does not apply |
The double heterozygote intercross looks efficient because it needs one fewer line, but it yields the experimental genotype at 12.5 percent rather than 25 percent. Halve again for a single sex requirement and you are holding a large number of animals to produce a small usable group. We recommend the Flox/+ Cre/+ crossed to Flox/Flox route whenever homozygous floxed breeders are available.
Send us the floxed allele and the Cre driver, and we will return the cross path with expected yields.
Request a Breeding SchemeFive things that break conditional knockout breeding
1. Linkage between the floxed allele and the Cre transgene
If the floxed locus and the Cre insertion site sit on the same chromosome, independent assortment does not apply. The expected frequency of the target genotype then depends on the parental haplotypes and recombination frequency. Programs that discover this after setting pairs lose a generation and sometimes a year.
We check the chromosomal position of the Cre insertion against the floxed locus during scheme design. If they are linked, the scheme changes before any pair is set.
2. Cre transmitted through the wrong parent
Some Cre lines deposit maternal Cre protein or RNA in the oocyte, which can drive recombination in the early embryo regardless of the offspring genotype. The result can be unintended recombination in tissues or cells beyond the intended Cre expression pattern, and it may not be detected by a standard tail genotype.
Where a driver has documented maternal effect, transmit Cre through the male. We set the scheme so the Cre carrying parent is the sire.
3. Germline recombination
Several tissue restricted Cre drivers show low level activity in the germline. Once recombination happens in a germ cell, the deleted allele is transmitted to the next generation as a constitutive null. That can alter the intended conditional genotype across generations.
This is why breeding stock genotypes need a deleted allele assay, not only a flox assay. We include it.
4. Controls bred separately
Controls from a different colony, room, or shipment can differ in microbiome, handling, and background. Those differences can affect behavior, metabolism, and immune phenotypes. Controls should ideally come from the same crosses and the same room.
5. Assuming induction equals deletion
For tamoxifen inducible CreER systems, recombination efficiency varies by tissue, by dose, by age at induction, and between animals. Tamoxifen itself has biological effects, so appropriate vehicle-treated controls are important alongside genotype controls. Plan the cohort around the number of animals you will need after confirming the expected recombination efficiency.
Choosing a Cre driver
Driver selection changes the biology of the experiment more than any other choice in the scheme. Points to settle before breeding:
- Expression pattern in your tissue of interest, confirmed by a reporter cross rather than assumed from the publication
- Off target expression in tissues that could confound the phenotype
- Constitutive versus inducible, and whether developmental deletion would be lethal or compensated
- Documented germline or maternal activity
- Chromosomal position relative to your floxed locus
- Availability, and whether the driver is a knockin at the endogenous locus or a random transgenic
A reporter cross before the main scheme costs one generation and prevents a study built on a driver that does not do what the paper reported.
See our Cre/loxP technology overviewGenotyping requirements
A conditional knockout colony typically requires three genotyping assays:
- Floxed allele assay distinguishing wild type, heterozygous, and homozygous floxed
- Cre assay confirming presence of the transgene
- Deleted allele assay detecting recombination that occurred where it should not have
We design and validate assays for new alleles, establish positive and negative controls, and document the protocol so your lab can run it after transfer.
See genotyping servicesPlan your scheme
The Breeding Scheme Architect calculates expected genotype ratios and generations to target for a single allele, at no cost and without an account. For two locus conditional schemes, send us the output and a scientist will extend it.
Try the Breeding Scheme ArchitectWhat researchers say
“iTL produced a new conditional mouse model for us and the quality of service was exceptional. The team is extremely knowledgeable and the work was completed at the highest possible standards. My project manager was excellent and always happy to answer technical questions and keep me up to date with progress and potential problems. I would recommend iTL highly and will use them again in the future if I need to generate a new mouse line.”
— Albert Basson, PhD
King's College London
“ingenious Targeting Laboratory is highly recommended for generating animal model generation. 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
Frequently asked questions
Request a breeding scheme
Send us the floxed allele, the Cre driver, your target n, and your study date. A PhD scientist will return a cross path with expected yields, a pair count, and a schedule.
Request a Breeding Scheme