TARGATT™ Site Specific Knock-in Mice

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Applied StemCell Inc.’s proprietary TARGATT™ knock-in mouse technology enables highly efficient site-specific gene integration in mammalian cells and animals. This technology uses PhiC31 integrase to insert any gene of interest into a specific docking site that was pre-engineered into an intergenic and transcriptionally active genomic locus. Applied StemCell can create a site-specific knock-in mouse for you in as fast as 3 months. Using our novel TARGATT™ system, a gene of interest can be inserted at a well-characterized, transcriptionally-active locus in the mouse genome with guaranteed transgene expression. Tissue-specific and/or ubiquitous expression options are available. Please contact us to discuss your project plan.

Advantages of TARGATT™ Technology for Knock-in and Knock-out Mice

  1. High integration efficiency mediated by PhiC31 integrase reduces time and cost
  2. Site-specific integration at a pre-selected genomic locus eliminates position effect and ensures high expression levels of the transgene
  3. Integration at intergenic region ensures that no internal genes are interrupted
  4. Single copy gene integration eliminates repeat-induced gene silencing and genomic instability
  5. Site-specific integration allows a precise comparison of the effects of the transgenes among different lines.

knock in mice schematic


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 knock-out models.

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.

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

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 et al., PNAS 2011)

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)

Watch This Technical Webinar to Find Out More

http://www.criver.com/customer-service/education-training/educations/2014/07/transgenic-mice-creation

CRE-LoxP Conditional Mouse Models for Tissue Specific Gene Knockout Using CRISPR/Cas9 and TARGATT™

Applied StemCell’s employs two complementary technologies to engineer conditional knockout Cre-LoxP mouse breeding pairs in a two-step process: the proprietary TARGATT™ and licensed CRISPR/Cas9 gene editing platforms.

  1. Using CRISPR/Cas9, the gene of interest can be floxed by knocking in LoxP sequences to flank the gene and to generate a conditional knockout mouse model (Figure 1).
  2. The TARGATT™ technology allows any gene of interest (up to 22 kb) to be inserted into preselected and engineered docking sites in the safe harbor locus (H11 locus) of the mouse genome. In this case, the Cre recombinase gene can be paired with a promoter of choice (tissue/ cell specific) and inserted into the safe harbor locus for guaranteed expression of the Cre gene driven by the chosen promoter (Figure 2).

When the Cre-mice are thus bred with conditional knockout mice, it results in mouse progeny with deletion of floxed gene in the specified tissue (Figure 3). 

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
 

Figure 1. The schematic describes the first stage in developing a conditional knock-out mouse model using two CRISPR strategies to generate a floxed (loxP flanked exon) mouse. In Strategy 1, a donor plasmid is used to deliver the loxP cassettes. The donor fragment contains two loxP cassettes flanking the targeted exon(s) along with 5’ and 3’ homologous arms necessary for directing a site-specific homology directed repair. In Strategy 2, two different loxP ssODNs are used along with its respective gRNAs designed for loxP insertions at the 5’ and 3’ regions of the target exons.

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. 


Support Materials

Supporting Materials:

Transgenic Animal Core Facilities

TARGATT™ Knock-In Mouse Generation Services are available at the following institutions.
  • Cedars Sinai Medical Center
  • Children's Hospital Boston
  • Cincinnati Children's Hospital Med Ctr
  • Columbia University
  • E.N.S. LYON / Plateau de Biologie Experi. Souris
  • Trinity College
  • Friedrich Miescher Laboratory
  • Harvard University
  • Johns Hopkins
  • Kyoto University
  • Kyusyu University
  • Moffitt Cancer Center
  • Max Plank
  • NIH
  • Northwestern University
  • Purdue University
  • Tokyo Medical and Dental University
  • UCLA
  • UCSF
  • University of Alabama
  • University of Kansas Medical Center
  • University of Massachusetts
  • University of Texas Health Science
  • West Virginia University

Let our Experts Create a TARGATT™ Mouse for You, or Do It Yourself!

FAQ
FAQ for TARGATT™ System (Selected)

1. Can I create models to over-express a gene of interest?

Yes, TARGATT™ system is ideal for gene over-expression. Different promoters, e.g., tissue-specific promoters or ubiquitous promoters, and inducible systems (Tet On/Off, loxP-stop-loxP) can be used for tissue-specific, ubiquitous, or inducible gene expression.

2.  Can I use TARGATT™ system to create transgenic mice with tissue-specific gene expression?

Yes, TARGATT™ system can be used to generate tissue-specific transgenic mouse models. Just use a tissue-specific promoter to drive the transgene expression. Alternatively, a loxP-stop-loxP cassette can be placed between a ubiquitous promoter and the transgene. Upon crossing with tissue-specific Cre mice, the transgene will be expressed in that particular tissue.

3. What promoters are used to drive gene expression?

Any defined promoters provided by the customer or published in the literature can be used.

4. What is the specific site that my gene of interest will be integrated into?

Your gene of interest will be specifically inserted at your choice of either of the two well-characterized loci: H11 and Rosa26. You can find more detailed information regarding the specific sites in the PNAS publication (Tasic B, et al., Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci U S A. 2011 Apr 4).

5. Besides H11 and Rosa26, can gene be inserted at other loci?

Yes. This would be a customized service.  We need to first insert the docking attP site into a desired locus using CRISPR/Cas9 and then insert the gene of interest into the attP site using TARGATTTM.

6. Can I integrate a reporter gene? What kind of reporter genes do you recommend?

Yes, you can express any reporter genes such as GFP, DsRed, mCherry, LacZ , Luciferase, and etc.

7. 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?

To date, the largest DNA fragment we were successfully with is 22 kb. Insertion efficiency appears to decrease with increasing DNA fragment size. Larger fragments (>10 kb)  require additional embryo injections to obtain positive animals.

8. Why does the TARGATT™ knock-in system have high efficiency?

Unlike other recombinases, such as CRE or FLP, the TARGATTTM integrase recognizes and recombines at two largely unrelated sites, attP and attB, in terms of their sequences. Once the integrase-mediated integration at attB and attP takes place, two new hybrid sites, attL and attR are created at the junctions. These new sites are unrecognizable by integrase; therefore integrase reaction is uni-directional. Once the DNA is integrated, it will not be excised, making the integration process highly efficient. With TARGATT™ integrase system, the gene is integrated in exactly the Rosa26 or H11 locus permanently.

9. How many copies of the gene will be inserted into the genome?

A single copy.

10. Do you have TARGATT technology available for Knock-in cell lines?

Yes.

Case Studies

Case Study  

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 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 ar H11.

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

knock in mice transmembrane gene

Four positive founders were identified from 27 newborn mice.

References
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)

  • 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.
  • Matharu, N., Rattanasopha, S., Maliskova, L., Wang, Y., Hardin, A., Vaisse, C., & Ahituv, N. (2017). Promoter or Enhancer Activation by CRISPRa Rescues Haploinsufficiency Caused Obesity. bioRxiv, 140426.
  • 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 investigation126(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)
Ordering
Let our Experts Create a TARGATT Mouse for You, or Do It Yourself!
In addition to our full service plan, you can purchase the materials to make a TARGATT mouse yourself.
 

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