Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.
Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.
Hacker
David L
DL
Laboratory of Cellular Biotechnology, Swiss Federal Institute of Technology Lausanne (EPFL), CH1015 Lausanne, Switzerland. david.hacker@epfl.ch
Bertschinger
Martin
M
Baldi
Lucia
L
Wurm
Florian M
FM
D017500:Adenovirus E1A Proteins / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017500:Adenovirus E1A Proteins; D007074:Immunoglobulin G; D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins