Knock-in Models

Request Consultation Genetically engineered mouse and rat models can be broadly grouped into three categories: knockouts, knock-ins, and transgenics. The intent of a knockout 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 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. A few examples of models that can be generated using a knock-in strategy include: 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. Given Taconic Biosciences' deep toolbox and extensive experience, the range of knock-in models that we can generate is vast. Depending on the model design, knock-in models may be used to streamline drug development, assess PK/PD of novel therapeutics, model human disease and explore pathogenic mechanisms, study basic biology, and more.

At Taconic we generate knock-in mouse models using either CRISPR/Cas9 in embryos or homologous recombination (HR) in embryonic stem cells (ESCs). We generate knock-in rat models using CRISPR/Cas9. 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 embryonic stem cells is better suited for long DNA sequence knock-ins (e.g., genomic replacement humanizations).

ESC-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. The use of ESCs allows for extensive molecular characterization early on in the project. Several examples of knock-in model targeting strategies utilizing HR in ESCs are shown below.

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 Knock-in Workflow