Humanized Mouse Models
Humanize your gene without limitations.
A humanized mouse is an animal model that expresses a human genetic sequence or serves as a host for living human cells or tissues.
At ingenious targeting laboratory, we create custom genetically humanized mouse models tailored to the specific research goals of scientists worldwide. We can design your ideal model without limitations – from small genetic changes to large replacements of genomic sequences, including the potential for conditional alleles, cassette knockins, and more.
Create a model precisely suited to your needs, rather than settling for pre-existing models.
Ingenious offers various strategies for genetic humanization, including our proprietary TruHumanization™ technology. Our range of options to choose from, based on your specific requirements, provides you with tailored, flexible, and technologically advanced solutions for creating models that advance human disease research and therapeutic development.
What Researchers Say
"iTL produced four conditional knockout mouse models on our behalf. They have been extremely helpful and informative at all stages of the project; all the way from construct design to breeding strategies and genotyping the new mouse models. I know where to turn when the needs comes up again for another mouse project; it is certainly faster and cheaper than doing this by ourselves."
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— William A. Coetzee, DSc, NYU School of Medicine
Why create a humanized mouse model?
The term "genetic humanization" covers a broad array of strategies for introducing and expressing human genetic sequences in an animal model. The genetic change may be as small as one altered nucleotide (to humanize a crucial site in a protein) or as large as hundreds of kilobases (potentially an entire gene or gene cluster including promoter regions).
Read on to learn more about different options for creating your next model.
Genetic Humanization by Random Transgenic Insertion
This method was first used in the 1980s to create mouse models with expression of human genetic sequences. DNA that's directly injected into mouse embryos can randomly integrate into the genome to create new transgenic lines.
Advantages: Speed and relatively low cost of establishing a new line containing human sequence.
Disadvantages: Mice must be bred until the inserted sequence segregates as a single allele, and the expression level of the added gene must be assayed. Also, expression of the transgene will depend on the location where it's inserted. Ideally it will land in a region of the genome without genes or regulatory sequences and the promoter driving the transgene will not be affected by nearby gene regulatory elements. Any of these problems will lead to a mouse line with unpredictable or unstable transgene expression.
Genetic Humanization by Safe Harbor Knockin
A knockin to a safe harbor locus such as the Rosa26 locus (Rapid-Rosa26™) offers predictable, stable, high expression from a defined location in the mouse genome. This strategy overcomes the disadvantages of random transgenic insertion by making a targeted insertion. Mice don't need to be screened and maintained for multiple generations to identify stable expressors, and transgene expression level is predictable.
The main advantage of safe harbor knockin is that the inserted gene can be expressed in any tissue by using a strong ubiquitously active promoter such as CAG. Expression can be made conditional with a LoxP-flanked STOP cassette, such that only cells expressing Cre will express the transgene.
Safe harbor knockin is best suited for overexpression of wild-type or mutant transgenes, expression of Cre or a reporter in a conditional manner, or other experiments where expression of the transgene in its normal genomic environment is not needed. Overexpression is not always the ideal experiment however, and the gene is expressed from the safe harbor locus rather than its native environment.
Genetic Humanization by Knockin to Endogenous Locus
Knockin to an endogenous locus can be used to place a human sequence under the regulation of the mouse promoter.
Full coding sequence knockin
To make a mouse version of a human variant, for example to create a mouse model of a human disease, it's possible to knock in the full coding sequence (cDNA). This places the human coding sequence under the control of the endogenous mouse promoter.
Point mutation knockin
Some coding changes between mice and humans are as small as a single base change. In these cases a point mutation knockin is the most straightforward option to create a mouse model. Disease-associated human variants can also be introduced into a mouse by point mutation knockin.
Advantages: This strategy places the human sequence under control of the homologous mouse gene promoter rather than a separate transgenic promoter.
Disadvantages: Only coding sequence can be humanized since the regulatory regions of the mouse gene are still present. Conditional alleles can be designed but the gene will be knocked out in cells where the mutation is not activated.
Genetic Humanization by Replacing an Entire Region of the Mouse Genome
The most thorough genetic humanization strategy replaces the mouse gene with its human homolog, or replaces a region of the mouse genome with the analogous region of human genomic sequence.
Essentially this combines knockout of the mouse gene and knockin of the human into a single allele, but that description doesn't capture the power of this model type.
