With the enhanced control of rat embryos and the use of CRISPR/Cas9 technology, the scope of applications for rats as experimental model animals in basic research, drug screenings, and preclinical drug evaluations has expanded. However, in regards to the Cre-LoxP system – an important conditional gene expression construct - the application of rats has been seriously hindered due to the limited availability of Cre driver rat strains compared with mouse strains. The Cre-LoxP system has been widely used in mice, and many genes have corresponding LoxP mice, as well as abundant Cre mouse resources with specific promoters. However, due to the delayed development of gene-editing technology in rats, there are much lower numbers of LoxP or Cre rats connected to a tissue-specific promoter compared to those that are available for mice.

Creating a Comprehensive Cre Repository

Cyagen continuously optimizes the production process to overcome multiple technical difficulties – such as the low fertilization rate of superovulated rat embryos, the high rate of abnormal embryos, the large differences between egg cell lines and individuals, and the thicker and more flexible nuclear and plasma membranes which make injections more difficult - to optimize our production efficiency with creating more complex conditional gene knockout rat models. In addition, Cyagen can provide a variety of gene-editing rat services, such as gene knockout (KO), point mutation, and gene knock-in (KI) across diverse background strains such as SD, Wistar, Long Evans, F344, and Brown Norway rats. Cyagen is committed to developing the best rat model for your research. Our efforts to support rat model research include the creating a repository of Cre models, including rat lines, to be available to researchers worldwide.

Rat Cre Lines Currently Under Development:

Note: Cyagen can provide pricing accommodations to researchers interested in Cre lines not currently available in our listings. Contact us to inquire about your conditional model needs and see how we can help develop a custom Cre strain to support your field of study.

Developing Conditional Rats Using Cre-Lox Recombination

Considering that Cre rats need to be crossed with LoxP rats for one to two generations to obtain conditional gene-editing animals alongside the fact that Cre enzymes have inefficiencies and probability of leakage – the breeding approach may affect the progress of scientific research. To address these potential drawbacks, virus-induced overexpression/knockout (KO) /knock-in (KI) /point mutation of rat models came into being.

Developing Conditional Rats Using Viral Vector Expression Systems

Currently, there are three main viral vector expression systems in use, namely adenovirus, lentivirus, and adeno-associated virus. These viral systems have their own advantages and disadvantages:

• Adenovirus has the advantages of high-efficiency expression, earlyinitiation of expression, relatively large packaged DNA fragments, but strong immunogenicity and toxicity. It is suitable for vitro transfection of cells and displays high mortality rates and short study window period when applied in vivo.

• Lentivirus can be reverse transcribed into DNA to achievepermanent integration into the host genome, facilitating long-term expression. Lentivirus is the most used gene delivery virus system, which has much smaller immunogenicity than adenovirus. Although it can also be used for in vivo transfection, it pales in comparison to the in vivo performance of adeno-associated viruses (AAVs) with regards to titer, efficiency, and immunogenicity.

• Adeno-associated virus (AAV) exhibits extremely low immunogenicity which can guarantee its long-term coexistence with the host, its primary advantage compared to other viral expression systems. Depending on the AAV construct, experiments can be carried out in laboratories with lower biosafety levels, up to a maximum of Biosafety Level 2 (BSL-2). The wide variety of serotypes enables unique affinity for different tissues, maintaining strong tissue specificity even in the case of systemic injection. Further, supplementing with localized injection can greatly improve the accuracy of integration into targeted tissues and cells for the experiment. AAV is the most suitable viral expression tool for in vivo experiments, although it remains limited by its insufficient cloning capacity.

Figure 1. Comparison of three common viral vectors

Based on the comparison of the above three viral vector expression systems for the generation of conditional gene expression animal models, it may be concluded that AAV vector-mediated tissue-specific expression has a better effect than the other two viral systems. 

Advantages of Adeno-associated Virus for In Vivo Gene Modification

It should be noted that the tissue-specific expression produced by adeno-associated virus (AAV) injection is not just a time-saving solution. Due to the uncertainty of in vivo experiments in life science research, cross-validation with multiple methods can also help to improve the significance of your research results.

