Humanize your gene without limitations.
Humanized mice are an excellent model system for studying human disease and for developing better therapeutics. Design your ideal model without limitations – from a single base change to replacement of a 200kb gene, including the potential for conditional alleles, cassette knockins, and more. A genetically humanized mouse model from ingenious will be the cornerstone of your research for years to come.
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. The advantages of this strategy are the speed and relatively low cost of establishing a new line containing human sequence. The disadvantages are that 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 function as desired. However it’s possible for a transgene’s expression to be affected by the surrounding genetic landscape, altering its expression pattern, and the transgene may also disrupt expression of nearby mouse genes. Identifying the location of transgene insertion is part of characterizing a new line and skipping this step adds an element of uncertainty to all experiments using the line. Newer methods enable targeting of a transgenic insertion to a known safe-harbor location in the genome to obviate many of these concerns. The additional time and cost necessary for the creation of a targeted transgenic line may outweigh the uncertainty of a random transgenic.
Targeted Genomic Humanization
This strategy is used to express a human genetic sequence in place of the mouse sequence. Different designs fall under this heading, from a point mutation of a single nucleotide to the insertion of a human cDNA to replace the mouse gene’s sequence. Such a replacement achieves two goals: preventing expression of the mouse sequence, and expressing the human sequence. Creating models like this is technically challenging but routine for anyone with experience in gene targeting methods. For many point mutation models the CRISPR/Cas9 method has made the process faster. In cases where that method is not appropriate the standard method of homologous recombination in embryonic stem cells can be used. Using such a line is simple as there is only a single allele to account for during breeding, and expression of the human sequence is driven by the mouse gene’s promoter.
Genomic Replacement by TruHumanization™ from ingenious
The TruHumanization™ strategy from ingenious represents the state of the art when it comes to genetic humanization. With this approach a region of the mouse genome is precisely replaced with 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. 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.
download our humanization white paperHow 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.
download our Humanization Comparison ChartHumanized 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.
Resources
Church DM, Goodstadt L, Hillier LW, Zody MC, Goldstein S, She X, Bult CJ, Agarwala R, Cherry JL, DiCuccio M et al. 2009. Lineage-specific biology revealed by a finished genome assembly of the mouse.PLoS Biol 7: e1000112.
Devoy A, Bunton-Stasyshyn RK, Tybulewicz VL, Smith AJ, Fisher EM. 2011. Genomically humanized mice: technologies and promises. Nat Rev Genet13: 14-20.
Macdonald LE, Karow M, Stevens S, Auerbach W, Poueymirou WT, Yasenchak J, Frendewey D, Valenzuela DM, Giallourakis CC, Alt FW et al. 2014. Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes. Proc Natl Acad Sci U S A111: 5147-5152.
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.
Zhang C, Xu Y, Chowdhary A, Fox D, Gurney ME, Zhang HT, Auerbach BD, Salvi RJ, Yang M, Li G, O’Donnell JM. 2018. Memory enhancing effects of BPN14770, an allosteric inhibitor of phosphodiesterase-4D, in wild-type and humanized mice. Neuropsychopharmacology43(11): 2299-2309.
Jun JC, Kertesy S, Jones MB, Marinis JM, Cobb BA, Tigno-Aranjuez JT, Abbott DW. 2013. Innate immune-directed NF-κB signaling requires site-specific NEMO ubiquitination. Cell Rep4(2): 352-61.
