Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis.
BACKGROUND
Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a powerful new approach for performing high throughput screens of gene function. An important application of cell-based microarrays is in screening for proteins that modulate gene networks. To this end, cells are grown over the surface of arrays of RNAi or expression reagents. Cells growing in the immediate vicinity of the arrayed reagents are transfected and the arrays can then be scanned for cells showing localised changes in function. Here we describe the construction of a large-scale microarray using expression plasmids containing human genes, its use in screening for genes that induce apoptosis when over-expressed and the characterisation of a number of these genes by following the transcriptional response of cell cultures during their induction of apoptosis.


RESULTS
High-density cell-based arrays were successfully fabricated using 1,959 un-tagged open reading frames (ORFs) taken from the Mammalian Gene Collection (MGC) in mammalian expression vectors. The arrays were then used to screen for genes inducing apoptosis in Human Embryonic Kidney (HEK293T) cells. Using this approach, 10 genes were clearly identified and confirmed to induce apoptosis. Some of these genes have previously been linked to apoptosis, others not. The mechanism of action of three of the 10 genes were then characterised further by following the transcriptional events associated with apoptosis induction using expression profiling microarrays. This data demonstrates a clear pro-apoptotic transcriptional response in cells undergoing apoptosis and also suggests the use of common apoptotic pathways regardless of the nature of the over-expressed protein triggering cell death.


CONCLUSIONS
This study reports the design and use of the first truly large-scale cell-based microarrays for over-expression studies. Ten genes were confirmed to induce apoptosis, some of which were not previously known to possess this activity. Transcriptome analysis on three of the 10 genes demonstrated their use of similar pathways to invoke apoptosis.
Palmer
Ella L
EL
MRC Rosalind Franklin Centre for Genomics Research (RFCGR), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SB, UK. ella.palmer@ucl.ac.uk
Miller
Andrew D
AD
Freeman
Tom C
TC
D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids; D011506:Proteins / Q000235:genetics*; D034622:RNA Interference; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
D011506:Proteins; D004247:DNA