How to Identify the Ideal Model Generation Service Provider

Introduction

Genetically engineered mice and rats are essential tools for basic biomedical research and drug discovery and development. Technical improvements and scientific advances in recent decades have made it easier to generate such animal models, but it remains a technically challenging process that requires significant effort, technical expertise, and access to the right equipment, licenses, and laboratory space. Because such resources and expertise are not readily available in every laboratory, many scientists choose to use a commercial service provider to generate new custom mouse or rat models rather than generating one by themselves. Outsourcing model generation to a commercial service provider offers several advantages, such as mitigating the risk of strategy design errors, preventing delays in project execution by using sufficient resources and the latest technologies, and allows for freeing up personnel for other important tasks in a research project. However, not all commercial providers offer the same range of project types, scope of services, and quality levels.  Therefore, a provider should be chosen wisely.

In this Insight, we will summarize some aspects that may differ between vendors and how these differences can impact not only the chances of successful model generation but also the entire in vivo research project or study. This may help researchers in the decision-making process when selecting a service provider for new model generation.

Scientific Support and Project Management

When outsourcing a mouse or rat model generation project, the project’s success depends on the experience and expertise of the selected vendor, both in the design of the model and in the execution of the project. Ideally, a new model should be designed specifically for the needs of a research program. One characteristic of a good model generation vendor is the access to a team of experienced scientists whose task is to help define the scientific requirements. The strategy to develop the model is designed to meet the scientific requirements, as well as to achieve the desired time and meet any budgetary constraints, while outlining potential biological or technical risks. This ensures that project design errors are mitigated and that the new model has the highest probability of success in serving as the optimal research tool for the planned experiments.

The execution phase of a new model should also be as effortless as possible for the customer. An ideal service provider should have an experienced team of PhD-level project managers to guide a customer through the entire process, from ordering the model to receiving the first animals, and to ensure that the model generation process moves forward without unnecessary delays. At the same time, an attentive project management team should keep the client well-informed about project progress by proactively providing regular updates and should be available to answer any questions that may arise during the execution phase. This will allow a customer to prepare in advance for all the steps that follow the generation of the new model, such as importing the animals to a new facility, developing on a breeding plan to generate study cohorts, and the following in vivo experiments, which often require coordination with collaborators or contract research organizations (CROs). Therefore, when selecting a commercial provider for a new model generation project, it is always fundamental to explore what level of scientific support will be provided and what level of project management support will be available.

Available Toolbox

Three main methods are used to generate genetically engineered mouse or rat models:

• Random integration transgenesis by pronuclear injection of DNA constructs into mouse or rat zygotes
• Gene targeting by homologous recombination in embryonic stem (ES) cells
• Gene editing of the genome by CRISPR/Cas9

All these technologies come with advantages and disadvantages that are beyond the scope of this Insight; nonetheless, it is important to highlight that no one method is perfect, and each new model generation project should be carefully evaluated. Researchers may find it tempting to select the method that promises the shortest timeline, is the least technically demanding or promises the lowest price, but for the success of a model generation project it is critical to choose the technology that best fits the specific needs of your project. Unconstrained selection in this manner is only possible if a commercial provider possesses a sufficiently comprehensive toolbox and the experience and equipment to use all three major methods for model generation, as mentioned above. Some vendors specialize in only one or two of these techniques and do not offer all of them. In such a case, all models will have to be generated with a limited toolbox, which means the design is often adapted to what is feasible using the specific technology available and not what fits best with the research project. This may result in a less than optimal solution and might lead to lower probability of success in generation of the model or in use of the model in the desired study.

Species and Genetic Background

Similarly, the species (either mouse or rat) and the genetic background should be selected based on the experimental needs. Rats are more expensive to house and maintain than mice due to their larger size, and the tools available to modify the rat genome are more limited compared to mice. However, CRISPR/Cas9-mediated gene editing has increased the possibilities for genetic modifications of rats, and rats do have certain advantages over mouse models for specific research fields or experimental setups. If a rat model is a better solution for a particular research question, then a provider that offers a wide range of genetic modifications in rats must be selected. For both mice and rats, there are certain strains that are used primarily for model generation projects. For the mouse, historically, the strain used depended on the technology being used, e.g., F1 hybrid strains for gene targeting is ES cells or F1 hybrid or FVB strains for microinjection, as these genetic backgrounds were more suitable for these techniques. Today, researchers often prefer congenic genetic backgrounds such as C57BL/6 or BALB/c to limit the influence of genetic variation on experimental results. For many areas of research, specific genetic backgrounds have some advantages, and therefore the genetic background of a new model should be chosen depending on the scientific needs of the planned experiments and not what is available at the specific vendor. Therefore, it is necessary to inquire which service provider can provide the most suitable mouse and rat genetic background for a specific model generation project. Some techniques like pronuclear injection or gene editing by CRISPR/Cas9 allow the use of a wider range of genetic backgrounds, where model generation via gene targeting in ES cells has certain restrictions based on the ES cell line availability.

