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Elizabeth Axton, Ph.D. and Dawn Tanamachi, Ph.D.
The term humanization is used, to much confusion, to mean several different things in mouse research. While broad in scope and technique, all humanized mouse models share a common goal – to better leverage the mouse model as a translationally relevant tool to model human diseases critical in drug discovery, and evaluating drug therapeutics for both efficacy and toxicity.
A genetically humanized model is one where a human gene, genomic sequence, or regulatory element is knocked into a mouse. Often, this is performed in tandem with a knockout of the endogenous mouse gene, functionally replacing the mouse gene with the human gene. Genetic humanization is useful when a specific gene or protein target is relevant to research outcomes. For example, one JAX strain expresses a G93A mutant form of human SOD1, a translationally relevant mutation that causes the mice to spontaneously develop Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig’s Disease) (JAX Strain #002726). Genetic humanization should not be confused with cellular humanization, where mice are engrafted with human cells such as hematopoietic stem cells (HSC) or peripheral blood mononuclear cells (PBMC).
With modern advances in genome engineering, one can make precise genetically humanized mouse models faster, more cost-effective, and on many different translationally relevant genetic backgrounds. However, the wide breadth of humanization strategies can be overwhelming, and it can be challenging to choose which method would be most suitable for your particular humanized mouse model. Here, we present key methods of genetic humanization, including an overview of the approaches, benefits and considerations, and examples of JAX mice that highlight the methodology’s utility.
Strategy 1: Insert human cDNA into the endogenous mouse locus
Overview: Human cDNA is inserted with CRISPR/Cas9 technology into the endogenous mouse locus. This strategy is advantageous for creating a knockout of the endogenous mouse gene and simultaneously replacing it with the human gene with a similar expression pattern. This is a valuable approach when the human sequence is relatively small (less than 5-6 kb), and the endogenous mouse gene does not result in viability or fertility issues when disrupted.
Benefits: Simultaneous one-step knockin of the human gene and knockout of the mouse gene, generating a single allele that is easier to breed and maintain than multiple alleles. Customizable by including reporters, and feasible on many diverse genetic backgrounds.
Considerations: Size limitations (insertions less than 5-6 kb), insertions are cDNA only. For some genes, knockout of the endogenous gene can cause viability or fertility issues.
JAX Example: Modeling SARS-CoV2 was initially a challenge because standard mouse models were not susceptible to COVID-19. To address this gap, researchers generated mice expressing humanized ACE2, the receptor used for cellular entry by several coronaviruses. One such strain was generated by inserting the human ACE2 cDNA into the endogenous mouse Ace2 while retaining mouse regulatory elements (JAX Strain #035000). These mice are susceptible to SARS-CoV2 and are now being used as a model for COVID-19 research.
Strategy 2: Gene Editing of the Endogenous Mouse Gene: SNP or Exon Swap
Overview: Gene editing methods are used to generate small point mutations or exon swaps in the endogenous mouse gene. This strategy is advantageous when there is high sequence homology between the mouse and human genes, and therefore small modifications will result in the expression of a human isoform.
Benefits: Small modifications are easy to accomplish with CRISPR/Cas9 and are typically cost-effective with shorter timelines. Retention of mouse regulatory elements drive translationally-relevant expression levels and can be accomplished in many genetic backgrounds.
Considerations: Must have high mouse-human homology as size limitations dictate the DNA length that can be replaced.
JAX Example: The human APOE gene is involved in lipoprotein metabolism, and human variants of this gene are implicated in both Alzheimer’s Disease and cardiovascular disease. To generate a humanized APOE, researchers first used homologous recombination to perform exon swapping of exons 2,3, and 4 of the Apoe mouse gene (JAX Strain #027894), then the resulting humanized APOE was further edited with CRISPR to generate point mutations to express the APOE3 isoform (R130C mutation, JAX Strain # 029018) and APOE2 isoform (R130C and R176C mutations, JAX Strain # 029017).
Strategy 3: Insert human cDNA into a safe harbor locus
Overview: Human cDNA is inserted with CRISPR/Cas9 or serine integrase technology into a safe-harbor locus that has widespread expression in the mouse, such as ROSA26. This robust, targeted strategy is advantageous for human gene knockins when replacing the endogenous gene is not feasible or wanted.
