With JAX, you can be confident your new model is precise and the genetic modification has been verified. We use a variety of technologies and strategies to deliver customized solutions that are specific to your needs.
Knock-out (KO) mice have either a deletion or loss-of-function modification of your gene of interest. Researchers use KO mice for a variety of applications, including investigating the function of a specific gene, creating models that spontaneously develop disease phenotypes, or conducting preclinical efficacy testing of new therapeutics.
Both global and conditional knock-out models are typically made using CRISPR/Cas9 in either mouse zygotes or ES cells, on the genetic background of your choice. Consult with our team of subject matter experts on the best design for your knock-out mouse today!
Global Knock-Out (Deletion)
A conventional global knock-out has a deletion of the mouse gene in all tissues throughout the developmental stages and the lifetime of the mouse. This is a fast and efficient approach to study the function of a gene, and this strategy works best for genes that do not produce a severe phenotype when disrupted. To generate the KO, JAX will delete one or more exons that will result in a frameshift of the mouse gene. This results in either a truncated or complete KO at the protein level in all mouse tissues.
A conditional knock-out (cKO) can be bred to a recombinase-expressing strain to generate tissue-specific and/or inducible knock-outs so that your gene can be deleted precisely when and where you need it. This is a highly customizable approach that is ideal for genes that may produce a severe phenotype when disrupted. Most conditional knock-outs use the Cre-lox system, where LoxP sites are inserted on either side of a specific region of the mouse gene. When exposed to cre-recombinase through breeding or an AAV, the region between the LoxP sites is removed resulting in a functional KO of the mouse gene. JAX can also generate conditional knock-out models for the Flp-FRT or Dre-Rox systems.
A Point Mutation Occurs When A Single Base Pair Is Added, Deleted Or Changed.
Researchers often make point mutations in mice to model spontaneous or hereditary disease-causing mutations that occur in humans, also known as single-nucleotide polymorphisms (SNP) or single-nucleotide variants (SNV). These mouse models allow researchers to study phenotypes that result from the mutation and perform preclinical testing for therapeutics intended to correct the mutation or resolve symptoms.
Point mutations are typically made using CRISPR/Cas9 in either mouse zygotes or ES cells, on the genetic background of your choice. Consult with our team of subject matter experts on the design for your point mutation today!
A point mutation is made directly in the mouse gene of interest. This approach is precise, fast, and efficient and works best for genes that do not produce a severe phenotype when disrupted. JAX uses CRISPR/Cas9 to engineer these single-nucleotide changes. Single-stranded oligonucleotide (oligo) templates are used to make precise modifications while reducing the impact on surrounding genetic regions.
Conditional Point Mutation
A conditional point mutation gives you the ability to express a point mutation in specific tissues or at specific time points. This approach is particularly valuable when working with a gene or mutation that results in a severe phenotype. A cre-dependent point mutation is inserted, which will only be expressed in the presence of cre-recombinase. To accomplish this, JAX inserts a targeting vector containing the point mutation, such that its expression is prevented by a LoxP-STOP-LoxP (LSL) cassette or dual LoxP system. JAX can also generate conditional point mutations using the Flp-FRT or Dre-Rox systems.
A knock-in is an insertion of a DNA sequence into a specific mouse locus. Knock-ins support a diverse array of research goals, including:
To generate a knock-in, the DNA construct is inserted at a specified mouse locus. This precise, targeted insertion helps prevent unintended consequences of the genetic modification. JAX uses CRISPR/Cas9 to make a cut at the insertion site, and homology-directed repair (HDR) is used to insert a cDNA template. There are many options for customizing a knock-in: insertions can be targeted to a mouse gene of interest, or inserted into a safe harbor locus for widespread expression. Expression can be made conditional by adding a LoxP-STOP-LoxP (LSL) cassette or TetO promoter, and epitope tags or fluorescent proteins can be included to monitor expression.
Mouse models are becoming increasingly sophisticated, and often large insertions are required to generate these complex models. If your mouse model requires an insertion that exceeds CRISPR/Cas9 size limits, you have options. JAX has developed advanced techniques that permit larger insertions into the ROSA26 safe harbor locus. Alternatively, we can use traditional transgenic approaches to randomly integrate large constructs into the mouse genome.
There are multiple strategies for accomplishing large insertions. We recommend consulting with a JAX subject matter expert to determine which approach and genetic background may be most suitable for your research goals. Contact JAX to discuss the design for your new mouse model today!
ROSA26 Locus Insertions
Gt(ROSA)26Sor (referred to as ROSA26) is a safe-harbor locus that has widespread, stable expression. ROSA26 is a non-coding gene, which means that it can be disrupted without phenotypic consequences. ROSA26 insertions are optimal for large, single-copy, targeted knock-ins. To generate a ROSA26 insertion, we use either CRISPR/Cas9 or Bxb1 technology to insert the cDNA donor template. We can include the promoter of your choice for either widespread or tissue-specific expression, or we can include LoxP-STOP-LoxP (LSL) cassette to make the expression cre-dependent.
