the transcriptional program in the response of human fibroblasts to serum iyer et al n.
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The Transcriptional Program in the Response of Human Fibroblasts to Serum ( Iyer et al). By: James Tirtorahardjo & Daphne Lin. Background Info. Fibroblasts important to tissue regeneration Serum: Liquid portion of blood without blood cells and clotting factors

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the transcriptional program in the response of human fibroblasts to serum iyer et al

The Transcriptional Program inthe Response of HumanFibroblasts to Serum (Iyer et al)

By: James Tirtorahardjo & Daphne Lin

background info
Background Info
  • Fibroblasts important to tissue regeneration
  • Serum: Liquid portion of blood without blood cells and clotting factors
  • Exposure of fibroblasts to serum initiates a variety of wound healing processes
fibroblast function in wound repair
Fibroblast Function in Wound Repair
  • Fibroblasts are located outside the bloodstream
  • Extracellular matrix formation
  • Secretion of collagen and fibronectin
  • Formation of a wound matrix
purpose
Purpose
  • Discover why fibroblasts are important to tissue regeneration
  • To study the transition from G0 to proliferation
  • To identify differences in gene expression over time during wound repair.
  • To group genes into categories based on expression pattern and function
  • To show that cDNA microarrays are a viable method of expression profiling
method
Method
  • After culture, quiescent fibroblasts are deprived of serum for 48 hours
  • Then stimulated by solution of 10% FBS (fetal bovine serum)
  • Cells harvested for cDNA microarray at 12 different times
cdna synthesis
cDNA Synthesis
  • mRNA is harvested from cells via organic extraction
  • cDNA clones were identified via sequencing and comparison to a database
cdna microarray
cDNA Microarray
  • Control cDNA made from G0 cells
method1
Method
  • 8613 different genes surveyed
    • 4000 named human gene
    • 2000 genes from other organisms related to the named genes
    • 4000 other genes from the human transcript map
  • Half were unnamed ESTs (expressed sequence tags)
verification
Verification
  • Gradual change in gene expression over sequential time points showed that the observations were valid
  • Inclusion of duplicates
  • Three independent mRNA hybridizations used
  • PCR assay for gene expression
analysis
Analysis
  • Genes within cluster are related/perform similar functions
  • Ex: Cluster D contains cell proliferation genes
fibroblast reprogramming
Fibroblast Reprogramming
  • Probably responsible for pulling fibroblast out of G0 phase and back into cell cycle
  • Reprogramming consists of turning off genes inducing quiescence and turning on genes that induce proliferation
fibroblasts in w ound h ealing
Fibroblasts in Wound Healing
  • Genes with similar function have similar expression patterns
significant findings
Significant Findings
  • Found 200 new genes
  • Other unknown genes can be placed into clusters based on expression pattern
  • cDNA microarray was shown to be an effective way to create an expression profile
issues with the method
Issues With the Method
  • Cannot perfectly replicate in vivo conditions
  • Cannot see exactly how the genes interact
  • Doesn’t tell the function of the individual genes (must be inferred)
  • Requires prior knowledge of the genes
clinical significance
Clinical Significance
  • Helps understand wound repair mechanisms
  • More info on G0 to proliferation transition
  • Identification of gene expression patterns for cell cycle
future studies
Future Studies
  • Testing fibroblast expression in response to other stimuli
  • Examining fibroblast expression in vivo wound repair response
  • Test serum response patterns in other cell types
summary
Summary
  • cDNA hybridization to examine gene expression pattern in response to exposure to serum
  • Identified gene clusters responsible for various cell activities
  • Identified 200 new genes