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"I’ve been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, […]” - Raghu Mirmira, MD, PhD University of Chicago.
Today a founder mouse for a new knockout line can be generated in as little as 3 months. The pups of that founder can be genotyped 3-4 months after that, which is an essential step to confirm that a potential founder will pass on the mutation to the next generation. These short mouse knockout timelines are made possible by the development of the CRISPR/Cas9 system of gene targeting. As discussed below the CRISPR method can directly modify the genome of a mouse embryo, skipping the intermediate step of gene targeting in ES cells which was previously the standard technique.
Recently a method called CRISPR has become popular for generating mouse knockout models. The technique takes advantage of a DNA-cutting enzyme called Cas9, which can be targeted to specific sites in the genome. If this enzyme is introduced into mouse embryos it introduces DNA breaks which are repaired by an error-prone process. When properly targeted this can alter a gene and disrupt its function, creating a knockout allele.
A CRISPR gene editing project begins with careful evaluation of the target gene to identify potential target sites for Cas9. The enzyme cuts at a site specified by a guide RNA molecule, and choosing the best sequence is crucial for success with this method.
Generation of targeting material:
A thorough analysis of the target sequence is performed by a skilled molecular biologist, and targeting materials are designed and produced. Whenever possible a test is performed to check for successful, targeted DNA cutting.
Total timeline: 3-6 weeks.
Targeting material introduced into embryos:
CRISPR reagents are introduced into fertilized mouse embryos. Cas9 cuts DNA at precise genomic locations as directed by the guide RNA. Once DNA double-strand breaks are made, the cell’s repair machinery takes over to mend broken ends. This process often introduces errors in the DNA sequence leading to non-functional alleles (knockouts). The treated embryos are born in a few weeks. If they are able to pass on the mutation to their offspring they will become the founders of a new mouse line.
Timeline for birth of potential founders: ~6 weeks.
From potential founders to germline-confirmed mice:
When the CRISPR method is used to target a gene in a mouse embryo it is possible for multiple mutations to be introduced. The mouse that is born is usually a genetic mosaic – different cells in the mouse may have different changes to the targeted gene. If a mutation is successfully introduced into the germ cells of the mouse it can usually pass on the allele to its pups. Each potential founder must be tested by breeding it with a wild-type mouse and genotyping the resulting pups. Once the mutation is confirmed in the next generation a new knockout line can be considered successfully established.
Timeline from birth of potential founders to confirmation of germline transmission: 12-14 weeks.
Total time to CRISPR mice for simple KO/KI alleles: 6 months.
Gene targeting in ES cells was first used to make a knockout mouse almost 30 years ago. Although many advances have made this technique more reliable and accessible since then basic strategy is unchanged. For each desired genetic modification a new, custom DNA molecule is created and introduced into ES cells where it can be used as a template for re-writing a region of the genome. The cells where this occurs are identified and used to generate gene-targeted mice.
Creation of targeting strategy and targeting materials:
The first step in the mouse knockout process is careful analysis of the target gene. A successful strategy will disrupt the function of the target gene while leaving other genes unaffected.
Next, a targeting vector is created. The vector includes DNA encoding the desired knockout allele for the target gene. Along with that the vector includes a marker sequence to identify successfully targeted ES cells. The mutant and marker sequences are flanked by long targeting sequences. This process can be challenging as the DNA vector is larger than most researchers typically use and it is vital that no unwanted mutations be introduced.
Analysis of the target gene and vector creation is usually completed in ~6 weeks.
Generation of gene-targeted ES cells:
Targeting vector DNA is electroporated into embryonic stem cells. The targeting sequences in the vector are recognized by cellular machinery because they match the sequences on either side of the targeted region. This induces the cell to use the vector sequence as a template to repair nonexistent DNA damage, inserting new DNA from the construct into the desired locus to replace a portion of the original genome. Correctly modified ES cells are first identified using the marker sequence, then carefully checked to ensure all desired modifications were introduced.
Targeted ES cells are usually identified in about 3-4 months.
Creation of founder mice and confirmation of germline transmission:
Confirmed positive ES cells are used to generate chimeric mice which may be able to pass the mutation on to the next generation. Chimeric embryos are made by using a very fine needle to inject ES cells into wild-type embryos. These embryos develop in surrogate mothers and are raised to breeding age. Genetic testing is then performed on the offspring (F1 generation) to determine which mice carry the genetic modification.
From injection to the identification of germline confirmed F1 mice takes ~ 4 months.
Total time to ES cell-mediated KO/KI mice, including complex alleles: ~ 8-9 months.
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