A transgene (plasmid or BAC) is inserted randomly into the mouse genome. Random transgenics can range in size from less than 1 kb, up to several hundred kb when using a BAC (Bacterial Artificial Chromosome). This approach is valuable when large insertions are required, and when a targeted insertion is not necessary. To generate a random transgenic model, we will synthesize or acquire the DNA construct, then microinject the construct into pronuclear embryos. Multiple founder mice will be produced, which may have different integration sites, copy numbers, and expression levels.
CRISPR/Cas9 Is A Versatile Gene Editing Technology That Is Precise And Efficient. CRISPR excels at making small genetic modifications such as knock-outs and point mutations, and can efficiently generate moderately sized knock-ins up to 4-6 kb. CRISPR works well in many different genetic backgrounds, so you can start in the genetic background or strain you need for your research goals – no more backcrossing for many generations! JAX’s CRISPR/Cas9 Gene Editing Service includes the design and generation of all reagents, gene editing in mouse zygotes or ES cells, generation and sequencing of founders, and one backcross to generate the N1 generation and confirm heritability. Most CRISPR projects have N1 animals ready to ship in 6-9 months, depending on the complexity of the model.
Mouse models generated by JAX’s CRISPR/Cas9 service are covered by CRISPR/Cas9 limited Use Licenses with The Broad Institute and Caribou Biosciences, Inc.
Bxb1 serine integrase is a new technology developed by JAX researchers that permits large, single-copy, targeted transgene insertions. If your construct exceeds the CRISPR size limits, you can consider using Bxb1 to insert your transgene into the ROSA26 safe harbor locus. Bxb1 has been reported to successfully make up to 40 kb insertions, and operationally JAX is guaranteeing insertions up to 10 kb. This strategy is currently available on C57BL/6J, NOD/ShiLtJ, and NSGTM mouse models. JAX’s Bxb1 Transgene Insertion Service includes the design and generation of all reagents, gene editing in mouse zygotes, generation and sequencing of founders, and one backcross to generate the N1 generation. Most Bxb1 projects have N1 animals ready to ship in 8-9 months, depending on the complexity of the model.
JAX can design and generate plasmids, or accept DNA constructs from your institution, to make random transgenic mouse models. JAX routinely achieves a 90% success rate for transgenic mouse generation! JAX's DNA Microinjection Service includes pronuclear microinjection of the transgenic DNA construct (plasmid or BAC) into pronuclear embryos of the genetic background of your choice. Embryos are then transferred into pseudopregnant females. Pups are genotyped to identify founder mice (3 mice on average) and shipped at six weeks of age. Your model will typically be ready 13-15 weeks after we acquire your DNA. Subsequent breeding to the N1 generation to confirm germline transmission can be performed through our Breeding Services.
JAX’s Embryonic Stem (ES) Cell Microinjection Service uses genetically modified mouse ES cells, either from your lab or commercial provider, to generate your new mouse model. We microinject the mouse ES cell clones into appropriate host blastocysts of the genetic background of your choice. Following microinjection, embryos are transferred to pseudopregnant host females, producing chimeric founder mice carrying the new genetic modification. Chimeric mice are identified by coat color and provided at 6 weeks of age. Mice are typically ready to ship 13-15 weeks after we receive the ES cells. Subsequent breeding to produce mice derived from the targeted ES cells (germline transmission) can be performed through our Breeding Services. If requested, we can include karyotyping of the ES cells and inject more than one ES cell clone as part of our full-service offering.
JAX is the sole source of the C57BL/6J mouse, which is the most popular genetic background for mouse model generation. JAX has successfully worked with over 200 mouse strains as a starting point for model generation projects! You can choose from common strains such as C57BL/6J or NSG™, or you can work with one of the 13,000+ strains currently in the JAX mouse collection. No matter what genetic background you choose, our genetically stable, germline-competent mouse models will meet your needs.
Every project starts with a free consultation to assess the feasibility of your new model. Our team of subject matter experts will work with you to design a model that will best achieve your research or drug discovery needs. Our expert mouse geneticists use a wide variety of model generation technologies to generate new mouse models, and can advise on which strategies will minimize risk. Most custom projects will be supported by our no-risk guarantee: if we can’t make your new mouse model, you will not pay.
You can have your mice shipped right to your institution, or you can utilize JAX’s expertise to have your colony expanded, preserved, characterized, tested, and analyzed. We invite you to explore the variety of breeding services and supporting technologies, or consider options for surgical or preconditioning services. You can be assured that all mice coming from JAX have a high health status and are specific-pathogen free (SPF), therefore quarantine is likely not needed before bringing them into your facility.
While working with JAX, your dedicated project manager will provide you with regular updates along the way, including a founder packet and N1 data packet. These packets will be discussed with you by your JAX partner. Beyond model generation, you can turn your newly created genetically modified JAX mouse into a validated model through expression analysis and/or phenotypic characterization. We invite you to explore the variety of in vivo preclinical testing and efficacy services that offer additional solutions.
Our experts are ready to help. Our team will work with you to find the best strategy to generate your new mouse model to support your drug discovery pipeline. Schedule a consultation today!