Cas9/GFP Safe-Harbor HEK293 Cell Line
Recombinant stable HEK293 cell line constitutively expressing a FLAG-tagged Cas9 nuclease and CopGFP (Pontellina plumata green fluorescent protein), which have been stably integrated into the AAVS1 safe harbor locus on chromosome 19. When transfected or transduced with single guide RNAs (sgRNAs), this cell line will sustain double-strand DNA breaks (DSBs) at targeted genome sites.
Expression of Cas9 is driven by a CBh promoter, whereas CopGFP is driven by an EF1A promoter. The combined DNA fragment (CBh-Cas9-bGH Poly A-EF1A-CopGFP-T2A-Puro-SV40 Poly A), shown in Figure 1, was integrated at the AAVS1 safe harbor locus using CRISPR/Cas9 technology. The construct also contains a puromycin resistance gene.
Cells were cloned by limiting dilution to obtain a monoclonal population. Without the adverse effects resulting from random integrations of Cas9/GFP into the HEK293 genome, this cell line behaves like parental HEK293 cells.
Figure 1: Transgene integration at the AAVS1 locus.
Purchase of this cell line is for research purposes only; commercial use requires a separate license. View the full terms and conditions.
| Name | Ordering Information |
| Thaw Medium 6 | BPS Bioscience #60183 |
The cell line has been screened to confirm the absence of Mycoplasma species.
AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a well-validated “safe harbor” site for hosting DNA transgenes. AAVS1 has an open chromatin structure and is transcription competent. Most importantly, disrupting the AAVS1 locus by inserting DNA transgenes has no known adverse effects on the cells. Specifically targeting the AAVS1 locus is a major advantage compared to the random integration obtained using other approaches such as lentivirus infection or cell transfection, which may cause insertional mutagenesis or disrupt important genes or cellular processes. GFP (green fluorescent protein) presents green fluorescence, and it was first identified in Aequorea Victoria. Pontellina plumata GFP (CopGFP) was later identified and is a superbright and fast maturation rate. The presence of fluorescent proteins allows for cell identification and quantification by flow cytometry or fluorescence microscopy, and localization studies in vivo, providing easy assay readouts.
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