Knockout Mouse Catalog | Cyagen APAC

From the creation of the first knockout (KO) mouse, scientists have come extremely far. CRISPR technologies have quickly become a leading method used by genetic knockout scientists today, in part due to the method’s short turnaround. The traditional embryonic stem cell (a.k.a. ES Cell, ESC)-based technology explained in this video is something of the past, mainly due to the length of time these projects take to complete (8-12 months). Although not covered in this video, Cyagen’s exclusive TurboKnockout® Gene Targeting service is based on traditional embryonic stem cell (ESC)-mediated targeting techniques and can be used to provide C57BL/6 or BALB/c mouse models in as few as 6 months.

Watch our newest video to learn about the development process used in generating traditional, ESC-mediated gene knockout mice used in a wide range of genetic and human disease research studies.


Video: What are Knockout Mice?

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Note: Any percentages mentioned in this video should be taken with a grain of salt as they are not exact values. For example, the targeting efficiency of vectors vary greatly not only from loci to loci, but also from vectors to vectors. 10% is just a rough average targeting efficiency.

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Establishing the First Knockout Mouse

In 1981 Sir Martin J. Evans managed to cultivate embryonic stem cells from mice, which paved the way to producing live mice with modified genomes. This advancement enabled Mario Capecchi and Oliver Smithies to breed live mice with specific genes inactivated, establishing the first knockout mouse model in 1989.   Ultimately, Capecchi, Smithies, and Evans were awarded the Nobel Prize in 2007 for establishing the first recorded knockout mouse.

Traditional Gene Targeting Method for Creating Knockout Mice

There are a few methods that are used to generate knockout mice, but in this video I will talk about the traditional gene targeting method that was used to develop the original knockout mouse models. Traditional gene targeting involves the use of embryonic stem cells - often abbreviated as ‘ES Cells’ or ‘ESC’.

These models start with mouse embryonic stem cells from a C57BL/6 mouse - The Blastocyst starts to form about 4 days after embryonic fertilization and consists of two main kinds of cells: the trophoblasts, or outer cells, and the inner cellular mass, known as the embryoblast. Once cells actually leave the embryoblast they are referred to as embryonic stem cells. First, cells are extracted from embryoblasts - in this case, from the black mouse embryo - and are plated using a special stem cell medium, then isolated and allowed to replicate. Once they leave the embryo they are referred to as embryonic stem cells.

Design and Insertion of a Targeting Vector

Researchers create specific vectors containing the targeted DNA constructs which have been altered to contain the neomycin phosphotransferase (neor) gene inside of them instead of the naturally occurring gene. The neomycin phosphotransferase which is labeled neor is a gene that codes for a protein that makes the cell resistant to neomycin, a common antibiotic. but only if the entire gene has been integrated into the stem cell genome via homologous recombination.

Transfection via Electroporation

Scientists use a machine called an electroporator to administer a high voltage electronic field, which creates openings in the phospholipid bilayer of the embryonic stem cell membrane.  allowing the vector containing the neor gene to enter the embryonic stem cells without doing damage to the cell itself. Electroporation is remarkably inefficient, so very few cells actually incorporate the vector past their walls. 

Homologous Recombination Mediated Cassette Exchange

Now that the vector has been electroporated into the embryonic stem cells, they will undergo homologous recombination which will give them a chance at gaining neomycin resistance while also undergoing a full gene knockout. Homologous recombination refers to the process of genetic information exchange between similar or identical molecules of DNA which takes place in prophase one of meiosis. In this case, beyond the gene of interest there are two similar exons which are identical on the vector genome. This similarity makes recombination at this loci possible.  

Efficiency of Homologous Recombination

Homologous recombination is not 100% efficient - so, after all is said and done, there will be cells that may have only partially knocked out the gene, some that have failed to knock out the gene all together, some that have may have recombined the neomycin resistant gene somewhere upstream and downstream of the gene of interest, and some cells that have completely knocked-out the gene in question and replaced it with the Neomycin resistance gene. 

Identifying Cells Containing the Complete Knockout Gene

At this point, there are some cells that have successfully completed the gene knockout and others that have not. It is up to the Scientists to determine which cells have completed the knockout and which have not. Using a medium containing neomycin, scientists can narrow down which cells contain the neomycin resistance as well as which cells have failed in the electroporation process or have undergone a partial recombination - these cells will die.

Around 90% of the remaining cells will contain a random integration of the resistance gene somewhere upstream or downstream of the target gene with the remaining 10 percent of the cells containing the knockout. Scientists must now perform polymerase chain reaction (PCR) and southern blot analysis in order to determine which of the cells contain the knockout. 

Generation of Chimeric (Founder) Mice

The now genetically modified embryonic stem cells are removed from the media and inserted into a fertilized mouse embryo at the blastocyst stage using micro injection. It is important to note that this blastocyst has been created by mating two wild type white mice together. The multicolored offspring from this surrogate mother are called chimeras - also known as founder mice.  They consist of cells from two different organisms, with different genetic profiles. The cells that make up the black fur in this mouse are the ones derived from ES cells which are heterozygous for the knockout while the white fur in the chimeras represents the cells that are homozygous for the wild type allele.

Breeding Founder Mice to Generate Heterozygous and Homozygous Knockout Mice

These chimeric animals are mated with a white mouse and the offspring will be either fully black or fully white. The fully black animals are called non mosaic heterozygous progeny, also known as F1 mice, they contain a genetic knockout which needs to be homozygous to be expressed. Homologous recombination refers to a process of genetic information exchange between two similar or identical molecules of DNA which takes place in prophase 1 of meiosis. 

In order to obtain the homozygous pair of knockout mice, two F1 mice are bred together. If we use a simple Punnett square, we are able to determine the offspring from these two heterozygous mice. One in four mice will be homozygous dominant for the knockout and thus one in four mice will fully express the knockout phenotype. Using PCR and DNA sequencing, scientists are able to tell which mice are homozygous dominant and which are still heterozygous. 


Ready-to-Use Knockout Mouse Models for Research

Since the Cyagen AI Knockout Mouse Model eBank repository was established, it has delivered ready-to-use heterozygous gene knockout (KO) mice to researchers worldwide in as fast as 3 months. In addition to these standard deliverables, we have begun offering homozygous breeding services to meet popular demand.

Homozygous knockout (KO) lines not only provide clear insights into phenotypic effects of a gene KO, but may also help identify genes essential for embryogenesis – an area of immense value in developmental biology. Understandably, many researchers have concerns about homozygous lines potentially compromising embryonic development. Using a combination of heterozygous and homozygous KO lines will provide the best tools for assessing human developmental disorders and significantly boost research into novel diagnostic and therapeutic approaches.


For a limited time, we are offering live KO & cKO mouse models, starting $999/mouse.  

>>Act now to lock-in your models - search for your gene of interest on the flash sales list this week.

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