Genetically engineered mouse and rat models can be broadly grouped into three categories: Gene knockouts, knock-ins, and transgenics. The intent of a knockout mouse model is to generate an animal with a loss-of-function (null) allele. In contrast, the primary intent of a knock-in model is to generate an animal with a gain-of-function or altered-function allele. Generally, a knock-in animal model is created by inserting a defined DNA sequence into the genome at a specific location. This manipulation may replace an endogenous DNA region or add an exogenous DNA segment.
Custom Model Generation
Knock-in Mice & Rats
Knock-in Model Overview
The generation of knock-in mouse models offers access to a variety of GEM models such as: Modeling human SNPs by creating a point mutation in the mouse or rat gene, genetic humanizations where the endogenous gene is replaced by the human gene, and fluorescent reporter models where the expression of a fluorescent protein is driven by endogenous regulatory elements. Depending on the model design, knock-in mice and rats may be used to streamline drug development, assess pharmacokinetic/pharmacodynamic (PK/PD) modeling of novel therapeutics, model human disease and explore pathogenic mechanisms, study basic biology at the whole animal, tissue-, and cell-level, and more. Given Taconic Biosciences' deep toolbox and extensive experience, the range of knock-in mice and rats that we can generate is vast.
At Taconic we generate knock-in mouse models using either CRISPR/Cas9 in embryos or homologous recombination (HR) in embryonic stem cells (ESCs), whereas knock-in rat models are generated using the CRISPR/Cas9 system. Broadly speaking, CRISPR/Cas9 methodology is better suited for short DNA sequence knock-ins (e.g., point mutations, small protein tags, fluorescent reporters) whereas HR in ESCs is better suited for long DNA sequence knock-ins (e.g., genomic replacement humanizations).
Embryonic stem cell-mediated mouse model generation remains the gold standard and best choice for complex projects. The fidelity of this process, along with our extensive experience, allows Taconic to routinely perform the largest genomic insertions in the industry.
Browse the menu below to see examples of how different targeting strategies lead to the generation of several different mouse models.
- Constitutive Knock-in
- Constitutive Knock-in with Conditional Knockout Option
- Conditional Knock-in
- Overview of the Taconic ESC-mediated Gene Knock-in Workflow
Constitutive Knock-in
This strategy is often used to express a gene of interest under the direct control of endogenous regulatory elements.
In this example, a human cDNA followed by the mouse 3' untranslated region and a polyadenylation (pA) signal is inserted into the first coding exon of the mouse ortholog. This targeting strategy is designed to allow for expression of the human protein under the control of endogenous mouse regulatory elements while also eliminating expression of the mouse protein.
Constitutive Knock-in with Conditional Knockout Option
This strategy is often used to express a gene of interest under the direct control of endogenous regulatory elements. The inclusion of flanking recombinase sites, however, allows for the conditional deletion of the knocked-in sequence after exposure to the appropriate recombinase.
In this example, the mouse gene including all exons and introns is replaced with the human ortholog including all exons and introns. Cre recombinase recognition sites (loxP sites) have been inserted to allow for the conditional deletion of most of the human gene coding sequence.
Conditional Knock-in
This strategy is often used to restrict expression of a specific modification when early or constitutive expression is undesired. Using a recombinase-dependent strategy similar to that employed for conditional knockouts, a knocked-in sequence may be designed such that expression only occurs after exposure to the appropriate recombinase.
In this example, loxP sites are inserted flanking exons 7 and 8 of the wild type gene and a duplication of these two exons incorporating a point mutation is inserted downstream. Following Cre recombinase activity, the wild type exons are removed and replaced by the duplicated exons which contain a point mutation.
Overview of the Taconic ESC-mediated Gene Knock-in Workflow
- Development of an animal model generation strategy
- Targeting vector construction and DNA sequence validation
- Transfection of targeting vector into ESCs
- Isolation of targeted ESC clones and molecular validation by extensive radioactive Southern blot analysis using multiple external and internal probes and multiple restriction enzymes
- Removal of selection cassette(s) by in vitro deletion (if necessary)
- Injection of validated ESC clones into mouse blastocyst-stage embryos
- Generation and molecular characterization of F0 founder chimeras
- Generation of F1 mice heterozygous for the knock-in allele
- Standard deliverable: 3 to 5 F1 heterozygous mice
- Optional ExpressMODEL® deliverable: 20+ F1 heterozygous mice
- Molecular characterization of F1 heterozygous mice
Featured Resources
“Taconic has been a great partner for us. We had previously relied on several other academic institutions for model generation, wasting tens of thousands of dollars and several years, with no success. Since starting our relationship with Taconic, the company has generated a Plk1-KI line that subsequently secured a major grant from the NIH. We are so pleased that we have already placed several additional orders and look forward to working with Taconic for many years to come.”
– Xiaoqi Liu, Biochemistry Department at major research institution
“We are very pleased to add Taconic to our list of licensees as it furthers our goal of making this technology more broadly accessible and Taconic is certainly a globally recognized leader in the animal model field.”
– Eric Rhodes, CEO of ERS Genomics
- Testimonial 1
- Testimonial 2
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