TARGATT™ Site-Specific Knock-in Rat H11

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Highlights of the Site-Specific Transgenic TARGATT™ Rat Model

  • Site specific gene insertion into a preselected, safe harbor locus locus
  • Irreversible integrase mediated integration
  • Single copy, large DNA fragment knock-in (up to 22 kb)
  • Direct injection of the DNA into rat zygotes, bypassing rat embryonic stem (rES) cells
  • Non-immunogenic TARGATT™ plasmid
  • Works independent of cell division
  • Sprague Dawley strain of rat which is used extensively in all areas of research
  • Founder rats in as little as 4-6 months
  • Advantageous in vivo model for researchers seeking better representation of human diseases

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:

 

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 to a technology specialist.

Generation of TARGATT™ Rats

Genotyping results from Sprague Dawley rats engineered with an attP site at H11 locus

TARGATT™ “attP” docking site was inserted into the well characterized, transcriptionally active, safe-harbor  H11 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™ rats ready for projects. (+: attP positive rat).

TARGATT-rat-genotyping-1

 

Use our TARGATT™ Rat for the generation of site-specific transgenic rats via pronuclear injection

TARGATT-rat-applicationconcept-2

 

TARGATT™ knock-in Rat Generation Timeline

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  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 




CRE-LoxP Conditional Rat Models for Tissue Specific Gene Knockout 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

Figure 1. The schematic describes the first stage in developing a conditional knock-out rat model using two CRISPR strategies to generate a floxed (loxP flanked exon) rat. 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 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. 

 


Support Materials
  • Watch our recorded webinar video  TARGATT™-Rat models
  • POSTER. 13th Transgenic Technology Meeting being held in Prague, Czech Republic. March 20-23, 2016
FAQ

FAQs for TARGATT™ Rat Model Generation

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 rat with tissue-specific gene expression?

Yes, TARGATT™ system can be used to generate tissue-specific transgenic rat 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 rat, the transgene will be expressed in that particular tissue. Tissue specific Cre rats can also be generated using TARGATT™ thereby providing a ideal Cre-Lox breeding pair.

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 the well-characterized safe harbor locus, the H11 locus.

5. Besides H11 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 TARGATT™.

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, 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 (>7 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 TARGATT™ 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 the exact 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. We have TARGATT™ technology available for human cell lines including iPSCs and mouse ESCs.

11. How many embryos do you microinject and implant?

We normally microinject about 100 embryos for implantation or as many needed as to achieve ≥ 30 pups on founder.

12. How many founders will you provide?

Atleast one founder. We encourage F1 breeding at our facility to test germline transmission and colony expansion.

13. Do you provide other proof of precise gene insertion other than genotyping, example, proof of gene expression, phenotyong, behavioral analysis, etc?

No. However, we do encourage our customers to test donor construct in vitro. Simple phenotyping or behavioral observations can be done upon customer’s request when the positive animals are house at ASC. Extensive studies will be treated as a separate project.
 

References

Description of the technology

Commentary, comparison with other transgenic methods

Tet inducible mice generated by TARGATT™

Advantage of Hipp11 (H11) locus

Applications for animal models generated by TARGATT™ (Applied StemCell's cited/ published articles)

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