• TARGATT™ Site Specific Knock-in Mice

TARGATT™ Site-Specific Knock-in Mouse

Applied StemCell’s proprietary TARGATT™ knock-in mouse technology enables highly efficient and site-specific gene integration to generate transgenic mouse models. This technology uses PhiC31 integrase to insert any gene of interest into a specific docking site that has been pre-engineered into an intergenic and transcriptionally active genomic locus for guaranteed transgene expression.

Advantages of TARGATT™ Technology for Knock-in Mouse Model Generation:

  • Safe harbor locus “large fragment knock-in” (up to 22 kb)
  • High efficiency insertion (up to 40%)
  • Single copy in an active locus: avoid gene silencing and genomic instability.
  • Germline transmitted F1 mice in 5-8 months
  • Tissue-specific/ ubiquitous, controlled/ inducible expression options

Please contact us to discuss your project plan.

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Application Notes

Key Points of the TARGATT™ Transgenic Mouse Technology

The H11 Locus supports a high level of gene expression in animal tissues (Liver, heart, brain)

knock in mice H11 Locus supports a high level of gene expression
Citation: (Tasic, B., et al. (2011). Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proceedings of the National Academy of Sciences108(19), 7902-7907.)

GFP expression in embryos carrying a single copy of pHb9-GFP transgene site-specifically integrated into H11

knock in mice GFP expression in embryos
Citation: (Arber et al. Cell 1999)

Conditional Knockout Mouse Models Using CRISPR/Cas9

Conditional knockout (CKO) animal models are gaining popularity as they circumvent the impediments of constitutive knockout models such as embryonic lethality, compensatory mechanisms and undesired phenotypes and model human diseases better. The most commonly used CKO system is the Cre-LoxP system, where the gene of interest (targeted exons) is flanked by two LoxP sequences (also called floxed allele). The flanking LoxP sequences are inserted at specific sites on either side of the gene of interest using CRISPR/Cas9 technology (Figure 1). The LoxP sites are a target for the Cre Recombinase which catalyzes the deletion of the floxed exon(s).

SCHEMATIC-CRISPR-CKO-mouse-1
Figure 1. The schematic describes the first stage in developing a conditional knock-out mouse model using CRISPR to generate a floxed (loxP flanked exon) mouse. A single stranded donor DNA (ssDNA) is used for delivering the floxed targeting exons to replace the wildtype form. The donor contains two LoxP sequences flanking the targeted exon(s) along with 5' and 3' homologous arms for directing a site-specific homology directed repair. The donor ssDNA is delivered along with Cas9 (mRNA or protein) and validated gRNAs via microinjection.

Cre-driver Transgenic Mouse Models Engineered Using TARGATT™ Technology

Cre- mouse models are generated by microinjection of an integration cocktail into the pronuclus of TARGATT™ mice engineered with "attP" docking sites at a preselected locus. The integration cocktail consists of the targeting vector (promoter+ Cre gene + attB sequence) and in vitro transcribed PhiC31 mRNA. The integrase catalyzes the recombination between the attB and attP sites, resulting in integration of the promoter-Cre transgene in a site-specific manner without any position effects associated with random insertion. The attB-attP recombination results in unique sequence (attL and attR) flanking the inserted transgene which is not recognized again by the integrase and thereby ensures an uni-directional, stable integration reaction.

knock in mice engineering of a Cre-driver mouse model

Figure 2. Schematic illustrates the engineering of a Cre-driver mouse model using TARGATT™ integrase technology. A cocktail of TARGATT™ donor vector carrying the integrase recognition sequence “attB” (orange arrow) and the Cre-driver transgene (promoter-of-choice; yellow triangle and Cre gene; dark blue), and the TARGATT™ integrase is microinjected into the pronucleus of a TARGATT™ mouse embryo that carries an “attP” docking site (purple arrow) inserted into a preselected safe harbor locus such as H11 (described earlier). The  Integrase catalyzes a recombination between the attP and attB sites, resulting in two new hybrid sites, attL and attR which are no longer recognized by the integrase enzyme. As a result, gene integration is stable and the process is highly efficient in generating transgenic Cre mice. 

