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Abstract

It is widely reported that the growth of recombinant bacteria and yeast is adversely affected by increased metabolic load caused by the maintenance of plasmid copy number and recombinant protein expression. Reports suggest that recombinant mammalian systems are similarly affected by increased metabolic load. However, in comparison to bacterial systems relatively little information exists. It was the aim of this study to test the effects of recombinant gene expression on the growth and metabolism of two industrially important cell lines. A BHK and CHO cell line were stably transfected with the human gastric inhibitory peptide (h-GIP)and glucagon receptor respectively. Selection was by way of the neomycin resistance (neo (r)) gene using G418.The growth and metabolism of both cell lines was affected by the presence of G418 in a manner indicative of increased metabolic load and which appeared to be caused by over-expression of the neomycin resistance protein. The two cell lines differed in their metabolic response to G418, which suggested that some cell lines or clones may be better able to tolerate a metabolic load than others. Growth under increased metabolic load was affected by medium composition with serum, insulin and glutamine concentration as influencing factors. Implications for the use of G418 are discussed.

An antibiotic selection marker for schistosome transgenesis

“…We determined that schistosomes were sensitive in culture to the aminoglycoside antibiotic geneticin (= G418), at doses similar to those reported for other eukaryotes including mammalian cell lines and free living nematodes (Tavoloni, 1997; Yallop and Svendsen, 2001; Giordano-Santini et al, 2010). Schistosomes that were transduced by retrovirus encoding neoR , the gene encoding resistance to neomycin, were rescued in culture from G418 toxicity, and the retroviral transgene copy number was enriched in comparison to transgenic schistosomes cultured in the absence of the antibiotic.…”

“…Given that the retroviral transgene constructs employed here carried the neoR gene, we investigated the sensitivity of schistosomes to geneticin (G418), resistance to which is encoded by the neoR gene. G418 was toxic for schistosomules at each concentration tested from 125 – 1,000 μg/ml, a toxicity profile comparable to C. elegans and several other free-living nematodes and mammalian cells (Tavoloni, 1997; Yallop and Svendsen, 2001; Giordano-Santini et al, 2010). G418 is derived from Micromonospora rhodorangea ; it is an aminoglycoside antibiotic similar to neomycin and kanamycin, all of which are widely used for selection of resistant cells.…”

“…This likely reveals enrichment of transgenic schistosomules within the population of transduced parasites subjected to G418 pressure (Yu et al, 1996). It likely reflects enrichment at the DNA level, not a conditional response to the antibiotic (Yallop and Svendsen, 2001). …”

“…Although widely used for genetic manipulation of cultured eukaryotic cells (Yallop and Svendsen, 2001), yeast and bacteria (Agaphonov et al, 2010), antibiotic selection systems have not yet been used for helminth parasites and were only recently reported with C. elegans and related free living nematodes (see Giordano-Santini and Dupuy, 2011). With C. elegans , most genetic markers for transgenesis are based on readily scored visual phenotypes.…”

Green fluorescent protein expression triggers proteome changes in breast cancer cells

“…Influence of the plasmid and selection by the way of the neomycin resistance (neo r ) gene using G418 has been previously studied in two cell lines (BHK, CHO) by Yallop et al [36,37]. They reported that while growth and metabolism of both cell lines was affected by the presence of G418 in the culture medium in a manner of increased metabolic load this effect was greatly compensated by addition of serum and glutamine to the culture medium [37].…”