Knockout mice serve as a powerful tool for genetic studies in in-vivo experiments. On the contrary, the systemic knockout mice can not be used for studying the function of specific genes found in specific tissues at the same time.
Another significant drawback of systemic knockout mice is that these may fail to deliver the desired results.
The reasons behind the limited use of systemic knockout mice include genes that alter the embryonic development of mice, premature death owing to severe postnatal physiologic defects, failure to reproduce, and the inability of clinical investigators to acquire pure-sibling animal models.
This has led to the development of conditional gene knockout. The Cre/loxP recombination system is a common conditional knockout technique. The principles and applications of this method are discussed in the subsequent sections.
1. Cre Recombinase and LoxP Sites
The Escherichia coli phage P1 consists of a Cre gene encodes for Cyclization Recombination Enzyme (Cre recombinase). This enzyme has a molecular weight of 38 kD and comprises 343 amino acids.
The enzyme has dual properties – catalytic property and recognition of loxP sites with subsequent recombination or deletion of genes located between loxP fragments.
The spacer region of the loxP locus determines loxP site orientation, while the reverse repeat sequence of this locus serves as Cre recombinase recognition sites.
2. Principle of Gene Recombination in the Cre/loxP System
The recombination techniques of this system are attributed to the interaction between loxP sites and Cre recombinase. The enzyme binds to either end of the loxP site and results in the formation of a dimer. The two dimers interact and form a tetramer.
Cre recombinase cuts off the DNA fragment located between two loxP sites which are then rejoined by the enzyme DNA ligase. The position and orientation of loxP sites determine the result of DNA recombination.
This can be explained by the three examples given below.
- The Cre recombinase catalyzes deletion of DNA fragments located between loxP sites, given that these sites are found on the same DNA and direction.
- The Cre recombinase catalyzes the inversion of DNA fragments located between loxP sites, given that these sites are found on the same DNA but in opposite directions.
- The Cre recombinase catalyzes Cassette change or chromosomal translocation if the two loxP sites are found on separate DNA sequences or chromosomes.
3. Application of Cre/loxP System to Obtain Specific Knockout Mice
The Cre/loxP system requires two transgenic mice to achieve the knockout of specific genes in-vivo under specific conditions. Embryonic stem cell technology is used for the first transgenic mice that involve the transfer of in-vitro gene sequences into embryonic stem cells.
The in-vitro gene sequence employs homologous recombination to replace the original sequence.
After transferring the in-vitro gene sequence, embryonic stem cells are placed in the pseudopregnant mice’s uterus, where these cells form an intact embryo and a transgenic mouse.
The second transgenic mouse is acquired via embryonic stem cell technique or oocyte injection. The Cre recombinase is introduced and is regulated by the specific gene promoter, allowing the expression of the gene only under certain conditions.
The two transgenic mice are allowed to mate and produce offspring that comprises both genotypes.
4. Advantages and Problems of the Cre/loxP Recombination System
This is the most commonly used recombination system and conditional knockout tool for the following advantages.
- High efficiency: After forming a dimer with a DNA sequence, the Cre recombinase triggers the DNA recombination process, which is time- and cost-efficient.
- High specificity: This is attributed to the unique sequence of loxP sites.
- Wide range of applications: Cre recombinase demonstrates optimal activity in different tissues and under variable physiological conditions or environments.
- Expression: The genetic expression of Cre recombinase is initiated by any type II promoter, which is seen in different cells, tissues, organs, developmental stages, and physiological conditions.
Despite the mentioned advantages of this recombination system, this technique is only limited to the integration and deletion of certain genes. Therefore, redundant DNA sequences are observed.
Summary
The sites-specific Cre/loxP recombination system is a promising conditional knockout technique for precise genetic modifications.
This system involves the thorough and accurate introduction of exogenous genes into the genome as well as modulates the genetic switches that are responsible for controlling both deletion and expression of the exogenous genes.
Despite being an effective conditional knockout technique, this system has several drawbacks in both clinical research and clinical application of this system.
However, with further improvement and development of technology, this recombination system has the potential to become more accurate, precise, accessible, and widely applicable.
About Cyagen company
Cyagen has served thousands of researchers and scientists all over the globe for up to 15 years after its development.
Cyagen products and technologies are associated with up to 4,5000 academic and research papers that have been published in the three major journals collectively known as the CNS – Cell, Nature, and the Science journals.
Cyagen also provides customization services for the conditional knockout, knockout as well as knock-in models. Moreover, Cyagen consists of a professional team that caters to surgical disease models of small animals.
Cyagen also has a mouse evaluation and drug screening platform that provides humanized mice for research related to cardiovascular disorders and Alzheimer’s disease, as well as immunodeficient mice that are obtained from American and European industry leaders.
This platform uses a sterile mouse technology that complies with international standards.
The products and services offered are related to the sterile animal models and include microbial colony transplantation services, sterile mice, and sterile animal customization services.
In addition, Cyagen also assists researchers and investigators in assessing the mechanism of interaction between genes and the colony.
Complete Model Animal Solutions
- The Turbo Knockout Mice – this model targets the embryonic stem cells and decreases the breeding time significantly, i.e., up to two generations.
- The CRISPR-Pro Knockout – this model mediates 20kb knockout and 10kb knock-in.
- The Humanized Mice – this model serves as a mature platform system that is used by famous pharmaceutical companies, including AstraZeneca, Hengrui Pharma, and Pfizer.
- The Transgenic Mice – this model has been cited in the top journal, including Nature.