Conditional knockout mice are generated by crossbreeding the two transgenic mouse lines: (a) the homozygous “floxed” (flanked by loxP) allele mouse model, and (b) the Cre-driver mouse model with tissue specific expression or ubiquitous expression (Figure 3). The Cre expression has minimal unwanted effects in the animal as the mouse genome does not contain endogenous loxP sites, providing an ideal background for site-specific recombination. 

knock in mice crossbreeding 

Figure 3. Crossbreeding the conditional knock-out mouse with a Cre-recombinase expressing mouse. The Cre expression is driven by a promoter of choice: tissue specific or ubiquitous promoter. As an example, a CNS-specific promoter is shown in the figure. The expressed Cre recombinase deletes the floxed exon(s) in a spatial specific manner there by causing a frame shift in downstream sequence. 
FAQs
Can I create models to overexpress a gene of interest?
Can I use TARGATT™ system to create transgenic mice with tissue-specific gene expression?
What promoters are used to drive gene expression?
What is the specific site that my gene of interest will be integrated into?
Besides H11 and Rosa26, can gene be inserted at other loci?
Can I integrate a reporter gene? What kind of reporter genes do you recommend?
What is the maximum size of a gene you can insert? Will the efficiency of your system be affected if the gene is too large?
Why does the TARGATT™ knock-in system have high efficiency?
How many copies of the gene will be inserted into the genome?
Do you have TARGATT™ technology available for Knock-in cell lines?
Technical Details

Applied StemCell's TARGATT™ site-specific knock-in mice service is advantageous over CRISPR/Cas9 for large DNA fragment insertion due to higher insertion efficiency and site-specificity.

TARGATT™ Technology can be utilized for a variety of applications including reporter gene expression, gene knockdown, disease models and even site-specific Cre expression for advanced conditional knockout models.

knock in mice schematic

We have compiled and reported data from our mouse model projects in a technical white paper titled TARGATT™ FOR TARGETED GENE INSERTION IN MOUSE MODELS. This paper highlights the advantages of this novel gene editing platform in generating site-specific knock-in mouse models, and Applied StemCell's expertise in successfully generating transgenic mice.

We also have off-shelf products to generate your own TARGATT™ transgenic mice as a do-it-yourself option:

TARGATT™ Mouse (C57BL6/H11)

TARGATT™ Plasmids

TARGATT™ Transgenic Kits

TARGATT™ Knock-in Mouse Generation Timeline

 Service Time Deliverables
1. TARGATT DNA Construction
(genes-of-interest in plasmid will be provided by the client)
 4-6 weeks A report on TARGATTTM vector cloning
2. Generation of TARGATTTM integrase
in vitro transcription and purification
 1 week A report on integrase mRNA synthesis and validation
3. TARGATT™ DNA Pronuclear Microinjection
(up to 150 embryos will be injected)
1-2 months  
4. Animal Care, Housing, and Genotyping
Pups generated and genotyping showing the proof of precise gene insertion
 3-4 weeks At least 1 founder with a single copy of transgene inserted into Rosa26 locus
A final report on the TARGATT™ knock-in project including the original targeting strategy and microinjection details

Link

Case Studies

Case #1. Site-specific knock-in of a coat color gene in mice

 knock in mice coat color gene

Case #2. Inducible glycoprotein gene knock-in mice

TARGATT construct: pBT-CAG-LoxP-Stop-LoxP-GeneX

Embryo donor strain: H11-C57BL6

 knock in mice unducible glycoprotein gene

Twenty-three pups were born from one microinjection experiment. *Two positive founders were identified using five primer pairs (PCR 1, 2, 3, 4, 5) to confirm that insertion was site specific at H11.

Case #3. Site-specific transmembrane gene knock-in mice.

knock in mice transmembrane gene

Four positive founders were identified from 27 newborn mice.

