Green fluorescent protein (GFP) is a meaningful instrument for cell-based assays owing to the intrinsic fluorescence of this protein that authorizes real-time examination of molecular phenomena in living cells. Seattle Genova cell line construction team has many years of expertise to facilitate unrivalled GFP tagging cell line creation. All of our protocols are well-documented, regularly evaluated by global regulatory agencies and extraordinarily competitive concerning titers obtained and speed of delivery.
Applications of GFP in cell-based assays for drug discovery
Several GFP variants have been created with optimal properties for both high-throughput screening and high-content screening. The use of GFP and its variants in this capacity gives a ‘fluorescent tag’ on the protein, which in vivo localization of the fusion protein.
The GFP chromophore
(GFP) undergoes an incredible posttranslational modification to create a chromophore out of its amino acids (S65, Y66, and G67) (1–3). GFP is small (238 aa), tolerates both N- and C-terminal fusions, and can be targeted to particular cellular locations.
Red-shifted GFP variants
Ser65Thr (which includes a Ser65 to Thr substitution in the chromophore). GFPmut1 or enhanced green fluorescent protein (which includes the same Ser65Thr change plus a Phe64Leu mutation).
Blue, cyan and yellow fluorescent proteins
Enhanced blue fluorescent protein (EBFP)
Enhanced yellow fluorescent protein (EYFP)
Enhanced cyan fluorescent protein (ECFP)
Use of GFP as a transcription reporter
In transcription reporter assays, alterations in the level of the reporter protein are presumed to reflect changes in mRNA levels arising from either induction or repression of cis-acting control elements linked to the reporter gene.
Destabilized EGFP and dEGFP biosensors
The creation of the dEGFP variant was accomplished by fusing a region of the mouse ornithine decarboxylase (ODC) gene encoding residues 422–461 to the C-terminus of EGFP. Western blot analysis utilizing anti-EGFP antibodies suggested that this decline in fluorescence following inhibition of protein synthesis is correlated with a similar decrease in d2EGFP protein levels.
Use of GFP to monitor protein localization
The specific biosensors utilized in such assays are several and include genetic fusions to monitor events such as cell surface receptor internalization, transcription factor trafficking, organelle and cytoskeletal dynamics during cell division, and protein kinase translocation.
This fluorescence-imaging platform is suited to exploit fully the utility of GFP technology for the protein localization assays utilized in drug discovery.
Cell line Construction Process
Cell Line Construction
Transfection is the process of initiating a foreign DNA (encoding the recombinant protein of interest) into a host cell. A small population of cells with foreign DNA integrated into their genome that strengthens their ability to express recombinant protein for long periods are applied to as stably transfected cells.
Positive clone screening & expression testing
Discovery and selection of high-quality clones from a transfected pool of cells. Screening vast populations by quantifying cell surface expression of protein-of-interest or secreting antibodies (titer ranking) will improve the probability of discovering rare high-affinity binders or high producers.
Single-cell isolation and cell viability
Single, viable cells must be isolated and cloned to confirm that the cell population is genetically identical, significantly decreasing the heterogeneity of expression
When creating cell lines for biotherapeutics, it is important from a quality and regulatory aspect to ensure that the cell line originates from a single progenitor and is thus monoclonal. Documentation of monoclonality (a regulatory metric for therapeutic cell lines) is generally image-based, whereby an image of a single cell is recorded and comprised in regulatory filings.
Clone productivity screening and titer
This is a test that observes the amount of recombinant protein or antibodies originating from the clonally-derived cell line.
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