TurboKnockout^® Humanized and Knockin Mice

Humanized knockin mice - mouse models with an endogenous gene replaced by a human gene or genomic sequence - are used in human gene function and disease research. While either nuclease-induced genome editing or traditional gene targeting may be used to develop humanized knockin (KI) mouse models, each method offers its own benefits for model development. The TurboKnockout® gene targeting service brings together the advantages of each method to provide complex gene modeling in C57BL/6 or BALB/c mouse models - including large-fragment knockins (LFKIs) up to 300 kb - on an accelerated timeline.

TurboKnockout^® delivers research-ready conditional knockout, reporter knockin, and humanization mouse models in as fast as 6 months.

>> Learn more about TurboKnockout

Cyagen TurboKnockout^® Humanized and Knockin Mice

Knockin (KI) animal models are developed by adding exogenous gene sequence(s), including transgenes, to the gene sequence of interest. Knockin mouse models are suited for many applications, including research of regulatory elements (e.g. promoter or enhancer sequences), expression quantitative trait loci (eQTLs), the production of therapeutically relevant humanized antibodies, and more.

Humanized Mouse Models

A humanized mouse is defined as a mouse model in which a portion or the full mouse gene sequence has been replaced by its human counterpart. The human gene knockin (KI) is expressed under the control of the wildtype mouse regulatory sequences. Humanized mouse models have been used in immunology, cancer research, drug discovery, transplantation research, infectious disease research, and other fields.

Humanization Knockin Strategy Example

Humanized Mouse Model Applications in Antibody Drug Development

With the rapid advancement of humanized antibody production, the use of genetic modification to develop mice that produce human antibodies has become an important facet of antibody drug development research. A humanized mouse expressing human antibody can be obtained through multiple rounds of gene targeting, respectively replacing the homologous mouse region with the human antibody heavy chain/light chain variable region. After being stimulated by the corresponding antigen, the humanized mouse can produce an antibody containing a humanized variable region portion against the antigen along with the constant region of a mouse antibody. Studies have shown that human antibodies obtained through humanized mouse screening have a more effective affinity and activity compared with human antibodies obtained by in vitro DNA recombination methods. 

Knockin (KI) Mouse Models – Point Mutation, Reporter, Rosa26 and more

By introducing point mutation(s) into homology arms or the targeting vector, desired mutation(s) can be incorporated into the target gene. The mutated gene is expressed under the control of the wildtype gene regulatory elements. Similarly, a visually detectable marker gene (such as GFP or lacZ) can be knocked into a gene locus to replace the coding sequence of the target gene for the purpose of monitoring the promoter activity of the gene. Alternatively, a marker or an epitope tag can be knocked into the end (or less frequently, the beginning) of the coding sequence to form a fusion protein, allowing examination of target gene expression and protein localization/trafficking. 

Marker (aka Reporter) Gene Knockin Strategy Example

ROSA26 Knockin – Safe Harbor Locus for Global Expression

ROSA26 is a safe harbor locus that has been widely used for achieving both global and conditional expression of the introduced gene in knockin (KI) mice. The gene of interest is targeted into the first intron of this locus. The expression of the gene can be driven by the endogenous ROSA26 promoter or by a promoter of choice that is integrated into the locus along with the gene. 

Workflow for TurboKnockout^® Humanized/Knockin Mice

1. TurboKnockout^® gene targeting strategy design

• Analyze relevant information for your target gene, including gene structure, neighboring genes at the genomic locus, known targeted animal models, etc.
• Develop a gene targeting strategy, including the method for screening targeted ES cells by PCR and confirmation by Southern blot.
• Propose the strategy to you for review and approval.

2. TurboKnockout^® targeting vector construction

• Clone relevant DNA fragments corresponding to homology arms into the targeting vector.
• Confirm construction of the final targeting vector by restriction digest and sequencing.

3. Targeting in embryonic stem (ES) cells

• Electroporate targeting vector into our super competent TurboKnockout^® ES cells, followed by appropriate drug selection and isolation of drug-resistant clones. By default, our ES cells are derived from the C57BL/6 strain. Additionally, we offer services using BALB/c-derived ES cells.
• Screen for targeted ES cell clones by PCR.
• Expand up to six PCR-positive clones, which are further confirmed by Southern blot.
• Karyotype ES cells to ensure correct chromosome number.

4. TurboKnockout^® mouse production

• Introduce targeted ES cells into host embryos, followed by transfer into surrogate mothers. The resulting TurboKnockout^® heterozygous mutants are 100% derived from the injected ES cells. We will aim to generate a minimum of 3 mice.
• Genotype founders by PCR to confirm their mutant status.
• Optional: Breed to F1.
• Delivery of mice to the customer

Pricing and Turnaround Time

^* You can choose to receive F0 founder animals and perform their own breeding to F1 mice. To ensure the successful generation of F1 mice and germline transmission, breeding of F0 animals require adherence to Turboknockout® breeding protocol, which will be supplied to you. You can also choose to have Cyagen perform F1 breeding, which adds 1-3 months to the projected turnaround time. You can choose to breed F0 founders to wildtype mice or to other stains such as Cre deleter strains of your choice; additional service fees may apply.

Note: For special knockout/knockin services not listed above, please inquire about availability and pricing. The turnaround time above does not include the time for obtaining host institution’s approval for mouse importation and transit time during shipping.

Donor Strain Information

Mouse: C57BL/6, BALB/c

Advantages of TurboKnockout^® Humanized & Knockin Mice

Compared with CRISPR-Pro-based techniques, TurboKnockout^® is free of patent disputes, provides precise models on a comparable timeline, and is the technology of choice among our clients performing drug development projects.

• Freedom to Operate: Technology of choice for drug development projects
• Success Rate: 100% guaranteed germline transmission (GLT)
• Turnaround Time: Founders as fast as 6-8 months
• Large Fragment Knockin: Insert fragments up to 300 kb
• Flexible Floxing: No size limitations for your floxed region
• 100% Money-Back Guarantee

Order Supporting Information 

Please see below for supporting information related to Cyagen animal orders.
（https://www.cyagen.com/us/en/support/order-information.html）

Inquiries and Quote Requests

Request a quote now. Alternatively, you can always email animal-service@cyagen.com or call 800-921-8930 to inquire about our services or obtain a quote for your project.