Knock-in mice

Knock-in mice

Knock-in mice

What are knock-in mice?

Knock-in mice have an insertion in a specific locus in order to produce genetically modified mice for customised research purposes. Reporter genes, human genes, related genes from the same organism, or point mutations can be introduced into the protein-coding region of a gene to produce knock-in mice. The difference between the knock-in and knockout approach is that knock-in mouse models are utilised to drive expression of an exogenous transgene or gene mutation, whereas knockout mice aim to disrupt the expression of a specific wild-type gene.

What are knock-in mice used for?

Researchers use knock-in mouse models to mimic human genetic disorders, understand embryonic development and evaluate therapeutics by assigning gene functions, dissecting genetic pathways and manipulating the cellular or biochemical properties of proteins. Read our research features and publications for examples of knock-in research.

Types of knock-in mouse designs:

  • Reporter – Track gene expression or the lineage specification and differentiation of desired cell types.
  • Point mutation – Study functional gain/loss of a protein with a subtle mutation by altering one or more nucleotides.
  • Humanization – Study antibody or small molecule drugs in mice expressing the human drug target.
  • Targeted transgenics at a ‘safe harbor’ locus – Study novel transgenes in the absence of random insertion effects.
humanized mice

How to make knock-in mice?

Knock-in mice are generated by gene targeting via homologous recombination of a targeting construct containing the desired knock-in sequence at the target genomic locus. Knock-in mouse models range from the introduction of a single point mutation within a gene to the replacement of an entire gene within its human orthologue or other large DNA sequence and can be generated via conditional or non-conditional approaches. Germline transmission has been achieved for all projects to date with leading timelines, thanks to our proprietary goGermline™ technology.

Non-conditional KI models

Non-conditional (constitutive) knock-in mutations are generated by knocking in a foreign sequence into a targeted locus which expresses the inserted sequence throughout the development and life of the model with no options or conditionality. Some of the common designs for non-conditional models are reporter-tagged mice and recombinase mice. However, we recommend the conditional approach whenever possible; the non-conditional approach is not recommended for transgene knock-ins which could affect development, as it can lead to lethality, sterility and developmental defects in ES cells, chimeras, and heterozygous mutant mice.

knock-in mice

Reporter/recombinase KI mice

Reporter knock-in mouse models can be used to track the expression of a targeted gene. Commonly used fluorescence reporters are green fluorescent protein (GFP) and red fluorescent protein (RFP).

Recombinase knock-in mouse models carry recombinases such as Cre. These mouse models are developed to cross with gene-targeted mice carrying the conditional knock-in or conditional knockout alleles.

Conditional KI models

Conditional knock-in mutations allow inducible and tissue-specific transgene expression in combination with Cre/Lox technology, which provides control over gene expression. This approach produces a stable gain of function as it has the ability to spatially and temporally inactivate or activate gene expression. We would typically choose to create our knock-in models (such as a point mutation, humanization or targeted transgenic model) using a conditional approach.

Point mutation/minigene KI mice

This conditional knock-in model uses the Cre/Lox recombination system to express the modified knock-in allele following breeding to a Cre-expressing line. Conditionality is conferred by the presence of a floxed minigene cassette. In the conditional allele, the wild-type mouse protein is expressed from the minigene. Following Cre-mediated recombination, the minigene is excised, permitting expression of the modified knock-in protein.

knock-in mice - Point mutation KI

How long does it take to generate knock-in mice?

knock-in mice

“No project queues – get started today!”

Ozgene’s Lean management style coupled with patented goGermline technology allows us to generate mouse models in the fastest possible time together with increased certainty of germline transmission.

OzBIG – Genomic replacements up to 240 kb

Ozgene also offers a service called OzBIG for robust creation of large, gene-targeted replacement models. This system enables routine generation of humanized mice with very large (20 to 240 kb) genomic replacements and insertions. OzBIG can also be used to generate knock-in mice with large non-humanized, transgenics like complex reporters and synthetic expression systems.

Read more about Ozgene’s Knock-in mice

Nat Metab.
2023 Feb 16. doi: 10.1038/s42255-023-00736-8. Online ahead of print.

PFKFB2-mediated glycolysis promotes lactate-driven continual efferocytosis by macrophages

Maaike Schilperoort, David Ngai, Marina Katerelos, David A Power, Ira Tabas
2023 Feb 15;42(4):e112118. doi: 10.15252/embj.2022112118. Epub 2023 Jan 3.

A critical period of prehearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells

Adam J Carlton, Jing-Yi Jeng, Fiorella C Grandi, Francesca De Faveri, Federico Ceriani, Lara De Tomasi, Anna Underhill, Stuart L Johnson, Kevin P Legan, Corné J Kros, Guy P Richardson, Mirna Mustapha, Walter Marcotti
PLoS One.
2023 Feb 9;18(2):e0280792. doi: 10.1371/journal.pone.0280792. eCollection 2023.

Mutation of regulatory phosphorylation sites in PFKFB2 does not affect the anti-fibrotic effect of metformin in the kidney

Geoff Harley, Marina Katerelos, Kurt Gleich, Mardiana Lee, Peter F Mount, David A Power
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‘I was impressed with the discussions with the Ozgene technical team, prior to us making a decision. Once the project started, regular updates made me feel like I knew what was going on at every step in the process.’

– Prof. Deborah Henderson, Newcastle University, UK