Mouse Model Generation

Step-by-Step Guide

We're the only mouse model company to perform all service steps in the USA.

What Researchers Say

"We recently generated a conditional knockout mouse model using iTL (inGenious Targeting Laboratory). We chose iTL based on time, cost, recommendation and location. We were pleased with the technical help we received in identifying the scope of the project. As well, information and services provided by the project manager were timely and extremely helpful and we were very impressed with the customer service. We were promptly updated throughout the project and upon completion, information was provided to help with genotyping and continuation of the project. We are very happy with the prompt services provided by iTL and would happily recommend them to our colleagues."

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— Jody Groenendyk, PhD, University of Alberta

Choosing Between Off-the-Shelf and Custom Models

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:

Advantages of Custom Models

• 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.

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.

Working with Core Facilities vs. Private Companies

Core Facility Advantages

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.

Private Company Advantages

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.

Hybrid Approach

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. For example, having a company start a project where complex genetic modifications must be introduced into ES cells, then shipping confirmed ES cell clones to your core facility to complete the project.

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 but extensive screening and validation is needed.

• 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.

• CRISPR/Cas9: Excellent technique for creating simple models. Complex models created with this method require thorough screening before use.

Step-by-Step Process: Gene-Targeted Model by HR in ES Cells

1. Design

Evaluate 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.

2. Targeting Vector Creation

Targeting vectors are larger than typical plasmids and it's more challenging to fix any mutations you find when making them. Take advantage of existing materials 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.

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.

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.

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.

4. Breeding F1s

Set up as many potential founders as you can to breed with wild-type mice, and genotype their pups. It's strongly recommended that F1 mice from different founders be treated as completely separate lines.

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.

About ingenious targeting laboratory

ingenious targeting laboratory 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.

Related Pages

• Transgenic Mouse Service
• Mouse Genotyping
• Targeting Vectors
• Targeted ES Cells
• Microinjection