When evaluating mouse models for your research you’re presented with a wide variety of options for obtaining new lines. The major decision to make is whether your experiments can be done using existing lines or if a new model is required. A tremendous variety of lines already exist and most are available from respected and well-run repositories. Such off-the-shelf models come with advantages: they’re already validated with published data and generally available right away for a nominal fee. They may even be established in the colony of a colleague who can send you breeding pairs immediately. However you may notice that although the existing lines are interesting they don’t exactly fit your research questions. Creating a new line requires a greater initial investment of time and resources but it’s worth considering the advantages:

• Work with a line that will answer your specific research questions. Designing your experiments around the mice you can order off-the-shelf can only take you so far. For example your research may benefit from a Cre line that’s only expressed in your tissue of interest rather than multiple tissues. Using the right lines will simplify your experiments at all stages from breeding through analyzing the results.
• Take advantage of the best possible model. Many widely-used lines were created decades ago using methods that have been greatly improved. Transgenic lines in particular are often made by randomly integrating expression constructs into the genome. This random approach can affect the transgene’s expression as well as affect surrounding genes in unpredictable ways. Current techniques can create a better version of an existing line to alleviate the potential inconsistencies that older methods introduce.
• The right model will pay for itself. The time invested in creating the right model can be paid back many times over. This is true not just in terms of being able to perform the experiments you really want to do, but also in simplified breeding and in confidence that the model will perform as designed. It’s true that custom models take time to create, but what you gain at the end makes up for the time it took.

With the right custom line you can feel confident that your results are truly due to your gene being specifically targeted and not something unexpected or out of your control.

Advantages and flexibility of performing some steps at your core, while having other steps performed by a mouse model service company

New custom mouse models represent a significant investment of time and resources no matter what approach you take. The best approach for your lab will depend on your research goals and the expertise and resources available to you.

If your lab doesn’t routinely generate models in-house then you’ll need a partner for some or all of the steps. This could be a collaborator but generally this means you’ll be working with a core facility or a private company. It’s common for labs to work exclusively with their local transgenic core or exclusively with a specific company but there are advantages to splitting up the work.

Core facilities have an undeniable edge in project costs for new mouse models. A core only needs to break even when it comes to operating costs and operations are subsidized by external funding. Therefore they’re able to offer much lower costs to labs that take advantage of their services. Another potential advantage is that the core is usually integrated with the same animal facilities you already use so there’s no need to worry about quarantining mice or obtaining health reports.

The typical trade-off for working with a core is that projects require oversight and sometimes specific steps of the project will have to be done by the lab that wants the new line. An example of this is screening potentially hundreds of ES cell clones to identify the ones that have been successfully genetically modified. The capabilities of different cores vary widely which can dramatically affect your project. If part of the project must be done in your lab and the core can’t help you troubleshoot an unfamiliar protocol you could see your project stall at a critical juncture.

Working with a private company to generate a new mouse model will inevitably cost more than working with a core facility. The trade-off for higher cost is that all steps can be done at the company without oversight. Any reputable company will be fully transparent at each step of the project. You should expect detailed reports with all the necessary information to describe the process in the methods section of a paper. You should also expect a company to troubleshoot a project if any step requires it. Finally, companies may have additional capabilities compared with core facilities, for example being able to generate more complex genetic modifications. This is particularly true in the age of CRISPR where core facilities are focused on simpler models.

In addition to the options of working exclusively with a core or exclusively with a company, a hybrid approach has many advantages. A bit more management is required by the lab that wants the model but with a little extra coordination it’s possible to take advantage of the relative strengths of companies and cores. The simplest example of this would be having a company start a project where complex genetic modifications must be introduced into ES cells. The company could handle the most challenging stages of the project, then confirmed ES cell clones could be frozen and shipped to your core facility to complete the project. The core can generate mice from the ES cells at a lower cost, and the mice that are generated won’t require quarantine.

Available technologies:

• Random transgenics: Straightforward method, lowest cost. Many steps such as vector creation and mouse screening can be done in your lab. Less work is required at the start to create this model type but extensive screening and validation is needed before a line can be used.
• Homologous recombination in ES cells: Method with longest track record for creating targeted knockins. The need for specific equipment, reagents, and skills usually means a lab will need to collaborate or outsource to create a model.
• CRISPR/Cas9: Excellent technique for creating simple models. Complex models created with this method require thorough screening before use.

