Characterization and gene expression profiling of a stable cell line expressing a cell cycle GFP sensor.
The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. These results confirm that engineering a stable cell line for low expression of a EGFP cell cycle sensor is minimally perturbing to the cell cycle and cellular gene expression.
Thomas
Nick
N
Discovery Systems Development, GE Healthcare Bio-Sciences, Maynard Centre, Whitchurch, Cardiff, UK. nick.thomas@ge.com
Kenrick
Mike
M
Giesler
Theresa
T
Kiser
Gretchen
G
Tinkler
Hayley
H
Stubbs
Simon
S
D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D016213:Cyclins / Q000032:analysis* / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D016195:G2 Phase / Q000235:genetics / Q000502:physiology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D046228:Microarray Analysis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018797:Cell Cycle Proteins; D016213:Cyclins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA