Transgenic Mouse Models
Precise and predictable gene targeting, only with ingenious.
|Transgenic Knockin to Your Gene of Interest||Safe-Harbor Transgenic Knockin|
|Express Cre, a reporter gene, or any sequence using your target gene’s promoter||Drive expression of a cDNA using a ubiquitous or tissue-specific promoter|
|Express a human cDNA in place of a mouse gene||Conditional cDNA overexpression, which you control using tissue-specific Cre lines|
|Express a modified version of a mouse gene to add a tag sequence or extra functional domain||Drug-inducible overexpression, controlled by administering doxycycline|
|Learn More||Learn More|
Transgenic mice have proven to be an invaluable research tool for studying human disease. At ingenious, we utilize precision gene targeting to generate your transgenic mouse. Our proprietary technologies allow for shorter production timelines and reduced costs when compared to other targeting methods, without compromising performance and quality.
Transgenic mice from ingenious are precisely targeted to create the model you need.
Faster production and lower prices.
Delivery of your germline-confirmed model is fully guaranteed.
Uses for Transgenic Models
Transgenic mice are utilized for an incredibly diverse range of experiments because this model type is so versatile. Using gene targeting to specifically target transgene insertion enables placement of your genetic construct at an exact location. Anything from the most faithful reporter line to a line for drug-inducible overexpression can be created using a transgenic knockin strategy.
- Transgenic Mice in Cancer Research
The generation and analysis of genetically modified mice, including transgenic and knockout animals, is an essential technology for cancer research. The mouse as a model for human cancer research has proven to be a useful tool due to the relatively genomic and physiological characteristics of tumor biology between mice and humans. Mice have several similar anatomical, cellular, and molecular characteristics to humans that are known to have critical properties and functions in cancer. Additionally, the proportion of mouse genes with a human ortholog is 80%, thus providing an excellent experimentally tractable model system as a research tool to investigate basic mechanisms of cancer development as well as responses to treatment.
Several advances have been made in modeling cancer in mice, and the new transgenic mice described in this review are now more capable of modeling human cancers with mutations that are controlled spatially and/or temporally. Cancer transgenic mice can be produced in different ways by introducing DNA into the mouse genome. One approach is via microinjection of DNA constructs or microinjecting endonuclease-based reagents) directly into the pronucleus of fertilized mouse oocytes. First, in the case of injecting DNA constructs, the transgene is randomly integrated in a small percentage of injected oocytes as one or more tandem copies into the mouse genome, and generally all the cells of such offspring possess the transgene. The method to produce transgenic progeny is relatively quick, but it includes the risk that the DNA may insert into a critical locus that can cause an unexpected, detrimental genetic mutation. In addition the transgene may be inserted into a locus that is subjected to gene silencing or inserts as multiple tandem copies, which produces extreme overexpression leading to non-physiological phenotypic effects, but more often such tandem transgene integrations are silenced in subsequent generations.
The other approach as described throughout this site is the gene targeted transgene approach. It includes the targeted manipulation of mouse embryonic stem cells at selected loci by introducing primarily loss-of-function mutations. Genetically modified ES cells are then microinjected into the mouse blastocysts and transferred to pseudopregnant recipient mice. The ES cells and donor blastocysts derive from mouse lines with different coat colors, and thus successful incorporation of targeted ES cells into the developing embryo of donor blastocyst results in chimeric offspring exhibiting variegated coat color. Chimeric offspring are further mated with wild-type mice of the blastocyst strain to assess the frequency with which ES cell derived genes are transmitted by a particular chimera.
Personalizing Humanized Mice
Humanized mice have shown great potential in preclinical oncology studies. To further increase the potential of these models for the study of human disease, there is a necessity for the immune system to be compatible with both its host environment and with the implanted tumor tissue to accurately model the patient’s immune response during treatment. Tissue incompatibility of humanized mice that are engrafted with an immune system from one person and implanted with the tumor of another could be the reason for the immune response observed in these models, which is thus not related to the specific treatment applied to the mice. When humanized models are produced from the engraftment of CD34+ cells, some of the mature xenoreactive T cells are also introduced into these mice. These T cells differentiate within the engrafted bone marrow, mature within the mouse and seem to display some xenoreactive tendencies (108). However, because the transplanted human immune system is weakened, it prevents complete rejection of the xenograft. One possible solution to this problem could be the production of a humanized xenograft model in which the CD34+ cells and implemented tumor tissue are derived from the same donor. Recently a new melanoma PDX model has been designed wherein tumor cells and tumor-infiltrating T cells from the same patient are transplanted sequentially in NOG/NSG knockout mice. This mouse model was developed to study the most advanced and most promising current anticancer therapies, immune checkpoint inhibitors and adoptive cell transfer of autologous tumor-infiltrating T cells that have demonstrated complete durable responses in a subpopulation of patients with advanced melanoma.
For other transgenic tumor models, human cell lines are injected into immunocompromised hosts such as athymic nude or severe combined immunodeficiency (SCID) animals. These are one of the oldest models used to evaluate cytotoxic therapies against cancer. These models have particular relevance for the development of chimeric antigen receptor (CAR) therapies, which can utilize either human cell lines or patient-derived samples to generate xenografts for antitumor efficacy evaluation.
