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TARGATT™ Human Induced Pluripotent Stem Cell (hiPSC) Master Cell Line & Knockin Kit

TARGATT™ Human Induced Pluripotent Stem Cell (hiPSC) Master Cell Line & Knockin Kit

$9,500.00

SKU AST-1600 Category Tag

Description

TARGATT™ Human Induced Pluripotent Stem Cell (hiPSC) Master Cell Line (Male) Knock-in Kit

Quickly create the most relevant cell line for your studies with iPSCs that are ready for TARGATT™ knock-in and then differentiation into your desired cell type. 

  • TARGATTT™ advantages—site-specific, single copy knock-in of 20+ kb of DNA at efficiencies approaching or exceeding 90% after selection 
  • Gene editing-ready iPSCs—save time with hiPSCs that come with a TARGATT™ landing pad engineered into the H11 safe harbor locus 
  • iPSC flexibility—differentiate into your desired cell type after integrating your transgene 
  • Male and female iPSCs available—both generated using episomal factors from well-characterized, karyotype normal iPSCs—find the female kit here 

Overview

The TARGATT™ hiPSC Master Cell Line (Male) Knock-in Kit (AST-1600) from Applied StemCell accelerates your cell line development by providing iPSCs with a pre-engineered TARGATT™ landing pad at the H11 safe harbor locus. Simply clone your DNA of interest into the TARGATT™ cloning plasmid, transfect into the TARGATT™ iPSC Master Cell Line (Male), select for integrant, and differentiate into your desired cell type.   

We also offer a TARGATT™ hiPSC Master Cell Line Knock-in Kit derived from a female donor (AST-1602)—find it here. 

Each AST-1600 kit contains sufficient cells and plasmids for 9 transfections and comes with: 

  • TARGATT™ hiPSC Master Cell Line (Male, ASE-9211-TGT-PH3)—engineered with a TARGATT™ landing pad at the H11 safe harbor locus of ASE-9211 hiPSCs (episomally reprogrammed fibroblasts, neonatal African American male donor).  
  • TARGATT™ 22.2 CAG-MCS-attB Cloning Plasmid (AST-3070) 
  • TARGATT™ CMV-integrase Plasmid (AST-3071) 

With the large-insert capabilities of the TARGATT™ platform, you can easily and efficiently insert large genes, multiple genes, and complex constructs—all types of editing that CRISPR/Cas9 is unable to achieve in a single reaction.

Integration is specific for sequences in the TARGATT™ landing pad, which we’ve pre-engineered into a well-characterized intergenic region—the H11 safe harbor locus—in our hiPSC Master Cell Lines. Gene expression from the H11 locus is consistent and robust, resulting in reliable protein production. In addition, because TARGATT™-mediated integration is unidirectional and irreversible, unlike insertion with cre-lox and flp-frt systems, gene expression is stable over time and with each passage. 

Learn more about TARGATT™ large knock-in technology

All Applied StemCell hiPSCs are characterized for pluripotency biomarkers, normal karyotype, and directed differentiation to three germ layers as a validation of functional pluripotency. In addition, the ASE-9211 parental iPSC line is also used by the NIST Genome Editing Consortium to generate benchmark materials to establish standards for genome editing and stem cell research data. 

How it works

TARGATT™ large knock-in technology leverages a site-specific serine integrase to unidirectionally insert plasmid DNA sequences into the genome at the TARGATT™ landing pad (Figure 1). The TARGATT™ integrase mediates recombination between the attB site in the cloning plasmid and the attP site in the genomic landing pad, resulting in insertion of the plasmid in the genome. During the recombination event, the attB and attP sequences are converted into attL and attR sequences which are not recombined by the integrase, ensuring irreversible insertion of the DNA. 

Figure 1. TARGATT™ integrase mediates recombination between the attB site in the donor plasmid (the cloning plasmid) and the attP site in the TARGATT™ landing pad in the chromosome.