Publications

Stanford University Researchers Find a Molecular Basis for Blond Hair Color using TARGATT™ Knock-In Mice

Recent genome-wide studies have linked blond hair color variants to regulatory regions, outside of protein-coding sequences, of genes. The human gene KITGL (mouse Kitl) encodes for a secreted ligand for the KIT receptor tyrosine kinase, which is essential for development of many cell types including melanocytes. A SNP (A>G substitution) located 350 kb upstream of KITLG (intergenic region) is associated with blond hair in Icelandic and Norwegian populations. A similar mutation in mice (caused by an inversion) causes a displacement of the orthologous mouse sequence to the blond-associated GWAS peak. The mice that are homozygous for this inversion are white, and mice that are heterozygous for the inversion are lighter than wild-type mice. In this publication, the researchers use TARGATT™ transgenic mouse technology to characterize the effect of the A>G substitution in the SNP and how it affects blond hair. TARGATT™ technology is superior to traditional methods of random transgene insertion because:

1.TARGATT™ allows precise, phenotypic comparison of a single nucleotide change. This would be difficult to achieve using random transgene insertion.

2.TARGATT™ allows efficient insertion of a large DNA fragment in a transcriptionally active locus, which would be very difficult to achieve using CRISPR, or other nuclease-based methods that are designed for smaller insertions or deletions.

To quantify changes in enhancer activity in vivo, Dr. Guenther, a scientist in Dr. Kingsley’s group created transgenic animals using the PhiC31 integrase, in animals expressing attP sites at the H11 locus (TARGATT™ approach). They made 2 types of transgenic mice: those carrying an A allele (ANC-Kitl) and those carrying a G allele (BLD-Kitl), corresponding to the variant sequence of the SNP. Each transgenic mouse only had one integrated copy of the allele, both expressed from the same locus. The resulting BLD-Kitl mice had significantly lighter hair than the ANC-Kitl mice.

knock in mice molecular basis for blond hair color 1

knock in mice molecular basis for blond hair color 2

Reference: Guenther et al., “A molecular basis for blond hair color in Europeans”, Nature Genetics, doi:10.1038/ng.2991.


Description of the technology

  • Zhu, F., Gamboa, M., Farruggio, A. P., Hippenmeyer, S., Tasic, B., Schüle, B., … Calos, M. P. (2014). DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Research42(5), e34. http://doi.org/10.1093/nar/gkt1290.
  • Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., & Luo, L. (2011). Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proceedings of the National Academy of Sciences of the United States of America108(19), 7902–7907. http://doi.org/10.1073/pnas.1019507108.

Commentary, comparison with other transgenic methods

  • Rossant, J., Nutter, L. M., & Gertsenstein, M. (2011). Engineering the embryo. Proceedings of the National Academy of Sciences108(19), 7659-7660.

Tet inducible mice generated by TARGATT™

Advantage of Hipp11 (H11) locus

Applications for mice generated by TARGATT™ (and cited/published articles)

