Egr1 and Gas6 facilitate the adaptation of HEK-293 cells to serum-free media by conferring enhanced viability and higher growth rates.
Animal-derived serum is an essential media supplement for mammalian cells in cell culture. For a number of reasons including cost, regulatory concerns, lot inconsistency, potential contamination with adventitious agents, and down-stream processing it is desirable to eliminate the use of serum. Existing protocols designed to adapt cells to serum-free media (SFM) are time-consuming and provide little insight into how the cells adapt. To better understand the physiological responses associated with serum withdrawal and to expedite the adaptation process, a Human Embryonic Kidney-293 (HEK-293) cell line was propagated in 10% fetal bovine serum (FBS) and was progressively adapted to SFM and analyzed at specific serum levels by oligonucleotide microarrays. Of the differentially expressed genes two, early growth response 1 (egr1) and growth arrest specific 6 (gas6), were selected for further analysis based on their level of differential expression, overall expression patterns, and proposed functionalities. HEK-293 cells, propagated in 10% FBS were transfected with egr1 or gas6 and then adapted to SFM. Results indicated that higher expression of either gene moderately enhanced the ability of both cell lines to adapt to SFM. Egr1 appeared to have a greater impact on adaptability than gas6. Results also indicated that specific protein production was unaltered when the expression of egr1 was increased. Flow cytometric analysis revealed increased expression of egr1 was associated with an increase in the percentage of cells in the G2/M phases. These results indicate that enhanced expression of egr1 or gas6 facilitate adaptation to SFM by improving growth and viability.
Jaluria
Pratik
P
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Biotechnology Unit, Building 14A, Room 173, Bethesda, Maryland 20892, USA.
Konstantopoulos
Konstantinos
K
Betenbaugh
Michael
M
Shiloach
Joseph
J
D000222:Adaptation, Physiological / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology* / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D000099255:Growth Arrest-Specific Protein 6
D016895:Culture Media, Serum-Free; D051766:Early Growth Response Protein 1; D036341:Intercellular Signaling Peptides and Proteins; D000099255:Growth Arrest-Specific Protein 6; C497325:EGR1 protein, human