Introduction
| GeneCopeia provides transgenic mouse services using CRISPR-Cas9. Many applications are readily within reach, including constitutive and conditional gene knockout, mutagenesis, correction of disease mutations, transgene knockin, and in-frame fusion tagging. |
Advantages
- Track record of success (Figure 1).
- Fast mouse line creation using CRISPR-Cas9 and pronuclear injection (Figure 2). F1 modified mice in as little as 6 months.
- In-depth project consultation.
- Expert CRISPR clone design, construction, and mouse injection
- Compatible with GeneCopoeia’s GeneHero™ ROSA26 Safe Harbor knockin clones.
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Case study: Gene tagging using GeneCopoeia’s CRISPR transgenic mouse services
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| Figure 1. In-frame GFP fusion of a chromosomal gene in mice using GeneCopoeia CRISPR technology. Cas9 mRNA, sgRNA, and a donor plasmid targeting the M. musculus P2xr2 gene (NCBI gene ID: 231602) were injected into 1-cell stage mouse embryos. CRISPR-Cas9 stimulates homologous recombination between the unmodified chromosome and the homologous donor, resulting in an in-frame, 3’ terminal GFP-tagged gene. Positive founder mice were identified by PCR and sequencing. Gray boxes: P2xr2 exons. |
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| Figure 2. Site-specific genome editing mediated by TALEN or CRISPR-Cas9, as well as its application on transgenic mouse generation. |
References
1. Knockout mice by TALEN-mediated gene targeting.Nature Biotrchnology.2012,31,23-24
2. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.Cell Research.2013,23,720-723.
3. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Geneme Engineeing.Cell 2013,153,1-9.
Applications
Constitutive knockout
Gene knockouts can be generated either by indel formation (without donor) or by recombination of a DNA donor with the chromosome via homologous recombination (HR). HR can be achieved using either a donor plasmid or a single stranded oligonucleotide. This results in the permanent, germline-transmissible knockout of the target gene in all cells of the body throughout development
Conditional knockout
In conditional knockout models, critical exons of the target gene are flanked by LoxP sites, to enable Cre-mediated removal, if desired. Modified mice carrying targeted LoxP sites are crossed with tissue-specific or inducible (e.g. Tet) Cre transgenic mice. Expression of Cre leads to deletion of the region between the loxP sites in desired tissues or developmental stages.
Mutagenesis or correction of disease mutations
Knockin mouse models can incorporate mutations into a target region, or correct disease-causing mutations. This is readily achievable using CRISPR-Cas9 and an oligonucleotide donor for HR.
Transgenic knockin
The mouse ROSA26 locus is a well-characterized “Safe Harbor”, which makes it useful as a transgene insertion site. Insertion of transgenes at ROSA26 causes no apparent adverse effects on fitness, and permits stable gene expression. We provide transgenic mouse lines with your gene of interest, and, if desired, a reporter gene intergrated in the ROSA26 safe harbor locus. Other genomic sites can be used for transgene knockin, if desired.
Humanization
Humanized mice refer to mouse models with a partially or fully functional human gene(s), cells, tissue or organ. The human gene is expressed under the control of the endogenous mouse regulatory sequences. Humanized mice are powerful tools for human disease studies.
To Order
We provide mouse lines with permanent, germline-transmissible knockouts, knockins and transgenes of interest. These modifications, which are produced using CRISPR-Cas9, are delivered as heterozygous F1 mice. GeneCopoeia also offers genome-editing consulting services. To get a quote, please fill out the inquiry form.
Delivery
- 3 or more F1 positive mice with TALEN- or CRISPR-Cas9-mediated trangenic modifications
- Service report
Estimated Time of Arrival
- TALEN- or CRISPR-Cas9-mediated trangenic mice: 6-9 months
Related Services
GeneHero™ CRISPR-Cas9 products and services – GeneCopoeia offers CRISPR-Cas9 sgRNA design and cloning services, Cas9 expression clones and other related products and services for simple and fast targeted genomic editing.
Genome-TALER™ TALEN and TALE-TF custom services – GeneCopoeia offers several levels of affordable custom services for designing, creating and validating TALENs, TALE-TFs and other TAL effectors-based targeted genomic modification tools.
Human AAVS1 & Mouse ROSA26 safe harbor gene knockin kits and clones – TALEN- or CRISPR-mediated gene knock-in kits designed to specifically transfer your gene of interest, selection marker or other genetic element into the human AAVS1 or mouse ROSA26 safe harbor site . More than 45,000 ROSA26 or AAVS1 compatible ORF donor clones are also available. The safe integration ensures transcription-competency of the transgenes and presents no known adverse effect.