Quality Control

A critical aspect when generating a new model with a commercial vendor is the quality control steps that are performed. If the quality control of a new model is not done adequately, the model may not perform as expected and in the worst case render itself unusable in the research project. Unfortunately, this may not be discovered until months or years later when experimental data generated with the model are analyzed. Ultimately this gives rise to false positive or misleading data that can cause an entire research program to be based on incorrect assumptions. 

Not all commercial vendors offer the same level of quality control and careful consideration should therefore be given to this when selecting the ideal vendor. The different techniques and technologies used to generate mouse and rat models require different types of quality control, and below we highlight some of the most critical assays that should be considered the minimum requirements.

For mouse models generated by gene targeting in ES cells, it is important to fully sequence the entire targeting vector to verify that no unwanted mutations are incorporated into the mouse genome. In addition, the correct integration of the targeting vector should be validated by Southern blot analysis using internal and external radiolabeled probes. Use of Southern blot analysis in these projects allows for the identification of incorrectly modified ES cell clones with random or partial integration of the DNA construct or with unwanted duplication of the target locus. This step is critical to the model generation process.

Mice or rats generated by CRISPR/Cas9 technology must be carefully analyzed due to the mosaic nature of founder animals and the potential occurrence of additional, unwanted modifications. Only fully sequencing the modified target allele(s) in both the F0 and F1 generations ensures the successful generation of a functional model. Additionally, gene editing by CRISPR/Cas9 might cause unwanted off-target modifications and there are different ways when and how these potential modifications can be analyzed. Some vendors only offer to check a few potential off-target sites in F1 animals, whereas others analyze a larger number of sites, or even use next generation sequencing (NGS) analysis of founder animals in place of standard sequencing in F1 animals. Next generation sequencing of both on-target and predicted off-target sites has the additional advantage that the optimal founder animal can be selected for germline transmission, further increasing the probability of successful model generation. 

Random integration transgenic mouse and rat models generated by pronuclear injection of bacterial artificial chromosomes (BACs), or plasmids into zygotes, are characterized by random integration of DNA constructs into the genome, which can result in different expression levels and patterns in each F0 founder animal. Characterizing different founder lines in parallel for transgene expression is labor intensive and it takes time to be obtain certainty if a line with the preferred expression level and pattern is identified. Some providers offer to take over part of the characterization of founder lines. Others even offer the possibility to analyze important parameters like expression level or genomic integration site already in the F0 generation, which allows for a much earlier assessment of the project success.

Timeline and Integration of Model Generation and Downstream Breeding

The time required to obtain first results from a new mouse or rat model is a combination of the time needed to generate a few animals with the correct genetic modification and the time it takes to expand these animals into study cohorts through breeding. Although the shortest possible timeline is often preferred, it is equally important that a vendor is able to provide a realistic timeline calculated specifically for each project, rather than a generic timeline estimate. Short timelines are appealing, but realistic timelines allow the receiving party to prepare everything for exactly when study animals will be available for experimentation. The transition from the initial model generation to the downstream breeding phase can have a significant impact on the overall project timeline. Therefore, a key criterion for selecting the ideal provider for model generation is to look beyond the model generation capabilities and extend the view into how the vendors offer to make the transition into breeding as seamless as possible. An ideal vendor should be able to produce the new model at a health standard that allows direct import into the facility where the breeding and/or experimentation will be performed to avoid costly and time-consuming rederivation of the animals. In addition, an ideal vendor should set up any genotyping assays required for a new line to avoid delays in breeding whether this is conducted in-house or at a third-party. 

One way to position a newly generated model for downstream production breeding is to allow custom sizing of the model generation deliverable. This way, the deliverable coming out of a model generation project can be scaled and adapted to the breeding plan by starting the downstream breeding with a larger number of F1 mice, thereby skipping one or several generations of breeding, which translates to saving several months on the breeding plan. The integration of the model generation plan with the downstream breeding plan is therefore a key selection criterion in identifying an ideal model generation vendor. This not only saves time and cost on the project, it also complies with the 3R’s principle as less animals are needed to achieve the goal in a well-designed end-to-end plan.

Important Questions to Ask a Model Generation Provider

With all of the different topics discussed above in mind, there are certain questions you might ask when selecting the ideal vendor for your next mouse or rat model generation project:

• What kind of scientific support is offered to find the best design for a custom model?
• What type of project management is provided?
• Is it possible to make an unrestricted choice among the three main methods used to generate genetic models?
• Is the provider fully licensed for all technologies and services offered?
• Which species and genetic backgrounds can be used to generate a new model?
• What quality control checks are performed during the model generation project?
• How often are the proposed timelines met?
• What will be the health status of the delivered animals and how will the health status be certified?