One of the critical factors that determine the utility of human xenograft models for other immunotherapeutic applications is the degree of immunodeficiency of the murine host. The classic athymic nude mice lack normal thymic development and therefore are deficient in T-cell function. However, because functional innate immune populations such as neutrophils and dendritic cells, as well as B cells and natural killer (NK) cells, remain, many aspects of the immune response, though perturbed, are present in athymic nude mice. Therefore, engraftment of human hematopoietic elements and other primary human cells is quite limited in this model. SCID mice are deficient in a DNA-dependent protein kinase required for T- and B-cell development, and Rag-deficient mice have defective Rag1 and Rag2 genes that are also deficient in T- and B-cell function. The knockout of the IL2rγ chain induces concurrent deficiencies in IL2, IL4, IL7, IL9, IL15, and IL21 receptor functions and generates mice that lack NK cells. By combining genetic mutations, the immunodeficiency in the resulting mice worsens, and with it comes an improvement in the engraftment of donor human immune cells. Thus, a transgenic model that has often been used are mice for the engraftment of human hematopoietic stem cells are derived from SCID, Rag1null, or Rag2null mice coupled with a targeted mutation in the IL2rγ gene. Combining different genetic modifications in the background of a transgenic mouse model is helping us generate different platforms to predict efficacy and resistance to cancer immunotherapies.
- Transgenic Mouse Models in Neuroscience
Genetically modified mice, particularly transgenic models, have proven indispensable for neuroscience research. Mice were already established as excellent models for the study of brain development and function even before genetic modification techniques became widely available. New technologies and methods developed in recent decades catapulted the mouse model to the forefront of neuroscience research. As an example, optogenetic tools are one of the newer and most exciting developments. To use this method, transgenic models are created that express light-sensitive proteins in specific neurons. The gene for the light-sensitive protein can be inserted randomly or in a targeted manner into the mouse genome. Shining a laser into the mouse’s brain allows the researcher to control the activity of those neurons with no other perturbation. The mouse could initially be put through a learning test with the laser off, then the experiment repeated with the laser on to test the role of the targeted neurons. Optogenetics is just one example of the new methods that are made possible by continued development of new transgenic mouse models.
A lab that uses mice to study human disease today is almost certainly using multiple transgenic models for their work. Thanks to decades of work on creating new kinds of genetic modifications a transgenic mouse model can be created for practically any conceivable experiment. It’s common to create a model that expresses the human sequence for a gene of interest. Taking this one step further, this sequence can include a specific disease-causing mutation. This will often recapitulate the course of the human disease to a great extent in the mouse. Such genetically engineered mice are essential in neuroscience research and are used to study a wide variety of diseases.
One transgenic mouse line, known as APPPS1, has transformed the study of Alzheimer’s disease. The original publication describing the line has been cited over 600 times since its publication in 2006. This line uses a neural-specific promoter to drive expression of two human genes, and each gene contains a known disease-causing mutation. By eight months of age APPPS1 mice begin exhibiting cognitive decline, for example as demonstrated by reduced performance at learning tasks. Some of the structural and molecular changes that occur in human disease patients are recapitulated in aged APPPS1 mice and this has enabled a wide variety of studies.
Newly-developed drugs can be tested using APPPS1 mice to determine whether or not they affect symptom progression. It’s also possible to introduce other genetic mutations in APPPS1 transgenic mice to understand how different genes interact. For example, a loss-of-function mutation can be introduced in the mouse genome, targeting a particular gene of interest. In healthy mice that mutation may have one effect on mouse health but a different effect may be observed in APPPS1 mice. Combining different genetic modifications in the background of a transgenic mouse model is helping us understand the causes of human diseases and enabling the development of new treatments.
History of Untargeted Transgenic Mice
Pronuclear injection of DNA can lead to low frequency integration of the sequence into the mouse genome at random sites. Extensive development of this method in the early 1980s made the creation of random-insertion transgenic mice a routine procedure. After potential founder mice are born they must be screened for germline transmission of the desired sequence. Ideally multiple alleles will be evaluated as expression of the transgene can be affected by its genomic position, and other genes can be disrupted if the transgene inserts within their sequence. Furthermore, multiple copies can randomly integrate into the genome – it may be necessary to screen a large number of mice until the appropriate founder has been identified.
In some cases the desired DNA sequence can be injected as a bacterial artificial chromosome (BAC) to alleviate some concerns with random insertion transgenesis. BACs can hold up to 200kb of sequence, which allows for large sequences such as entire genes or large promoters to be introduced into the mouse. Inserting an entire gene including introns, exons and regulatory sequences is beneficial when the coding sequence alone may not be sufficient for proper expression. The use of BAC transgenic constructs can result in more faithful gene regulation and processing as well as the potential to perform functional and regulatory studies on multiple transcripts.
Things to keep in mind when deciding if a pronuclear injection transgenic model is right for you:
- Random and unpredictable transgene integration and expression.
- Variable copy numbers of the transgene.
- Small transgenes may lack the cis-regulatory elements needed for proper expression .
- BAC constructs are harder to work with and require more molecular biology expertise.
- Need to screen founder mice (as opposed to ES cells). May not be 3Rs-friendly .
Consider Targeted Transgenics
Targeted transgenic approaches reduce or eliminate the downsides of random integration by pronuclear injection. Safe-harbor loci can be targeted by ingenious’ technologies to efficiently introduce your overexpression construct into the genome in a location where it will not interfere with other genes or be affected by the surrounding genetic environment.
To learn more about transgenic mice, speak with one of our scientific consultants today.
1) Sadelain M, Eirini PP, and Frederic DB. 2012. Safe Harbours for the Integration of New DNA in the Human Genome. Nature Reviews Cancer 12(1): 51-58.
2) Timeline for Transgenic Mice Analysis, University of Michigan
3) BAC recombineering and transgenesis, Vanderbilt University
4) What are the 3Rs? NC3RS