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Evaluation of transfected populations in batch culture conditions

All of the NSO puromycin resistant transfected populations (A-E pEF, A-E Y28A and A-C Bcl-2) were analysed for Bcl-2 content by western blot analysis. Of these NSO transfected populations only four (A, D and E Y28A; and C Bcl-2) were found to contain human Bcl-2 and subsequently those cell lines expressing Bcl-2 and there equivalent empty vector populations (that should not and did not contain Bcl-2) were isolated and grown to stock cultures. Western blotting results on the mixed populations of A, D and E Y28A and pEF did not show any significant difference in Bcl-2 expression levels between batches. Bcl-2 expression was either present in the populations or not and those where expression was found did not vary in the extent of expression level between populations of cells.

Upon screening all of the populations in batch culture only population EY28A showed significant protection of apoptosis. Protection from apoptotic cell death was demonstrated in serum-deprived conditions and apoptosis induction by staurosporine compared to the control population (EpEF) and the ild type untransfected population (NSO WT). These populations of cells were then cloned and studied further.

Cho cells transfected with the mutant Bcl-2 gene (Y28A bcl-2) offered no protection to apoptosis in similar conditions as those used for the NSO cells in any of the batches tested even though Bcl-2 expression was evident. However, CHO cells transfected with the wild type Bcl-2 (puromycin resistance) showed significant protection from apoptosis and also increased growth rate compared to the wild type and control cells. This population of cells was also sent for cloning to Lonza Biologics but work was suspended due to IP issues.

CHO22H11 cells containing the IgG construct were transfected with the wild type neomycin resistance Bcl-2 gene. A control population was also constructed containing the neomycin vector without the Bcl-2. CHO22H11 wild type, CHO22H11 neo and CHO22H11 Bcl-2 populations were tested in batch culture in standard growth conditions (DMEM/F12 10%FCS). The Bcl-2 cell line showed significant protection from apoptosis in normal batch and staurosporine induced conditions compared to the wild type and control vector transfected populations. Strangely, as previously found with the NSO 6A1 populations, the control vector offered some protection in the death phase compared to the wild type population. As previously found with the NSO cell line, it appears that the total antibody production for the three cell lines is relatively equivocal and that towards the end of the batch when you would expect the Bcl-2 cell line to be producing more compared to the control it is in fact producing slightly less antibody. This may be due to metabolic overload of the cells by the Bcl-2 thus the cells are driving their energy resources towards cell division rather than protein production.

In order to investigate the protection offered by the neomycin vector alone at the death phase, cell samoles were taken during the batch culture for Bcl-2 expression analysis. Although no Bcl-2 expression was observed for this cell line as the only levels present should be endogenous we observed an increase in Bcl-2 expression throughout the batch culture. This cl-2 expression is thought to be endogenous as it exhibits the traditional two band pattern not found with the transfected Bcl-2 cell lines. No such Bcl-2 expression is found in the wild type cell line. It appears that by an unknown method the neomycin vector is increasing the expression levels of the Bcl-2 within the cells thus offering the cells increased natural protection to the onset of apoptosis.

Reported by

University of Birmingham
Dept. of Chemical Engineering
B15 2TT Birmingham
United Kingdom
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