The Transcriptional Program in the Response of Human Fibroblasts to Serum-Reference-Cited by-同舟云学术

The Transcriptional Program in the Response of Human Fibroblasts to Serum

Published:1999-01 Issue:5398 Volume:283 Page:83-87
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Iyer Vishwanath R.¹,Eisen Michael B.¹,Ross Douglas T.¹,Schuler Greg¹,Moore Troy¹,Lee Jeffrey C. F.¹,Trent Jeffrey M.¹,Staudt Louis M.¹,Hudson James¹,Boguski Mark S.¹,Lashkari Deval¹,Shalon Dari¹,Botstein David¹,Brown Patrick O.¹

Affiliation:

1. V. R. Iyer and D. T. Ross, Department of Biochemistry, Stanford University School of Medicine, Stanford CA 94305, USA. M. B. Eisen and D. Botstein, Department of Genetics, Stanford University School of Medicine, Stanford CA 94305, USA. G. Schuler and M. S. Boguski, National Center for Biotechnology Information, Bethesda MD 20894, USA. T. Moore and J. Hudson Jr., Research Genetics, Huntsville, AL 35801, USA. J. C. F. Lee, D. Lashkari, D. Shalon, Incyte Pharmaceuticals, Fremont, CA 94555, USA. J. M....

Abstract

The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference25 articles.

1. Serum- and Polypeptide Growth Factor-Inducible Gene Expression in Mouse Fibroblasts

2. A normal human diploid fibroblast cell line derived from foreskin (ATCC CRL 2091) in passage 8 was used in these experiments. The protocol followed for growth arrest and stimulation was essentially that of (16) and (17). Cells were grown to about 60% confluence in 15-cm petri dishes in Dulbecco's minimum essential medium containing glucose (1 g/liter) the antibiotics penicillin and streptomycin and 10% (by vol) FBS (Hyclone) that had been previously heat inactivated at 56°C for 30 min. The cells were then washed three times with the same medium lacking FBS and low-serum medium (0.1% FBS) was added to the plates. After a 48-hour incubation the medium was replaced with fresh medium containing 10% FBS. mRNA was isolated from several plates of cells harvested before serum stimulation; this mRNA served as the serum-starved or time-zero reference sample. Cells were harvested from batches of plates at 11 subsequent intervals (15 min 30 min 1 2 4 6 8 12 16 20 and 24 hours) after the addition of serum. mRNA was also isolated from exponentially growing fibroblasts (not subjected to serum starvation). mRNA was isolated with the FastTrack mRNA isolation kit (Invitrogen) which involves lysis of the cells on the plate. The growth medium was removed and the cells were quickly washed with phosphate-buffered saline at room temperature. The lysis buffer was added to the plate transferred to tubes and frozen in liquid nitrogen. Subsequent steps were performed according to the kit manufacturer's protocols.

3. The National Center for Biotechnology Information maintains the UniGene database as a resource for partitioning human sequences contained in GenBank into clusters representing distinct transcripts or genes (18 19). At the time this work began this database contained about 40 000 such clusters. We selected a subset of 10 000 of these UniGene clusters for inclusion on gene expression microarrays. UniGene clusters were included only if they contained at least one clone from the I.M.A.G.E. human cDNA collection (20) so that a physical clone could easily be obtained (all I.M.A.G.E. clones are available commercially from a number of vendors). We attempted to include as complete as possible a set of the “named” human genes (about 4000) and genes that appeared to be closely related to named genes in other organisms (about an additional 2000). The remaining 4000 clones were chosen from among the “anonymous” UniGene clusters on the basis of inclusion on the human transcript map (www.ncbi.nlm.nih.gov/SCIENCE96/) and the lack of apparent homology to any other genes in the selected set. A physical clone representing each of the selected genes was obtained from Research Genetics. This “10K set” is included in a more recent “15K set” described at www.nhgri.nih.gov/DIR/LCG/15K/HTML/p15Ktop.html. Of these clones 472 are absent from the current edition of UniGene and were presumed to be distinct genes. The remainders represent 8141 distinct clusters or human genes in UniGene. These clones thus presumed to represent 8613 different genes were used to print microarrays according to methods described previously (21 22).

4. One microgram of mRNA was used for making fluorescently labeled cDNA probes for hybridizing to the microarrays with the protocol described previously (23). mRNA from the large batch of serum-starved cells was used to make cDNA labeled with Cy3. The Cy3-labeled cDNA from this batch of serum-starved cells served as the common reference probe in all hybridizations. mRNA samples from cells harvested immediately before serum stimulation at intervals after serum stimulation and from exponentially growing cells were used to make cDNA labeled with Cy5. Ten micrograms of yeast tRNA 10 μg of polydeoxyadenylic acid and 20 μg of human CoT1 DNA (Gibco-BRL) were added to the mixture of labeled probes in a solution containing 3× standard saline citrate (SSC) and 0.3% SDS and allowed to prehybridize at room temperature for 30 min before the probe was added to the surface of the microarray. Hybridizations washes and fluorescent scans were performed as described previously (23 24). All measurements totaling more than 180 000 differential expression measurements were stored in a computer database for analysis and interpretation.

5. The nominal identities of a number of cDNAs (currently about 3750) on the microarray were verified by sequencing. The clones that were sequenced included many of the genes whose expression changed substantially upon serum stimulation as well as a large number of genes whose expression did not change substantially in the course of this experiment. About 85% of the clones on the current version of this microarray that were checked by resequencing were correctly identified. In all the figures gene names or EST numbers are given only for those genes on the microarrays whose identities were reconfirmed by resequencing. In the cases where a human gene has more than one name in the literature we have tried to use the name that is most evocative of its presumed role in this context. The remainder of the clones have been assigned a temporary identification number (format: SID######) and a putative identity pending sequence verification. The correct identities of these genes will be posted at our Web site (genome-www.stanford.edu/serum) as they are confirmed by resequencing.

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