AAVS1 Safe Harbor Integrated Landing Pad Cell Lines: Simplified Targeted Genome Engineering


 

A pre-engineered cell line with integrated genetic elements that facilitate simple payload exchange for your specific promoter and gene of interest. The landing pad construct is targeted to the AAVS1 safe harbor locus for stability and contains a florescent marker for downstream screening applications. Landing pad cell lines are offered as off the shelf products that have undergone rigorous testing and validation upfront. The product is available in a variety of cell backgrounds.

The landing pad construct features a promoter and mKATE2 payload flanked by homology arms. Each segment is separated by unique loxp sites that do not recombine with one another. Exchange of the promoter or payload requires a simple nucleofection with cre recombinase and  a DNA donor containing complimentary loxp sites. Cells can then be screened for integration via loss of mKATE signal (FACS). This design makes engineered cell lines accessible to more researchers than ever before. Additionally, our Cell Design Studio™ Team can make use of landing pad cell lines in our custom engineering services.

Jurkat T Lymphocytes

Jurkat T lymphocytes are a human, acute T cell lymphoma cell line isolated in the late 1970s from the peripheral blood of a young male patient suffering from T cell leukemia. The cells possess a pseudodiploid karyotype and have been characterized as expressing CD3 and, upon stimulation, interleukin-2. This line has also been used to determine the cytotoxicity of the cytolethal distending toxin (CDT) holotoxin and its components and the study of expression of bisphenol A exposed estrogen receptor-β (ERβ) and estrogen-related-receptor-α (ERRα) in vitro.

Benefits

  • Single copy integration into the AAVS1 Safe Harbor locus; confirmed by junction PCR (jPCR) and CNV analyses.
  • Continuous stable expression of the far-red fluorophore mKATE2.
  • Rapid and robust Cre mediated exchange with matching lox2272 and loxP plasmid or PCR amplicon with your gene of interest (GOI).
  • Select for mKATE2 negative population by FACS sorting.
  • Have an enriched mKATE2 negative population within 2-4 weeks.

FACS analysis of Jurkat Landing Pad clone prior to Cre-mediated exchange.

FACS analysis of Jurkat Landing Pad clone Cre-only nucleofection control, one week post-nucleofection

FACS analysis of Jurkat Landing Pad clone nucleofected with Cre mRNA and targeting DNA vector containing EGFP for exchange, one week post-nucleofection

A549 Cancer Cells

A549 is a human lung carcinoma cell line isolated in 1972 from a lung tumor of a male patient suffering from carcinoma. The cells possess a hypotriploid karyotype, express EGFR and mutant KRAS. A549 cells have been used to study proliferation induced by IGF1 as well as growth inhibition by BChTT. In addition, these cells have been used to model alveolar type II pneumocyte differentiation.

Benefits

  • One copy of the cassette integrated into the AAVS1 Safe Harbor locus; confirmed by junction PCR (jPCR) and CNV analyses.
  • Continuous expression of the far-red fluorophore, mKATE2.
  • Rapid and robust Cre mediated exchange with matching lox2272 and loxP plasmid or PCR amplicon containing your gene of interest (GOI).
  • Select for mKATE2 negative population by FACS sorting or other fluorescence assisted instrument.

A549 10X

Imaging of A549 cells landing pad cell line. Left, image demonstrates the homogenous expression of mKATE2 in the cell line. Middle, a brightfield image of the same culture showing 90% confluent A549 culture. Right, merged image showing all the cells in the culture are stably expressing mKATE2.

A375 Cancer Cells

A375 cancer cells are a human malignant melanoma cell line isolated from a 54 year old female. The cells possess a hypotriploid karyotype. The cell line produces rapidly growing amelanotic melanomas in anti-thymocyte serum treated NIH Swiss mice. A375 cells have been used to obtain paracrine factors for prolonged culture of mesenchymal stromal cells (MSCs). It has also been used to study oncolytic activity of the peptide LTX-315.

Benefits

  • One copy of the cassette integrated into the AAVS1 Safe Harbor locus; confirmed by junction PCR (jPCR) and CNV analyses.
  • Continuous expression of the far-red fluorophore, mKATE2.
  • Rapid and robust Cre mediated exchange with matching lox2272 and loxP plasmid or PCR amplicon containing your gene of interest (GOI).
  • Select for mKATE2 negative population by FACS sorting or other fluorescence assisted instrument.
  • A375 landing pad cell lines can be used to accelerate the study of transporters and other ADME/tox related genes.

A375 10X

Imaging of A375 cells landing pad cell line. Left, image demonstrates the homogenous expression of mKATE2 in the cell line. Middle, a brightfield image of the same culture showing 90% confluent A375 culture. Right, merged image showing all the cells in the culture are stably expressing mKATE2.

HCT-116 Cancer Cells

HCT-116 cancer cells are a human colorectal carcinoma cell line isolated from the colon of an adult male. The cells possess a near diploid karyotype and express carcinoembryonic antigen. Cells are tumorigenic in nude mice and form colonies on agarose. HCT-116 cells have been used to study the importance of cyclin D1 for the activity of lithocholic acid hydroxyamide (LCAHA) In addition the cells have been used in studies pertaining to iron uptake.

Benefits

  • One copy of the cassette integrated into the AAVS1 Safe Harbor locus; confirmed by junction PCR (jPCR) and CNV analyses.
  • Continuous expression of the far-red fluorophore, mKATE2.
  • Rapid and robust Cre mediated exchange with matching lox2272 and loxP plasmid or PCR amplicon containing your gene of interest (GOI).
  • Select for mKATE2 negative population by FACS sorting or other fluorescence assisted instrument.
  • HCT-116 landing pad cell lines can be used to accelerate the study of transporters and other ADME/tox related genes.

HCT-116 cells 72 h post nucleofection with GFP control exchange plasmid and cre mRNA. The Left image demonstrates the mKate2 expression of the non-exchanged cells. The middle image shows the cells in which GFP cDNA has been exchanged into the AAVS1 landing pad locus. The image on the right shows the overlay of the GFP with the mKATE showing that the cells have switched and can be isolated using FACS sorting or by other single cell cloning methodologies.

THP-1 Cells*

Derived from the peripheral blood of a 1 year old male with acute monocytic leukemia. THP-1 cells have Fc and C3b receptors and lack surface and cytoplasmic immunoglobulins. These cells also stain positive for α-napthhyl butyrate esterase, produce lysozymes and are phagocytic (both latex beads and sensitized erythrocytes). THP-1 cells can also restore the response of purified T lymphocytes to Concanavlin A, show increased CO2 production on phagocytosis and can be differentiated into macrophage-like cells using for example DMSO.

Benefits

  • THP-1 cells are highly sensitive to exogenous DNA making gene editing challenging.
  • Cre mediated DNA exchange allows for more efficient integration of genes of interest using mKATE negative selection.
  • Expands capabilities of THP-1 cells for genetic engineering.  

*Available only as a custom project.


 

Materials

Product No. Description
CLL1219 Safe Harbor Landing Pad Cell Line A375 Cancer Cells
CLL1220 Safe Harbor Landing Pad Cell Line A549 Cancer Cells 
CLL1222 Safe Harbor Landing Pad Cell Line HCT-116 Cancer Cells
CLL1221 Safe Harbor Landing Pad Cell Line Jurkat T-Lymphocytes 
CLL1223 Safe Harbor Landing Pad Cell Line THP-1 Monocytes