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Fig. 5. Retroviral insertion mutagenesis of CHO cells and genetic screening for HASPB export mutants. (A) Intercellular spreading was monitored by growing CHO_MCAT-TAM2 cells ('CHO wild type') with CHO_MCAT-TAM2 cells retrovirally transduced with the HASPB-N18-GFP construct in a mixed culture. HASPB-N18-GFP expression was induced by 1 µg/ml doxicycline for 48 hours at 37°C. Cells were then processed for FACS sorting using affinity-purified anti-GFP antibodies and APC-coupled secondary antibodies to detect exported HASPB-N18-GFP by cell surface staining. CHO_MCAT-TAM2 cells and CHO_MCAT-TAM2 cells expressing HASPB-N18-GFP were gated based on GFP fluorescence and APC-derived fluorescence of CHO_MCAT-TAM2 cells (green curve) and CHO_MCAT-TAM2 cells expressing HASPB-N18-GFP (blue curve) was measured as depicted in the histogram. Autofluorescence was measured using CHO_MCAT-TAM2 cells that were treated with antibodies. (B) CHO_MCAT-TAM2 cells expressing HASPB-N18-GFP were treated with retroviral particles encoding the cell surface protein CD4. Following transduction, CD4-positive cells were selected based on anti-CD4 cell surface staining using FACS sorting. (C) CD4-positive cells as enriched in the FACS experiment depicted in B were subjected to three rounds of FACS sorting. Cells were monitored in dot blot mode with GFP-derived fluorescence shown on the y-axis and HASPB-N18-GFP cell surface staining shown on the x-axis. The left-hand panel shows the population grown in the absence of doxicycline, the right-hand panel shows the population grown in the presence of 1 µg/ml doxicycline for 48 hours at 37°C. To select for HASPB export mutants the sorting window was adjusted as depicted in the right-hand panel to isolate cells characterized by high GFP fluorescence and low APC cell surface staining.

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