Uni-Select™ Antibody Discovery and Screening
Uni-Select™ Mammalian Cell-Based Membrane Proteins Antibody Screening! Applied StemCell provides Custom TARGATT™ Master Cell Generation in your cell lines, and ready-to-use TARGATT™ HEK293, and TARGATT™CHO to build protein libraries using site-specific transgene integration at a preselected safe harbor locus.
This is a fast, unique and efficient platform for biopanning, including bispecific mAbs, membrane proteins, CAR-T cell screening, and for bioprocessing.
- Single copy integration
- Homogenous expression of protein variants
- Preselected safe harbor locus
- Stable protein expression
- No bacterial backbone insertion
- Inducible expression compatible for membrane proteins (optional)
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TARGATT™ System in HEK293T Cells
Figure 1. Schematic representation of site-specific gene insertion using TARGATT™ Master Cell Lines. The TARGATT™ Master Cell Line is engineered with the attP landing pad (or docking site) at a preselected safe harbor locus (hROSA26). The TARGATT™ PhiC31 integrase catalyzes an irreversible reaction between the attP sites on genome and the attB sites on the donor vector, resulting in integration of a single copy of the GFP reporter gene at the locus.
Figure 2. GFP expression in hROSA26 locus in TARGATT™ HEK293T Master Cell Lines. The CAG-GFP vector was used to verify a fast knock-in in the TARGATT™-HEK293T Master Cell Line. An enriched GFP signal was shown under fluorescence microscopy. (Left) bright field microscopy. (Right) Immunofluorescence; GFP channel.
Figure 3. TARGATT™-HEK293T Master Cell Line transfected with donor plasmid containing GFP and attB. (a) Parental HEK293T cell line only (control); (b) Parental HEK293T cell line was transfected with donor plasmid containing GFP reporter by random insertion (+GFP); (c) TARGATT™-HEK293T Master Cell Line was transfected with GFP donor plasmid before ganciclovir (GCV) selection (GFP+); (d) TARGATT™-HEK293T Master Cell Line was transfected with GFP donor plasmid after GCV negative selection to eliminate clones without gene of interest (GFP+GCV). The data indicates that the TARGATT™-HEK293 Master Cell Line system provides a robust, fast and efficient integration platform for generating a uniform cell population with stable transgene expression in the hROSA26 locus. This platform paves the way for homogeneous expression of gene of interest and subsequent protein production.
TARGATT™ System in CHO Cells
Figure 4. TARGATT™-CHO Master Cell Line with GFP transgene integration into the hH11 locus. GFP signal was detected by fluorescence imaging after transfection with donor construct by random insertion in bright field (a) and GFP channel (b), and TARGATT™ integration plus GCV selection in bright field (c) and GFP channel (d).
- Venken, K. J. T., Sarrion-Perdigones, A., Vandeventer, P. J., Abel, N. S., Christiansen, A. E., & Hoffman, K. L. (2016). Genome Engineering: Drosophila melanogaster and beyond. Wiley Interdisciplinary Reviews. Developmental Biology, 5(2), 233–267.
The TARGATT™ Knock-in Master Cell Line Kit uses PhiC31 integrase to insert any gene of interest into a preselected intergenic and transcriptionally active genomic locus (hROSA26, hH11, or other pre-defined safe harbor loci). The TARGATT™ technology can be utilized for generating knock-in cell lines and libraries for a variety of applications including reporter gene expression, gene knockdown, conditional/ inducible gene expression, gene overexpression, antibody expression libraries, bioproduction of recombinant protein, and disease modeling.
Comparing TARGATT™ with lentivirus-based mammalian cell libraries
Applications for Cell-Based Library Screening
Antibody discovery:
- Biopanning (scFv screening)
- Off-target screening with membrane protein library
- Bioprocessing
Immuno-oncology (CAR-T cells):
- CAR affinity/ efficiency screening
- CAR specificity screening for safety assessment
- “Universal” CAR-T cell
- Novel immune target discovery: biomarkers and checkpoints
Receptor identification
- Ion channels
- GPCR
Protein evolution
- Enzyme activity and specificity
- AAV capsid specificity and efficiency
Others:
- Off-target screening
- Non-membrane, non-secretary protein library
1. How does the TARGATT™ gene integration technology compare to the Flp-In™ System?
The TARGATT™ gene integration technology involves site-specific gene integration mediated by the PhiC31 integrase. The PhiC31 integrase an irreversible, integration of a single copy of the gene of interest into a preselected, intergenic and transcriptionally active genomic locus. There are similarities between the two systems such as site-specific gene integration, transcriptionally active locus, single copy knock-in, and stable cell line generation. In the Flp-In™ system however involves the co-integration of elements of the bacterial (plasmid) backbone along with the gene of interest. The inclusion of the prokaryotic elements in the vicinity of the gene of interest could negatively affect the expression of the transgene. The Flp-In™ system also does not select against random gene integration at sites other than the FRT docking site (Callesen et al., 2016).
