GloNeuro

Virtual laboratory / DNA and genes

Breeding a conditional knockout line

A Cre–loxP breeding programme planned week by week, from founder crosses to an age-matched cohort with littermate controls, with a count of the surplus animals.

Guided experiment: Breeding and genotyping a mouse line.

Breeding a conditional knockout line

A conditional knockout removes a gene only in chosen cells. The gene, or an essential part of it, is flanked by loxP sites (a floxed allele), and the Cre recombinase of bacteriophage P1 cuts out the DNA between two lox sites that lie in the same orientation; Sauer and Henderson showed that Cre promotes this site-specific recombination between lox sites in a mammalian cell line (Sauer and Henderson, 1988). When Cre is expressed from a cell type-specific transgene, the deletion happens only in those cells: Gu and colleagues deleted a segment of the DNA polymerase beta gene in T cells in this way, whereas deleting it in the germline is lethal, so the conditional approach allowed viable mutants to be studied (Gu et al., 1994). Producing animals that carry both the Cre transgene and two floxed alleles, together with suitable controls, requires a planned breeding programme over several generations.

Design the experiment

Questions to investigate

  1. How many weeks does it take to reach 8 experimental and 8 control mice of both sexes with each strategy, and how much does this vary between programmes?
  2. Compare Cre+; fl/+ × fl/fl with first breeding Cre+; fl/fl animals. Which gives the cohort sooner, and which produces fewer surplus animals?
  3. Narrow the age window. Why does the time to the cohort rise steeply, even when enough mice of each genotype have been born in total?
  4. Why can Cre+; +/+ littermate controls not come from a Cre+; fl/+ × fl/fl cross, and what does the Cre+; fl/+ × Cre−; fl/+ cross cost instead?
  5. Link the Cre transgene to the floxed locus. How does the recombination fraction change the proportion of Cre+; fl/fl pups?
  6. Switch on germline deletion with an assay that cannot detect the deleted allele. Which mice are misclassified, and how can the problem be detected?

In a real laboratory

A breeding programme starts with a written plan approved by an Institutional Animal Ethics Committee (in India, under the CCSEA and the Prevention of Cruelty to Animals Act 1960; elsewhere, the equivalent national body): the cohort needed, the crosses, the number of breeding cages and when breeding will stop. Founders come from colonies of known health status, and each line is kept on a defined genetic background. Every pup is identified and genotyped, usually from an ear or tail sample at weaning, and the register records its parents, litter, birth date, sex, genotype and fate. Experimental animals are compared with littermate controls, tested in random order and by an experimenter blind to genotype. New Cre lines are checked for germline recombination and for effects of the sex of the Cre parent. Colony records are reviewed to reduce the number of surplus animals, as the 3Rs require, and studies are reported following the ARRIVE guidelines (Percie du Sert et al., 2020).

How the simulation works

Each mouse carries two copies of the floxed locus, a hemizygous Cre transgene (unlinked, or linked with a chosen recombination fraction) and an optional reporter allele. Each pup receives one gamete from each parent, and its sex is assigned at random. The expected proportions and the Punnett square are computed exactly from the same gametes. The colony is simulated day by day: conception within a few oestrous cycles of pairing, gestation of about 19–21 days, optional conception at the post-partum oestrus, litter sizes around the chosen mean, and loss of single pups or whole litters before weaning. A weekly check weans and genotypes litters (from about 21 days), sets up and retires cages, and searches for a cohort, preferably of littermates, born within the age window. Breeding stops when the cohort is complete. Germline deletion is modelled only as loss of the floxed allele passed on by a Cre+ parent, with an illustrative probability. A χ² test compares the Cre and floxed-allele calls in production litters with Mendelian expectation. All colony values, including the default litter size of 6, are approximate; litter size and age at maturity (about 6–8 weeks) depend on the strain. Changes in fertility with age, health problems, fighting, sterility of individual animals and the cost of stock colonies are not simulated.

  1. Sauer, B., & Henderson, N. (1988). Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proceedings of the National Academy of Sciences of the USA, 85(14), 5166–5170. https://doi.org/10.1073/pnas.85.14.5166
  2. Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., & Rajewsky, K. (1994). Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science, 265(5168), 103–106. https://doi.org/10.1126/science.8016642
  3. Percie du Sert, N., Hurst, V., Ahluwalia, A., et al. (2020). The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLoS Biology, 18(7), e3000410. https://doi.org/10.1371/journal.pbio.3000410

About the simulations

Each simulator is a simplified version of a published model of cells, signals, instruments or behaviour, and runs in the web browser. The simulations show how the techniques work and how design choices change the results; they do not replace training in a laboratory.

All data are simulated and generated afresh on each run, so results vary between runs, as in real experiments. Nothing is stored or sent: designs and results are lost when the page is closed, unless the protocol is copied or the data file is downloaded.

Last updated