  • Matharu, N., Rattanasopha, S., Tamura, S., Maliskova, L., Wang, Y., Bernard, A., ... & Ahituv, N. (2018). CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science, eaau0629.
  • Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY
  • Barrett, R. D., Laurent, S., Mallarino, R., Pfeifer, S. P., Xu, C. C., Foll, M., ... & Hoekstra, H. E. (2018). The fitness consequences of genetic variation in wild populations of mice. bioRxiv, 383240.
  • Ibrahim, L. A., Huang, J. J., Wang, S. Z., Kim, Y. J., Li, I., & Huizhong, W. (2018). Sparse Labeling and Neural Tracing in Brain Circuits by STARS Strategy: Revealing Morphological Development of Type II Spiral Ganglion Neurons. Cerebral Cortex, 1-14.
  • Kumar, A., Dhar, S., Campanelli, G., Butt, N. A., Schallheim, J. M., Gomez, C. R., & Levenson, A. S. (2018). MTA 1 drives malignant progression and bone metastasis in prostate cancer. Molecular oncology.
  • Jang, Y., Broun, A., Wang, C., Park, Y. K., Zhuang, L., Lee, J. E., ... & Ge, K. (2018). H3. 3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development. Nucleic acids researchhttps://doi.org/10.1093/nar/gky982
  • Tang, Y., Kwon, H., Neel, B. A., Kasher-Meron, M., Pessin, J., Yamada, E., & Pessin, J. E. (2018). The fructose-2, 6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC. Journal of Biological Chemistry, jbc-RA118.
  • Chen, M., Geoffroy, C. G., Meves, J. M., Narang, A., Li, Y., Nguyen, M. T., ... & Elzière, L. (2018). Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS. Cell Reports, 22(13), 3587-3597
  • Kido, T., Sun, Z., & Lau, Y.-F. C. (2017). Aberrant activation of the human sex-determining gene in early embryonic development results in postnatal growth retardation and lethality in mice. Scientific Reports7, 4113. http://doi.org/10.1038/s41598-017-04117-6.
  • Nouri, N., & Awatramani, R. (2017). A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons. Development144(5), 916-927.
  • Li, K., Wang, F., Cao, W. B., Lv, X. X., Hua, F., Cui, B., ... & Yu, J. M. (2017). TRIB3 Promotes APL Progression through Stabilization of the Oncoprotein PML-RARα and Inhibition of p53-Mediated Senescence. Cancer Cell31(5), 697-710.
  • Jiang, T., Kindt, K., & Wu, D. K. (2017). Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells. eLife, 6, e23661.
  • Booze, M. L., Hansen, J. M., & Vitiello, P. F. (2016). A Novel Mouse Model for the Identification of Thioredoxin-1 Protein Interactions. Free Radical Biology & Medicine99, 533–543. http://doi.org/10.1016/j.freeradbiomed.2016.09.013.
  • Feng, D., Dai, S., Liu, F., Ohtake, Y., Zhou, Z., Wang, H., ... & Hayat, U. (2016). Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration. The Journal of clinical investigation, 126(6), 2321-2333.
  • Sun, N., Yun, J., Liu, J., Malide, D., Liu, C., Rovira, I. I., … Finkel, T. (2015). Measuring in vivo mitophagy. Molecular Cell60(4), 685–696. http://doi.org/10.1016/j.molcel.2015.10.009.
  • Devine, W. P., Wythe, J. D., George, M., Koshiba-Takeuchi, K., & Bruneau, B. G. (2014). Early patterning and specification of cardiac progenitors in gastrulating mesoderm. eLife3, e03848. http://doi.org/10.7554/eLife.03848.
  • Fogg, P. C. M., Colloms, S., Rosser, S., Stark, M., & Smith, M. C. M. (2014). New Applications for Phage Integrases. Journal of Molecular Biology426(15), 2703–2716. http://doi.org/10.1016/j.jmb.2014.05.014.
  • Chen-Tsai, R. Y., Jiang, R., Zhuang, L., Wu, J., Li, L., & Wu, J. (2014). Genome editing and animal models. Chinese science bulletin59(1), 1-6.
  • Park, K.-E., Park, C.-H., Powell, A., Martin, J., Donovan, D. M., & Telugu, B. P. (2016). Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins. International Journal of Molecular Sciences17(6), 810. http://doi.org/10.3390/ijms17060810.
  • Guenther, C. A., Tasic, B., Luo, L., Bedell, M. A., & Kingsley, D. M. (2014). A molecular basis for classic blond hair color in Europeans. Nature Genetics46(7), 748–752. http://doi.org/10.1038/ng.2991.
  • Villamizar, C. A. (2014). Characterization of the vascular pathology in the acta2 r258c mouse model and cerebrovascular characterization of the acta2 null mouse. UT GSBS Dissertations and These (Open Access)Paper 508 (2014)
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