Note that some Applied StemCell TARGATT™ products are configured with a dual integrase cassette exchange (DICE) system to eliminate insertion of cloning vector sequences into the genome. 

Figure 2. DNA knock-in strategy for AST-1600 TARGATT™ hiPSC Master Cell Line (Male).

Supporting data

Integration is efficient and site-specific 

Figure 3: We achieved 46% (hygromycin only) and 67% (hygromycin + GCV) knock-in efficiency using TARGATT™ to insert a 22.2 CAG-MCS-attB plasmid into the hiPSC master cell line ASE-9211-TGT-PH3. A. Genotyping strategy that uses 2 sets of primers to confirm site-specific knock-in at the 5’ and 3’ junctions (575 and 848 bp, respectively) of the TARGATT™ landing pad. B. Image of the PCR gel electrophoresis. C. Summary of the genotyping results.

Integration and transgene expression are stable over multiple passages 

Figure 4: Integration and transgene expression are stable over multiple passages. We verified the stability of integration and gene expression over 10 passages using (A) the 3’ junction PCR assay described in Figure 4 and (B) GFP fluorescence.

TARGATT™ hiPSCs differentiate into all three germ layers 

Figure 5. TARGATT hiPSCs differentiate into all three germ layers. We differentiated the ASE-9211-TGT-PH3 hiPSC line into specific lineages of the germ layers and immunostained for biomarkers of each lineage to confirm lineage commitment and co-stained with the nuclear marker, DAPI (blue). Images show co-localization of biomarker with DAPI. Endoderm (EN) marker: SOX17 (green); Mesoderm (ME) marker: GATA4 (green); Ectoderm (EC) marker: β-Tubulin (red).

Frequently asked questions

No, the TARGATT™ iPSC Master Cell Line is for research use only. We offer GMP-grade TARGATT™ iPSCs (AST-9480) for clinical applications.

The TARGATT™ iPSC Master Cell Line is engineered from ASE-9211, a well-characterized, karyotype-normal iPSC line. ASE-9211 was reprogrammed from fibroblasts using episomal reprogramming.

TARGATT™ technology enables highly efficient, single-copy, site-specific integration at the H11 safe harbor locus. Integration is stable and results in long-term expression—advantages that other random or multi-copy insertion methods cannot consistently provide.

TARGATT™ technology is ideal for: 

  • Reporter knock-in cell lines 
  • Drug discovery screening 
  • Library screening 
  • Large transgene knock-ins for therapeutic studies 

TARGATT™ technology provides high knock-in efficiency with stable and site-specific gene integration. After drug selection, efficiency can approach 90%. 

The following materials are not included and must be procured separately: 

Material Vendor Catalog Number
mTeSR™ Plus
Stem Cell Technologies
05825
Matrigel® hESC-Qualified Matrix
Corning
354277
Rock Inhibitor Y-27632
Segment
04-0012-02
CryoStor® CS10 Freeze Media
Stem Cell Technologies
210102
0.5mM EDTA in PBS
Life Technologies
15575-020
TrypLE™ Express Enzyme (1X)
Gibco
12604013
Neon™ Transfection System Kit
ThermoFisher
MPK10096
Hygromycin
InvivoGen
Antihg-1
Penicillin-Streptomycin (100X)
Gibco
15140-122
Ganciclovir (GCV)
Selleck Chemicals
S1878

If you are using similar or alternative reagents, we recommend ensuring they align with the TARGATT™ culture protocols for optimal results.

The TARGATT™ system uses a patented serine integrase that specifically recognizes TARGATT attB sites in the cloning plasmid (also referred to as the donor plasmid) and attP sites in the TARGATT landing pad. Because unbound double strand breaks are not created, random integration using the host double strand break repair machinery does not occur.

TARGATT™ can efficiently insert payloads of 20 kb in a single reaction and larger payloads with nested TARGATT™ landing pads, making the technology suitable for a wide range of genetic engineering applications.

Yes, the TARGATT™ cloning plasmid allows you to clone and amplify your desired gene of interest for site-specific insertion.