• TARGATT™ Site-Specific Knock-in Rat

TARGATT™ Site-Specific Knock-in Rat

Applied StemCell’s proprietary TARGATT™ technology enables generation of physiologically relevant transgenic rat models suitable for a variety of applications including reporter gene expression, gene knock-down, conditional gene expression and disease models. This technology uses the Phic31 integrase to mediate an irreversible integration of large transgene(s) into a preselected, safe harbor locus with guaranteed gene expression.

  • Site-specific, single copy transgene integration overcomes challenges associated with random integration
  • TARGATT™ knock-in rats in 6-9 months
  • Direct microinjection of the TARGATT™ reagents into rat zygotes

Using the TARGATT™ technology, ASC has also developed Neural Specific Cre-Rat Lines in a Sprague Dawley rat background. Please inquire.

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Application Notes

TARGATT™ Technology for Transgenic Knock-in Rat Model Generation

1. Generation of TARGATT™ “attP” Master Rats

TARGATT-rat-genotyping-1

Figure 1. Genotyping results from Sprague Dawley rats engineered with an attP site at rH11 locus. TARGATT™ “attP” docking site was inserted into the well characterized, transcriptionally active, rH11 safe-harbor locus using CRISPR/Cas9. The attP positive (+) founder was bred to F1 and F2 generations respectively. Applied StemCell now has a well-established colony of homozygous TARGATT™ ”attP” Master rats ready for projects. (+: attP positive rat).

2. Generation of CAG-GFP Rats using TARGATT™

TARGATT-rat-genotyping-1

Figure 2. Schematic representation of TARGATT™ technology to generate transgenic rat expressing your gene of interest. TARGATT™ “attP” master rat embryos containing the attP “docking site” at rH11 lcous are used for microinjection of the integrase mRNA, and the TARGATT™ donor plasmid containing the attP recognition sequence, attB and the gene of interest. The integrase catalyzes an irreversible integration of the transgene into the rH11 locus to generate transgenic rats expressing your gene of interest.

Figure 3. Design and construct of CAG-GFP rat using TARGATT™. A CAG-GFP transgene was inserted by integration of the gene cassette using PhiC31 integrase at the rH11 docking site (locus) in the TARGATT™ attP "Master" rats.

Figure 4. Generation of transgenic CAG-GFP rats. Two founder pups (red: #17 and #19) were identified to carry the gene of interest by PCR using 4 sets of genotyping primers: 5’ GoI (412 bp), 3’ GoI (750 bp), 5’ BB (708 bp), and 3’ BB (919 bp). Note: -: negative control; GeneRuler™ 100 bp plus DNA ladder.

Application for TARGATT™ Transgenic Rat Models

CRE-LoxP Rat Model Breeding Pairs for Tissue Specific Conditional Gene Knockout/ Expression Using CRISPR/Cas9 and TARGATT™

Applied StemCell’s employs two complementary technologies to engineer conditional knockout Cre-LoxP rat 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 rat 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 rat 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-rats are thus bred with conditional knockout rats, it results in rat progeny with deletion of floxed gene in the specified tissue (Figure 3). 

Conditional Knockout Rat 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-Rat-1

Figure 1. The schematic describes the first stage in developing a conditional knock-out rat model using CRISPR to generate a floxed (loxP flanked exon) rat. 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 Rat Models Engineered Using TARGATT™ Technology

Cre- rat models are generated by microinjection of an integration cocktail into the pronuclus of TARGATT™ rats 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.

SCHEMATIC-TARGATT-Cre-Rat

Figure 2. Schematic illustrates the engineering of a Cre-driver rat 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™ rat 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 rats. 

Conditional knockout rats are generated by crossbreeding the two transgenic rat lines: (a) the homozygous “floxed” (flanked by loxP) allele rat model, and (b) the Cre-driver rat 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. 

SCHEMATIC-CKO-CRE-Breeding

Figure 3. Crossbreeding the conditional knock-out rat with a Cre-recombinase expressing rat. 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 rat 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 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?
How many embryos do you microinject and implant?
How many founders will you provide?
Do you provide other proof of precise gene insertion other than genotyping, example, proof of gene expression, phenotyping, behavioral analysis, etc.?
Technical Details

Site-specific transgenic rats are gaining importance as a preferred in vivo model for researchers who seek better representation of human genetics and physiology. Their similarity to human physiology and pathology makes them a better in vivo model than mice for the study of cardiovascular, diabetics, neurological, psychiatric and autoimmune dysfunctions. Major improvements in site-directed mutagenesis using techniques such as CRISPR, TARGATT™, ZFNs and TALENs have made generation of genetically modified rat models possible. In particular, the CRISPR/Cas9 system and our proprietary TARGATT™ technology, complement each other to provide comprehensive gene editing platform for the generation of customized transgenic rats.

Applied Stem Cell Inc.’s proprietary TARGATT™ technology enables highly efficient site-specific gene insertion in mammalian cells and animals. This technology uses PhiC31 integrase to insert any gene of interest into a docking site that has been pre-engineered in an intergenic and transcriptionally active genomic locus with guaranteed transgene expression.

Applications for TARGATT™ Rats

 The TARGATT™ Rat is an ideal platform for generating rat models for applications such as:

  • Transgene overexpression models
  • Humanized rat models (large gene insertion)
  • Reporter gene insertion models
  • Inducible expression rat models (Example: Tet-regulatory systems)
  • Cre - driver models - We can generate Cre models using promoters listed below or ANY promoter provided by customers (with available sequence) using our versatile TARGATT™ technology (See Technical Details Section)

 Neural Specific Cre- Rat Lines Using TARGATT™ Technology:

Neuronal promoters:

Nes

 PAG

CaMK2a

Syn1 

Wnt1

Thy1 

HB8

PDGF 

MOR23

Crh 

Pomc

GFAP 

Drd1a

Six3 

TH

Plp1 

GAD67

Tie2

Other Promoters:

VE-Cad

CA

CA-LoxP-STOP-LoxP-GFP-LacZ

If you do not see a promoter-of-interest in this table, please contact us to talk to a technology specialist.

TARGATT™ knock-in Rat Generation Timeline

Service Time Deliverables
1. TARGATT DNA Construction 
(genes-of-interest in plasmid will be provided by the client)
 4-8 weeks A report on TARGATT™ vector cloning
2. Generation of TARGATT integrase  2 weeks A report on integrase mRNA synthesis and validation
in vitro transcription and purification    
3. TARGATT™ DNA Pronuclear Microinjection
(up to 150 embryos will be injected)
 1-2 months  
4. Animal Care, Housing, and Genotyping
1-2 months At least 1 founder with a single copy of transgene inserted into H11 locus
Pups generated and genotyping showing the proof of precise gene insertion
  A final report on the TARGATT™ knock-in project including the original targeting strategy and microinjection details
Recommended but Optional: F1 breeding
2-3 months  
 
 
Publications

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)
Have Questions?

An Applied StemCell technical expert is happy to help, contact us today!