Transgenic Animal Models: Mouse, Rat, and Rabbit

Let’s say your reviewers would like you to confirm the results of your recent in vitro experiments in an animal model in order to publish your most recent paper or fund your grant proposal. Or maybe you’ve exhausted your in vitro testing capabilities to investigate your current hypothesis and it’s time to embark on studies in live animals. Or are you a researcher that frequently works with transgenic animal models, but the models have always been readily available, and now you need to create something specifically designed for your research goals?

We get it. Transgenic animal model technology can at first seem intimidating, but it doesn’t have to be. For over 20 years, ingenious targeting laboratory has been a leader in custom animal model generation, and has provided quality transgenic mouse, rat and rabbit models for thousands of research studies. Whether you are new to the transgenic model field or you have decades of experience, our helpful, knowledgeable team can guide you through the process of animal model development based on your unique experimental goals. It is our job to demystify the science and offer solutions you never thought possible.

For those of you less familiar with or needing a refresher on transgenic animal models, we’ve answered some basic common questions you may have regarding transgenic models below. We’ll describe the three transgenic animal models ingenious offers, our techniques, and introduce some of their collective advantages.

1) What are animal models used for?

Animal models are used for a variety of purposes, including production of recombinant proteins, pharmaceutical and consumer products, and tissue implants. In biomedical research, animal models are primarily used in experiments to learn about the anatomy, genetics and physiology of humans without putting human life at risk. Smaller animal models that can be easily housed make experimental conditions easier to control and can be used in quantities necessary to sufficiently power research studies. Because animals don’t often suffer the same genetic diseases humans do, researchers must replicate these disease states in animal models to reveal the causative mechanism of the disease, and hopefully, develop effective therapies or even a cure.

There are two basic approaches when replicating human genetic disease in animal models: directed and nondirected [1]. The nondirected approach induces genome-wide mutations utilizing chemicals and radiation, and the resulting animals are screened for characteristics that resemble those of known human diseases. The directed approach instead uses established gene mutations and aims to replicate that mutation directly in the model organism, whether by single gene knockout (rendering a gene nonfunctional), single gene knockin (adding a transgene, or foreign DNA, into the organism at a specific locus), conditional expression of single gene knockouts or knockins (an “on” or “off” switch for the desired mutation), transgenesis (random integration of foreign DNA into the genome of another organism), or chromosomal rearrangement. Here at ingenious, we specialize in directed approaches to efficiently generate transgenic animal models and guide our customers through every step of the process.

2) How do you create a transgenic animal model?

Transgenic Mouse

Transgenic mice are used extensively to model a wide variety of human diseases. Cancer research, in particular, utilizes a large number of conditional knockout mice, where transgene expression can be turned on at a specific time or location or both, to mimic the gene loss that occurs during tumor development. Conditional knockout mice have been created for Kras, Myc, and p53 genes, in order to better understand their contribution to tumor growth and development.

ingenious utilizes a targeted embryonic stem (ES) cell approach to create transgenic mouse models. Briefly, we integrate your transgene of interest into the genome of mouse ES cells in cell culture using homologous recombination between the desired location in the mouse genome (genetic locus) and your transgene construct. The ES cells are then screened for your specific transgene at the desired locus, and positive cells are injected into recipient blastocysts (day 5 mouse embryos). These chimeric (possessing both transgene-positive and -negative cells) blastocysts are implanted into pseudo-pregnant female mice that give birth to chimeric pups. If the transgene-positive ES cells injected into the recipient mouse blastocyst contributed to the resulting mouse germline (cells that differentiate into eggs or sperm), the resulting chimeric mouse can then pass the transgene onto future generations of mice.

