Genetically Humanized Mice

Genetically humanized mice play important roles in many different stages of the development of treatments against human diseases. They are used to study basic biological functions, to model and understand the pathophysiological processes in human disease, and are pivotal during later stages of drug development when lead therapeutics have to be refined and tested in tolerization and toxicity assays.

Depending on the project scope and the experimental goals, various kinds of genetic humanizations are possible, ranging from single substitutions of conserved amino acids, to the expression of human cDNAs from endogenous or safe harbor loci, or the partial or complete replacement of the mouse gene by its human counterpart.

Humanization Projects

Described below are some of the most common types of genetic humanizations and point out specific advantages and limitations. For selection of the most appropriate strategy for your research, please reach out to one of our Model Generation Experts. Below are a three different examples of humanization projects; each carry their own set of advantages and disadvantages.

Full/Partial Genomic Replacement
Minigene Knock-in
Random Integration Transgenics

Full/Partial Genomic Replacement:

In this strategy, the coding region of the mouse gene is replaced by its human counterpart, including human intronic sequences. This is intended to result in the expression of the human protein isoforms regulated by the endogenous mouse promoter and enhancers. Untranslated exonic regions at the 5’ and 3’ ends are often, but not always, unchanged to support proper transcription. Partial humanization (e.g., of only an extracellular domain of a transmembrane protein) is also possible by this approach, resulting in a chimeric mouse-human protein.

Advantages:

Expression controlled by endogenous regulatory regions
Transcript diversity is preserved
Simultaneous knockout of mouse gene

Disadvantages:

Potential for altered expression levels (i.e., non-physiological)
Often requires gene targeting in embryonic stem (ES) cells

Minigene Knock-in

In this strategy, a human minigene is inserted into the orthologous mouse region. The minigene contains the cDNA of one of the gene’s transcripts and a poly A signal. To increase transcript stability and expression, the minigene can additionally contain an intron and the mouse or human 3’ untranslated region.

Minigenes may also be inserted into safe harbor loci, though this precludes the use of regulatory sequence from the orthologous region in mouse, and depends instead on the use of a pre-defined promoter.

Advantages:

Expression controlled by endogenous regulatory regions
Simultaneous knockout of mouse gene
Potentially eligible for in vivo CRISPR targeting for faster timeline

Disadvantages:

Transcript diversity is not preserved
Elevated risk for altered expression levels (i.e., non-physiological) when compared to genomic replacement

Random Integration Transgenics

In Random Integration Transgenics, plasmid constructs or genomic bacterial artificial chromosome (BAC) vectors (of up to 200kb length) encoding a human gene sequence are microinjected into fertilized oocytes, resulting in the random insertion of the transgene at a single or multiple sites in the genome. The BAC vectors can also carry modifications like the insertion of a reporter gene or mutations in the coding regions for the expression of disease-related protein variants. Importantly, this type of humanization may be pursued in both mice and rats.

Advantages:

Expression controlled by human regulatory regions (variable depending on transgenic expression construct used)
Fast generation of founders using pronuclear injection

Disadvantages:

Orthologous mouse gene still present
Integration can disrupt endogenous genes, leading to unwanted phenotypes
Structure and copy number at each integration site will vary, and is unpredictable
Significant characterization of expression levels and patterns needed

Experience & Expertise You Can Trust

Taconic Biosciences' model generation team has produced about 5,000 models in the last 15 years, developing a globally-recognized reputation for advancing the work of in vivo researchers. Our scientific program managers are here to help you navigate the complexities of model generation.

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