Step-by-step process – Creating a gene-targeted model by HR in ES cells

1. DesignEvaluate as many factors as possible: experimental plans for the model, potential effects that the genetic modification will have on the gene, strategies for creating the model and screening down the line. Good planning can make the difference between a model that’s good for one paper and a model your lab will use for 20 years.

2. Targeting Vector CreationTargeting vectors are larger than the plasmids you’re probably used to working with and it’s more challenging to fix any mutations you find when making them. Take advantage of existing materials that people have used in the past but be sure to sequence each component before adding it to your targeting vector.

3. Electroporation The targeting vector is forced into ES cells by zapping them with electricity, a process requiring particular skills, equipment, and careful handling.

4. ES Cell Screening Multiple steps must be carefully performed to identify ES cells that carry the desired genetic modifications. It’s also crucial that ES cell viability be maintained during this process.

5. ES Cell Injection / Birth of Chimeras ES cells are injected into blastocysts from wild-type mice and these embryos are implanted into foster mothers. Some of the resulting pups will be chimeras, derived partially from the wild-type host embryos and partially from the injected ES cells.

6. Breeding F1s Chimeras are set up for mating with wild-type mice. Some of the resulting pups will carry the desired mutation – when this occurs it’s referred to as germline transmission.

7. Breeding / Colony Management Initially your new line will comprise just a few F1 heterozygotes. Expanding your colony to propagate the line and generate mice for experiments requires careful planning and management until a larger number of mice are available.

8. Cryopreservation All lines should be protected against unexpected problems. Cryopreservation of sperm is a simple and cost-effective way to ensure that a line can be recovered quickly if something unexpected happens to your mice. Make it a goal to cryopreserve your new line within a year, potentially by setting aside a couple of male mice that have already bred successfully. A bonus for cryopreservation is that the sperm can be used for IVF if you ever need a large number of mice in a hurry.

Step-by-step process – CRISPR

1. Targeting Material Design and Creation Online tools simplify the design process if you’re making a simple model with the CRISPR/Cas9 method, and ordering the reagents is easy.

2. Embryo Injection / Electroporation Multiple methods are available to deliver Cas9 and your targeting materials into embryos. The embryos are then implanted into foster mothers.

3. Screen Potential Founders The efficiency of genome editing by CRISPR/Cas9 varies widely. To successfully create a new line you need a founder mouse where the germ cells are viable and contain the desired mutation. One way to check for successful editing is to look at DNA from the tail or ear. However this method isn’t ideal because it doesn’t conclusively show that a mouse can pass on the mutation.

4. Breeding F1s Set up as many potential founders as you can to breed with wild-type mice, and genotype their pups. Hopefully the mutation will be passed on efficiently. It’s strongly recommended that F1 mice from different founders be treated as completely separate lines. The specific mutation in each founder is likely to be different and may affect expression. Validate each founder’s offspring separately before deciding what to do with the mice.

5. Validation At minimum you must sequence around the site of the mutation. It’s a good idea to validate the genomic integrity around the mutation as well because unexpected rearrangements and deletions are a possibility. The lines must also be functionally validated to show that, for example, a knockout allele is actually disrupting expression.

Your Next Steps: Mouse Model Generation

Now that you’ve reviewed ingenious targeting laboratory’s step-by-step process for generating a custom mouse model, this is a good time to seriously consider how to approach your next project. While off-the-shelf models tend to be easier to obtain and are available at a lower cost, they may not be the best fit for your exact experiment. As a result, you may have to limit the research you actually want to do.

A custom mouse line that has been tailored specifically to your experiments can give you the certainty you seek in your research. With precise targeting of your gene of interest, you can expect accurate data for your experiments. Compared to an existing line, a custom model may have more upfront costs and require more time and resources to generate. However, in the long-run, it’s an investment for your research that will save you time and money. Use the same model time and time again for future experiments or even generate multiple derivative models.

In addition, you can decide which steps you’d like to perform in the lab, give to your core, or outsource to a reputable, private company like ingenious targeting laboratory. This can not only help you stay within your budget, but also give you more control over your research and more time to work on other tasks in your lab.

Using the right mouse line will help you answer your research questions for years to come.

About ingenious targeting laboratory

ingenious targeting laboratory (ingenious) specializes in generating custom genetically modified mouse, rat, and rabbit models. Since 1998, we’ve completed over 2,500 projects for researchers at universities, institutions, and companies around the world. Our model capabilities include knockins, knockouts, humanization, point mutations, targeted transgenics, and more. Generated using both traditional and cutting-edge technologies, our animal models have been published in notable journals such as Science, Nature, and Cell.