Transgenic Rat

Rats have greater physiological similarity to humans than mice, and these similarities extend to cognitive and behavioral characteristics, making rats an excellent animal model to study human neurological disorders such as Alzheimer disease and Parkinson disease. As an example, TDP-43 (TDP) gene mutations have been associated with two neurodegenerative diseases, amyloid lateral sclerosis (ALS) and frontotemporal lobe degeneration associated with motor neuron disease. Researchers have successfully created transgenic rats containing a mutant TDP protein with a M337V substitution that produces ALS phenotypes (characteristics) in the transgenic rats, creating an effective model for current and future ALS studies.

ingenious offers a unique technology to create transgenic rat models, reducing the time necessary to generate a founder (the first animal that develops from, in this case, a transgenic sperm) population compared to other commercially available techniques. Specifically, we incorporate your transgene of interest into rat Spermatogonial Stem Cells (SSCs) by utilizing the highly efficient and accurate CRISPR/Cas9 genome editing system. We insert your gene of interest into rat SSCs, and inject positive SSCs into rat hosts. These injected SSCs later differentiate into sperm cells in the host, which allows the transgene to be inherited by future generations. By directly targeting rat SSCs, our approach avoids the mosaicism (possessing both transgene-positive and -negative cells) experienced when using CRISPR/Cas9 directly on rat embryos. In other words, with the ingenious approach, all founders that test positive for your transgene of interest will contain your transgene in every cell, saving you both time and money.

Transgenic Rabbit

Transgenic rabbit models have several unique advantages over rodent models in modeling acquired and inherited human disease. Importantly, rabbits have a longer lifespan and are larger than rodent models, which allow researchers to acquire more tissue and perform in vivo monitoring procedures that would either be impossible or require specialized equipment for equivalent studies in mice or rats. Rabbits are also inherently sensitive to dietary cholesterol, and several strains have abnormal lipid metabolism, making them excellent models for human atherosclerosis and lipid metabolism. Human hepatic lipase, human lipoprotein lipase and human apo A-I transgenic rabbit models have been created to study the effects of these genes in atherosclerosis and lipid metabolism.

Our laboratory microinjects the CRISPR/Cas9 genome editing system into rabbit embryos to generate transgenic rabbit models. Using your transgene of interest, we determine which guided RNA sequences will be most effective for proper integration of your transgene, and inject that sequence along with the sequence required for Cas9 expression into pronuclear-stage embryos. Embryos are implanted into host mothers to allow birth of transgenic founders. Our usage of the CRISPR/Cas9 system greatly increases the efficiency of accurate transgene incorporation in the embryo over technologies relying on homologous recombination alone.

3) What was the first transgenic animal?

The very first transgenic animal was created back in 1974, when Rudolf Jaenisch generated the first transgenic mouse by injecting foreign DNA into an early mouse embryo. Eight years later, transgenes would be inserted in the germline of transgenic mice that could be passed onto future generations. In 1984, the very first oncomouse was produced by inserting the Myc oncogene into the mouse genome to predispose tumor growth in the animal. This transgenic animal model of cancer revolutionized the field of biomedical research and paved the way for an explosion in the scientific understanding of disease and disease treatment.

4) Why do researchers use mouse models for studying human genes?

Mice and humans are physiologically quite similar, and the mouse’s small size and large breeding capacity make them an excellent animal model for studying the function of mouse genes and their orthologous (equivalent) human gene counterparts. While mice and humans are phylogenetically related, a large number of evolutionary differences have accumulated between our two species, a factor researchers must keep in mind when extrapolating the results of genetic experiments in mice to humans. Use of mouse models has greatly advanced our understanding of physiology in both health and disease, paving the way for therapeutic drug development and safer clinical trials for disease therapies and cures.

5) What are some animal models that have been used to study genetics?

Mice, nematode worms (Caenorhabditis elegans) and fruit flies (Drosophila species) are historically the most commonly used animal models to study genetics. Scientists study genetics by experimentally manipulating the genes of model organisms in order to better understand the function of genes in vivo. Because genetic studies require a large number of progeny (offspring) to assess rules of inheritance, animal models that are small, mature quickly, and produce many offspring are ideal. The more phylogenetically related a model organism is to humans, the more confident we can be in the conservation of orthologous gene function in humans. Given their phylogenetic relationship to humans, mice, rats and rabbits are among the most commonly used model organisms used in genetics research today. ingenious targeting laboratory specializes in generating the customized transgenic mouse, rat and rabbit models required for today’s complex genetic studies and can assist researchers in developing conditional transgenic animal models for transgenes that inhibit normal development of offspring in utero.

References

1) Hardouin SN, Nagy A. 2000. Mouse models for human disease. Clin Genet57(4): 237-244.