Homologous Recombination Conditional Knockout Mouse Model and Knock-in Mouse Models
Conventional knock-in and knock-out mouse generation service by homologous recombination.
Project: Germline-transmitted, conditional knock-out mice by tetraploid complementation
"Overall, I am very satisfied with the quality of your service. After we received the mice, we did extensive studies to characterize them. All of the PCR reactions confirm the gene modifications are in the correct sites. We have also crossed mice with Cre mice to demonstrate that the Cre deleted the exon in heterozygous pups as expected. All data obtained showed that the mice were correctly targeted. In addition, I am very happy with the frequent communication with your scientists during the process. I highly recommend your services."
- Testimony of Dr. Zhenheng Guo, PhD, Assistant Professor of Internal Medicine, Division of Endocrinology and Molecular Medicine, University of Kentucky
Service Milestones for the Generation of Knock-in Mouse / Knockout Mouse Models:
- Gene targeting vector construction and sequencing
- ES cell targeting, screening and expansion of positive ES cell clones
- Karyotyping of targeted ES cells
- Cre/FLP recombination (optional)
- Chimera production by blastocyst injection
- Breeding of chimeras for germline transmission
- Genotyping of offspring
- Transfer of heterozygous targeted mice to the customer
- Optional: breeding to generate homozygous mice
- Applied StemCell has years of experience and an exceptionally high success rate in generating conventional gene-targeted mice with our proprietary mESC lines.
- Extensive experience in gene targeting and genetic mouse models.
- Rigorous quality control with multiple check-points to ensure your projects are completed with highest accuracy
- All mice are generated in and shipped from our California facility (NIH guidance certified).
|Express gene "X"||Replace gene "X" with gene "Y"||Delete gene "X"|
|BAC Engineering||Conditional/Inducible Knockout|
Knock-in Mouse Models
The introduction of a gene into a specified location of the mouse genome can be used for various applications. Mice can be homozygous or heterozygous for the inserted gene.
- Reporter genes (e.g. GFP, lacZ) are used for expression analysis of a gene of interest. The reporter gene is inserted in-frame (non-disruptive) into the gene of interest, allowing for visualization of temporal and spatial gene expression pattern.
- Humanized disease models can be generated by inserting a human mutant gene or gene fragment into the corresponding mouse gene. The diseased human allele is thus transcribed with the appropriate genomic context and can be analyzed at the behavioral, pathological, cellular and/or molecular level.
- DNA recombinases (CRE, FLP) can be inserted into a gene of interest. The cell type-specific expression of the recombinase then allows for gene inactivation in the desired tissue after crossing this knock-in mouse with a conditional knockout mouse.
Knockout Mouse Models
The knockout technology is most commonly used for gene inactivation, either in a constitutive or conditional fashion.
- Constitutive knockout mouse models are widely used to study gene function. The gene of interest is permanently inactivated in all cells in the animal. However, this ubiquitous knockout may cause lethality and is therefore not always recommended.
- Conditional knockout models are inducible - the targeted gene is excised after crossing the mouse with a Cre-transgenic mouse line. Gene inactivation can be cell type-specific and/or chemically induced.
|1. Targeting Vector Construction (varies depending on complexity of cloning
Amplification of homologous arms, cloning, selection marker cassettes, and sequence confirmation
|2. ES Cell Targeting
ES Cell culture and electroporation
Positive and negative selection
Single colony picking and transfer to 96-well plates
Germline transmission tested ES cells will be used on C57BL/6 inbred background (optional)
|3. ES Cell Screening for up to 500 Clones per DNA Construct
PCR screening and sequencing confirmation
On C57BL/6 inbred background
|0.5 - 1.5 months|
|4. Cre/Flp Recombination (Optional, depending on the project)
Cre/Flp transfection into ES cells
ES cell screening
|5. Karyotyping to identify good ES clones
Up to 6 targeted clones will be karyotyped by chromosome counting
|6. ES cell microinjection (2 to 4 ES clones per construct, blastocyst injection)
ES clones will be injected into blastocysts to create chimeric mice
For C57BL/6 inbred ES cells, up to 200 blastocysts will be injected
|7. Housing and Breeding (breeding of 1-3 good chimeras with wild-type mice)
Breeding of chimeras for F1 heterozygotes, confirmation of germline transmission of gene targeting by genotyping
6 inbred ES cells
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Applied StemCell's cited/ published article:
- Zhao, M., Tao, F., Venkatraman, A., Li, Z., Smith, S. E., Unruh, J., ... & Marshall, H. (2019). N-Cadherin-Expressing Bone and Marrow Stromal Progenitor Cells Maintain Reserve Hematopoietic Stem Cells. Cell reports, 26(3), 652-669.
- Li, C., Zheng, Z., Ha, P., Chen, X., Jiang, W., Sun, S., ... & Chen, E. C. (2018). Neurexin Superfamily Cell Membrane Receptor Contactin‐Associated Protein Like‐4 (Cntnap4) is Involved in Neural EGFL Like 1 (Nell‐1)‐responsive Osteogenesis. Journal of Bone and Mineral Research https://doi.org/10.1002/jbmr.3524.
- Geraets, R. D., Langin, L. M., Cain, J. T., Parker, C. M., Beraldi, R., Kovacs, A. D., ... & Pearce, D. A. (2017). A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies. PloS one, 12(5), e0176526.
- Miller, J. N., Kovács, A. D., & Pearce, D. A. (2015). The novel Cln1R151Xmouse model of infantile neuronal ceroid lipofuscinosis (INCL) for testing nonsense suppression therapy. Human Molecular Genetics, 24(1), 185–196. http://doi.org/10.1093/hmg/ddu428.