TARGATT™ CHO-K1 (H11) Master Cell Line & Knock-in Kit

SKU :
AST-1400
Catalog # :
AST-1400
Size :
1 kit
* MTA required The TARGATT™ CHO-K1 Master Cell Line and transgenic kit was designed for fast and site-specific knock-in in Chinese Hamster Ovary (CHO) cells using an easy-to-use gene knock-in approach. The master cell line provided in this kit contains both the “attP” docking-site and the PhiC31 integrase expression cassette engineered into the CHO H11 safe harbor locus in the genome. Any gene of interest can be cloned into the provided TARGATT™ “attB” clonin .....
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Description

* MTA required

The TARGATT™ CHO-K1 Master Cell Line and transgenic kit was designed for fast and site-specific knock-in in Chinese Hamster Ovary (CHO) cells using an easy-to-use gene knock-in approach. The master cell line provided in this kit contains both the “attP” docking-site and the PhiC31 integrase expression cassette engineered into the CHO H11 safe harbor locus in the genome. Any gene of interest can be cloned into the provided TARGATT™ “attB” cloning plasmid (under control of the strong CAG promoter or promoter-of-choice), and transfected into the master cell line for generating a stable knock-in cell line. The TARGATT™ integrase-based technology enables efficient DNA integration and high-level gene expression without disrupting internal genes. The TARGATT™ CHO-K1 cell line can therefore be used for uniform, site-specific gene knock-in, generation of isogenic cell lines, and amplification strategies for isolating high expression cells, without the need for single cell cloning.

The TARGATT™ CHO-K1 master cell line and transgenic kit are suitable for research applications involving gene overexpression and high-level expression of recombinant proteins and other biologics in a rapidly expanding bioproduction industry and for other applications*.

Advantages of using TARGATT™ Master Cell Lines for gene knock-in:

  • High efficiency, unidirectional integration
  • Site-specific, stable knock-in cell line generation
  • Single copy gene integration into safe harbor locus
  • Gene expression from an active, intergenic locus
  • Easy-to-use protocol requiring transfection of only one (donor) plasmid

*TARGATT™ master cell lines can be generated in any cell line including stem cells. Please contact Applied StemCell for TARGATT™ cell line engineering services to generate a master cell line in a specific cell line of choice.

This product is for research purposes only.


Contents:

All cell lines and reagents provided in this kit are sufficient for transfection according to the given protocol:

  • TARGATT™ CHO-K1 Master Cell line (AST-1400-1)
  • TARGATT™ 20.1 (CAG-MCS-TK) cloning plasmid (AST-3068)
  • TARGATT™ 21 (CAG-GFP-TK) positive control plasmid (AST-3061)

Schematic Representation of the Transgene Integration in the TARGATT™ CHO-K1 (H11) Master Cell Line

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Figure 1. Schematic representation of TARGATT™ site-specific transgene integration mediated by integrase in TARGATT™ CHO-K1 (H11) master cell line. The TARGATT™CHO-K1 (H11) Master Cell Line was engineered with the attP landing pad at the Hipp11 (H11) safe harbor locus. The TARGATT™ plasmid containing the integrase recognition site, attB is used to clone the transgene. The integrase catalyzes an irreversible reaction between the attP site in the genome and attB site in the donor vector, resulting in site-specific integration of the gene of interest at the selected locus. The cells containing the gene of interest can be enriched using ganciclovir negative selection.

Genotyping of 5’-arm and 3’ arm in TARGATT™ CHO-K1 (H11) Master Cell Line

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Figure 2. Genotyping of 5’-arm and 3’-arm in TARGATT™ CHO-K1 (H11) master cell line. RT-PCR analysis shows the expected sizes for 5’ arm and 3’ arm junction PCR product in a 1.5 % agarose gel: 386 bp and 568 bp, respectively. Primer sequences used: 5’-arm-Forward: 5’-ACCTGGAAAAAGCAGTCCCAAC-3’; 5’-arm-Reverse: 5’-ACACCTCCCCCTGAACCTGAA-3’; 3’-arm-Forward: 5’-GGCGGGCCATTTACCGTAAGTTAT-3’; 3’-arm-Reverse: 5’-AAGGCTCTTGTCTCCTCAAAGCTG-3’.

GFP Expression after Transfection

landingpage-ast-1400-cho-k1-h11-microscopy 

Figure 3. Uniform and Stable GFP expression 21 days post-transfection using TARGATT™ CHO-K1 (H11) Master Cell Line and Knock-in Kit. The TARGATT™ CAG-GFP plasmid vector was used to evaluate gene integration in the parental CHO-K1 cells (a-b) and TARGATT™ CHO-K1 (H11) master cell line (c-d). An enriched GFP signal was detected by fluorescence imaging in TARGATT™ CHO-K1 (H11) Master Cell Line. (Left) bright field microscopy. (Right) Immunofluorescence; GFP channel.

Flow Cytometry Analysis of GFP Expression after Integration and Selection in TARGATT™ CHO-K1 (H11) Master Cell Line

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Figure 4. Flow cytometric analysis of GFP expression level in CHO-K1 cells. GFP expression was measured by flow cytometric after transfection of TARGATT™ CAG-GFP plasmid in parental CHO cell line by random integration (c-b) and in TARGATT™ CHO-K1 (H11) Master Cell Line by site-specific gene integration (c-e). (a) CHO-K1 parental cell line without transfection; (b) CHO-K1 parental cell line randomly transfected with TARGATT™ CAG-GFP plasmid; (c) TARGATT™ CHO-K1 (H11) master cell line without transfection; (d) TARGATT™ CHO-K1 (H11) master cell line transfected with TARGATT™ CAG-GFP plasmid before GCV selection; (e) TARGATT™ CHO-K1 (H11) master cell line transfected with TARGATT™ CAG-GFP plasmid after GCV selection.


TARGATT™ Master Cell Line Publication:

  • Chi, X., Zheng, Q., Jiang, R., Chen-Tsai, R. Y., & Kong, L. J. (2019). A system for site-specific integration of transgenes in mammalian cells. PLOS ONE14(7), e0219842.