TruHumanization™ Technology
ingenious' TruHumanization™ process can place up to 200kb of human genomic sequence into a desired genomic location, in a single step. The method is precise, so you can design exactly the model you need with the exact amount of human sequence required for your experiments.
For example, you may wish to humanize only and exactly the sequence that encodes an extracellular domain but leave part of the gene with mouse sequence. Such a region might span part of exon 1, all of exons 2 and 3, and part of exon 4. ingenious' TruHumanization™ method can replace exactly this region with human exonic and intronic sequences.
Alternately you may want an entire gene humanized, from a promoter element 5kb upstream of the start site to the end of the 3' UTR. With TruHumanization™ the perfect model for your research is within reach.
Every essential region of the gene can be included in the humanized region, including regulatory features you may not be aware of. This is the true power of a TruHumanization™ model: by more faithfully recapitulating the human gene it can be used for any future study of that gene rather than being limited by what is known today.
A TruHumanization™ model created by ingenious will include all regulatory features in the replaced region such as promoters, splice sites and UTRs. The human sequence replaces the homologous mouse gene so it's more likely to have the same genomic landscape as the gene does in human cells.
Random-insertion transgenic strategies do not place the human sequence in the correct genomic landscape which could result in altered gene regulation. ingenious' TruHumanization™ strategy delivers the most faithful models available today for studying the function of a human gene in a living animal.
Humanization Strategies Available at ingenious
Coding Sequence Knockin
- Expression of human coding sequence under the control of the mouse promoter
- Can incorporate mutations, co-express reporter gene
- Can include LoxP design for tissue specific or temporal control
Point Mutation
- Change a key amino acid to match human sequence
- Duplicate a mutation identified in human patients
- Conditional point mutations give you enhanced control
Gene Replacement (TruHumanization™)
- Replace up to 200kb of genomic sequence in a single step
- Humanize a single exon or an entire gene including noncoding regions
- The ultimate in genetic humanization
How are genetically humanized mice created?
Currently the majority of new genetically humanized models are point mutants where a critical nucleotide in the mouse sequence is changed to match the human sequence. These point mutations can often be introduced using the CRISPR/Cas9 method, a popular way to make targeted genetic modifications.
In some cases a gene can't be targeted using CRISPR/Cas9 however. The most common reason to avoid using CRISPR/Cas9 is when a homozygous knockout of the gene produces a strong phenotype. In such cases the point mutation would be introduced by the more established method of homologous recombination using embryonic stem cells.
ES cell approaches are also used when making a cDNA knockin or a TruHumanization™ genomic replacement. To make a knockin using ES cells it's necessary to first create a genetic construct called a targeting vector. The targeting vector has the knockin sequence in the middle with targeting sequences on either side, and is introduced into ES cells.
The targeting sequences are recognized by enzymes in the cell and essentially trick the cell's DNA repair machinery into making the knockin sequence a part of the genome. This method can make precise and targeted genetic modifications because the targeting sequences are specific to a single location in the mouse's genome.
The modification could be as small as changing a single nucleotide or as large as replacing 100kb of mouse genomic sequence with human sequence.
Humanized Mice as Human Disease Models
The mouse has a long history as a model organism for the study of human disease and basic biology, based on the similar genetics and physiologies of the two organisms.
In some cases a key difference between mice and humans can interfere with a particular experiment, for example a slight change in a protein domain or active site may interfere with antibody or small molecule binding. Differences at the tissue level can also be significant, preventing studies of systems such as the human immune system.
Humanized mice are one option for overcoming these challenges – mice altered at the genetic or cellular level to more closely match human biology.
Genetic humanization strategies range from mutating a few key bases for a critical protein domain to replacing entire genes including noncoding regions. Gene replacement models are an exciting system enabling the study of splicing and other regulatory mechanisms in the context of a living animal.
Genetically humanized mice are straightforward to use in experiments as they carry the modification in their genome and pass it on to their offspring. A mouse that expresses a disease-causing mutant form of a human gene can be the cornerstone of an extensive research program.
For more details contact ingenious today to discuss your next humanized mouse model.
References
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ENCODE (ENCyclopedia Of DNA Elements): The goal of this international database is to map functional elements within the human genome to the biological functions they regulate.
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