Figure 2. Tissue specificity of different Adeno-associated viruses (AAVs)

Additionally, adeno-associated viruses (AAVs) are often used in combination with Cre or LoxP rats. Although scientists continue to develop more LoxP and Cre rats, this process does not happen overnight – especially with the problems posed by Cre enzyme leakage in rats, which may lengthen the development time. The construction of viral vectors takes much less time than breeding animals to meet the volume required for experiments. Therefore, the most efficient strategy is to use AAV virus to perform tissue-specific operations on existing rats to obtain preliminary results, while developing Cre and LoxP rats. This approach can be used to study and verify the phenotype of animals quickly, saving time and allowing rapid evaluation of the research strategy and direction.

Combining AAV with Cre/LoxP for Generation of Conditional Models

In recent years, Cre-LoxP animals have matured in the study of the spatial specificity of genes, and there are even cases where the transferred promoters are expressed in multiple tissues simultaneously. For tissue-specific Cre expression models, the expression of Cre in other areas would interfere with the accuracy of research results. This problem can be solved with fixed-point injection of AAV, which demonstrates a very strong regional specificity, into the target tissues.

To make better use of the gene-editing mice on hand, the AAV injection protocol can be divided into two types: 1) if you have LoxP rats already, you can inject with AAV packaging tissue-specific Cre; 2) conversely, if you have Cre rats, the virus can be injected with the target gene or other elements connected to the LoxP site. In general, tissue specific promoter for Cre recombinase with AAV Packaging is more commonly used.

Figure 3. AAV viral packaging DNA combined with mice for conditional expression

Disadvantages of AAV-mediated Conditional Gene Editing

Although AAV-mediated conditional gene editing has the many advantages mentioned above, its disadvantages should not be ignored:

• Size limitations: The packaging fragments of AAV injection are short in length, which limits their application scope forlarge fragment genes. This size limitation often leads to trade-offs on accessory gene elements such as reporter genes and restricts the selection of promoters to precede the Cre genes.

• Injection training: High-precision AAV injection will necessitateappropriate training to ensure consistent modeling, which incurs additional cost and time before starting experiments. For brain injections that need to accurately target specific brain regions, the relatively small brain size of mice makes the AAV administration more difficult. As the virus is an external source that not produced by the cell itself, uneven concentration distribution at the injection site will occur, which will affect the experimental results. It is well understood that the virus content in the center of the injection is higher than the surrounding area, but it may still take additional efforts to accurately grasp the location of gene modification.

• Cost: Although the cost of using AAV for conditional expression is lower than that of Cre-LoxP mice in most cases, there are two exceptions:

1. Systemic Injection - requires huge amount of viral vectors, cost increases linearly with the increasing number of animals injected.
2. Hybrid Mice - large initial investment and animal breeding stage costs, which decrease marginally with the successful construction if no accidents occur.

Figure 4. Comparison of viruses and gene-edited mice for developing conditional knockout models

*Positional injection and short-term experiments have advantages.

Considering the pros and cons of the two methods for developing conditional animal models can help ensure their optimal use suited to your research plan. When conditions permit, AAV-assisted conditional knockout animals and traditional Cre-LoxP animals can be complementary and mutually validating in vivo models. With the advantages that rats provide for human disease research and recent advancements in gene editing among the species, rats are becoming an indispensable tool for biomedical research.

References:

1. Dietrich, M.R., R.A. Ankeny, and P.M. Chen, Publication trends in model organism research. Genetics, 2014. 198(3): p. 787-94.
2. Haery, L., et al., Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation. Front Neuroanat, 2019. 13: p. 93.
3. Tsien, J.Z., Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting. Front Genet, 2016. 7: p. 19.
4. Meek, S., T. Mashimo, and T. Burdon, From engineering to editing the rat genome. Mamm Genome, 2017. 28(7-8): p. 302-314.
5. Balakrishnan, B. and G.R. Jayandharan, Basic biology of adeno-associated virus (AAV) vectors used in gene therapy. Curr Gene Ther, 2014. 14(2): p. 86-100.
6. Kim, H., et al., Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res, 2018. 34(4): p. 147-159.