My First Knockin Mouse Model
Creating a new mouse model is a major step in any research program. Some things may have come to mind such as, “Can’t I finish this project using models that already exist?” or “Will I ever graduate if I undertake a knockin mouse model project?”
If you have reached a stage where a new knockin mouse model is necessary to advance your research then this blog is for you. Here are 3 things to know before getting started.
1- Random insertion will require more screening than targeted insertion
Your first question will probably be “Can I get the answers I need from a random-insertion knockin or should I consider a targeted knockin at the endogenous gene?” The initial costs for random-insertion transgenics can be quite low if an academic core facility can perform the embryo injections. It’s vital to consider the total cost and effort that will be required before the new line is ready for experiments, however. For random transgenics multiple lines are generated and there will be variability between lines. First, a large number of embryos will need to be injected with your DNA construct in order to get potential founders, then the founders will need to be screened by mating to see if they pass on the transgene. After positive founders are confirmed by mating, you will then have to look at the expression levels of your transgene in the pups, as there will be variability here as well. This will need to include Southern blot analysis to determine how many copies of the transgene integrated, how many chromosomal sites the transgene inserted into, to verify transgenic status, and to determine if the transgene is intact. Once you determine that your transgene is present and intact, you may find mice you can use – but how can you say it is a “real” representation of what you are seeing in human disease? After all, your transgene inserted into a random location that could be affected by epigenetic and positional effects. Further validation is needed to prove your new line is a good model for your research.
Another option is to knock in your cDNA in a targeted manner at the ATG site of the known gene that you are studying. With targeted knockin mice, you can replace expression of the endogenous gene with the cDNA if you so choose, or if you want to maintain expression of your endogenous gene, then a 2A sequence can be used along with the cDNA insertion so both genes are expressed. Since this is a targeted insertion at a known site identifying germline transmitters is more straightforward, saving time and cost in colony management. All pups will have the same expression pattern since this is a targeted, single copy insertion. Also, your cDNA will be regulated by the natural promoter, so expression patterns and features will be as they are in the native context of your gene of interest, saving questions on whether what you are seeing is real or not.
Since creating a knockin mouse model is a major step in your research program, carefully thinking about the longer term goals may help you to decide if random insertion is best or if a targeted approach will better advance your research for years to come.
2- A constitutive knockin could be lethal, and there is a work around for this
Just because a mutation can be studied in tissue culture cells does not mean that live animals carrying the mutation will be born when you move into in vivo work, as some mutations will be lethal during embryonic development. Say you want to study a mutation that affects the brain. No good if your mutation kills embryos before the brain develops. If you choose to proceed with a constitutive mutation model, that’s fine as long as the risk of not having anything to study, and the time and cost this took away, is understood. There is a work-around for this from the very start though: creating an inducible mutation model.
The design of your model can be critical to having something at the end to study. In the case of mutations, it is possible to design a strategy that allows your gene to be wild type at first, and then the mutation can be “turned on” at a point specified by you – whether that’s in a tissue- or time-specific manner. This flexibility can allow you to bypass any embryonic lethal phenotype from a constitutive mutant. One way to achieve this is to incorporate inverted mutation cassettes and loxP sites into your design. Your gene will function like wild type at first and the floxed mutation cassette will not be in-frame until acted upon by a specific Cre. You can choose from a large repository of Cre animals for tissue specific and temporal control of your mutation cassette. For more on these types of designs, visit Point Mutations.
3- Expanding my colony will take longer than I think
When first established, your new line will number a few precious mice. Your ultimate goal will be to generate a cohort of mice of a certain age and sex, which you can then utilize for your experiments. Depending on the model you made, this may require multiple mating steps, genotyping for various genetic elements, and calculated scaling up to obtain the specific number of mice with the appropriate sex, background and genotype. This can take longer than you think. From the basics of colony management, to perfectly timed breedings, to genotyping strategies – many components have to come together in order to be successful. Careful planning will let you use the new line for experiments ASAP while also scaling up the size of your colony.
Luckily there are resources available to help plan and streamline the scale-up process . For example, ingenious targeting laboratory offers a free online tool to help you estimate breeding costs and timelines. To try it out, visit: Free Breeding Planner or click below
Keep these facts in mind and your new line will be a great resource for years to come.