Benefits: Well-established and validated methods, minimal unintended phenotypic consequences, customizable by including tissue-specific promoters or reporters, can be made inducible (Cre or Tet), and feasible on many diverse genetic backgrounds.
Considerations: Size limitations (insertions less than 5-10 kb), insertions are cDNA only. If a knockout of the endogenous mouse gene is needed, then the humanized strain would need to be bred to a knockout strain which can add 1-2 generations of breeding.
While several safe-harbor locus options exist, the most common one is Gt(ROSA)26Sor, more commonly referred to as ROSA26. ROSA26 is a non-coding gene that does not produce any proteins, which means that it can be disrupted without any phenotypic consequences. ROSA26 has widespread expression in the mouse and has been well-evidenced to maintain expression levels of the target protein from genetic insertions into this region.
JAX Example: One of the most frequently mutated genes in human breast cancer is PIK3CA. Researchers sought to investigate the PIK3CA H1047R, which accounts for approximately 40% of breast cancer PIK3CA mutant alleles. This strain was generated by inserting a Cre-dependent PIK3CA H1047R mutation into the Rosa26 locus on an FVB/nJ genetic background (JAX Strain #016977). When bred to a strain that expresses Cre in mammary cells, females spontaneously develop mammary tumors and can be used to investigate therapeutic interventions targeting the PIK3CA H1047R mutation.
Strategy 4: Plasmid or BAC Transgenic with Random Integration
Overview: Plasmid or bacterial artificial chromosome (BAC) constructs can be microinjected into the pronucleus of fertilized mouse eggs, resulting in random integration of the transgene into the mouse genome. BAC transgenesis is an advantageous approach when large insertions of human gene sequences are required or when it is crucial to retain human introns and regulatory elements.
Benefits: Can accomplish large-sized insertions (up to 300 kb), maintains human intronic regions and regulatory elements, can use various tissue-specific promoters to drive expression, can customize with fluorescent reporters to track expression.
Considerations: Random integration is unpredictable and can result in the disruption of endogenous genes (potentially impacting viability or fertility), copy number variations, and germline transmissibility issues. If a knockout of the endogenous mouse gene is needed, then the transgenic humanized strain would need to be bred to a knockout strain which can add 1-2 generations of breeding.
JAX Example: Humanized FCRN mice were developed as a platform to assess the half-life and PK of human monoclonal antibodies. To generate these mice, a BAC construct expressing the full-length human FcRn alpha-chain (FCGRT) was microinjected into C57BL/6J embryos and subsequently bred to an Fcgrt knockout mouse strain. Multiple lines were generated (JAX Strains #004919 and #014565, among others), resulting in mouse strains with different pharmacokinetics. These mice exhibit a physiologically relevant expression of the human FcRn, which more accurately model antibody PK in humans.
Making Custom Humanized Mice at JAX
Like most questions in mouse research, there is no one-size-fits-all approach to generate a new genetically humanized model. JAX scientists are available to guide your decision and can discuss the benefits and considerations for each approach as they apply to your humanized mouse project. Every custom model generation project starts with discussing your model and feasibility assessment by our study directors. Some questions that are helpful to consider in this process are:
- What are your scientific goals?
- What is the size of the human gene insertion?
- Does the endogenous mouse gene need to be knocked out?
- What genetic background do you prefer for your model? Does it need to express a disease phenotype, be immunodeficient, etc.?
- Is it essential to maintain the human gene sequence, including introns and regulatory elements?
- Do you need the humanized allele to be inducible or co-express a reporter such as GFP?
The JAX Model Generation team utilizes multiple mouse model generation techniques to generate humanized mice, including CRISPR/Cas9, ES cell microinjections, and DNA microinjections. Learn more about JAX model generation capabilities and what it’s like to work with JAX. Beyond the model generation phase of the project, JAX can also assist with downstream applications of your new humanized mouse model, including expression analysis, breeding or speed expansion to produce cohorts of mice, surgical and pre-conditioning services, and a wide range of phenotyping through our in vivo services group.
Speak to a model generation scientific expert about your genetically humanized mouse model needs today!