Custom Model Generation

Knock-in Mice & Rats

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. 

black mouse

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

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 chart

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.

conditional knock in chart

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

White Paper

Succeeding with Mouse Model Generation: Choosing the Right Genetic Background

Backcrossing, Breeding Colonies, C57BL/6, C57BL/6J, CRISPR/Cas9, Embryonic Stem (ES) Cell, ES Cells, GEMs, Genetic Background, Genetically Engineered Mice, Genotype, Immunodeficient, Outbred, PNI, Preclinical, Rat, Single Nucleotide Polymorphisms (SNPs)
succeeding with mouse model generation whitepaper cover

Get In Touch

Book a complimentary consultation or get support for an existing order

If you need immediate assistance, please contact Customer Service:

Taconic Corporate Offices

Phone: +1 (518) 697-3900
273 Hover Ave., Germantown, NY 12526

North American Customer Service

Phone: +1 (518) 697-3915 
Toll-free:  +1 (888) 822-6642

Hours: (Monday - Friday): 7 a.m. - 6 p.m. ET

European Customer Service

Phone (Europe and Denmark): +45 70 23 04 05 
Phone (Germany): +49 214 50 68 023 
Hours: (Monday - Friday): 7 a.m. - 5 p.m. CET

Let's Get Social!

How can we help you?
Fill out the form to connect with us:

Welcome! Tell us a little about yourself