Retrospective assessment of clonal origin of cell lines.

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.

Disruption of ShcA signaling halts cell proliferation--characterization of ShcC residues that influence signaling pathways using yeast.

Shc adapter proteins are thought to regulate cellular proliferation, differentiation and apoptosis by activating the SOS-Grb2-RAS-MAPK signaling cascade. Using the small hairpin RNA (shRNA) technique, we found that decreasing ShcA mRNA reduced the proliferative ability of HEK293 mammalian culture cells. We then recapitulated phosphorylation-dependent Shc-Grb2 complex formation in Saccharomyces cerevisiae. Immunoprecipitation followed by Western analysis demonstrated that activated TrkB, composed of the intracellular domain of TrkB fused to glutathione S-transferase (GST-TrkB(ICD)), promoted the association of ShcC and Grb2 in yeast. The Ras-recruitment system (RRS), in which a myristoylated (Myr)-bait and son of sevenless (hSOS)-prey are brought together to complement the defective Ras-cAMP pathway in a thermosensitive cdc25H mutant yeast strain, was used to validate a phenotypic assay. Yeast cells transformed with both Myr-ShcC and hSOS-Grb2 (referred to as scheme 1) or Myr-Grb2 and hSOS-ShcC (scheme 2) did not grow at non-permissive temperature; the additional transformation of GST-TrkB(ICD) enabled growth. GST-TrkB(ICD) also enabled growth with hSOS-Grb2 and either Myr-ShcA or Myr-SHP2. Mutational analysis of TrkB showed that its kinase activity was essential for complementation, while its docking site for Shc proteins was not. Mutational analysis of ShcC showed that the PTB and SH2 domains were not essential for complementation but phosphorylation at Y304 in the CH1 domain was. Phosphorylation at Y304 could not be substituted by an acidic amino acid. The RRS provides a genetic system to probe Shc proteins and potentially identify member specific protein partners and pharmacological reagents.