Download A Gene Expression Screen

A Gene Expression Screen
Zhou Wang and Donald D. Brown
Presented By: Neema Izadi
Introduction
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In Amphibian Tadpole
1) Thyroid hormone is known to cause tail
degeneration.
2) Study done to figure out which genes are up
regulated and down regulated.
3) Used genetic screening
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Estimate number of genes involved
Identify genes involved
Guarantee genes are involved in process
Background
1) Microarray techniques are presently
used to identify differing gene expression
2) Instead of ~20 genes, DNA Microarray
can find hundreds.
3) Microarray techniques did not become
available until 1995.
4) This study was done in 1991
Materials and Methods
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Preparing the cDNA
1) Amputated tadpole tails and created:
(-) control and (+) T3-treated
2) Oligo(dT) used to prime first cDNA strand
3) Average size insert was 2 to 3 kb, largest
was 5kb.
Materials and Methods
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Preparing the cDNA driver
1) cDNA digested with: Alu I and Alu I plus
Rsa I seperately.
2) Ligated with oiligonucleotide linker with one
blunt end and one protruding end.
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Linker Contained a EcoRI site near the end
3) Driver was biotinylated so that they could be
detected later by adding streptavidin.
4) Drivers are PCR amplified
Materials and Methods
Subtractive Hybridization
• Involves many cycles of the following:
1) Long Hybridization – supress common
complex rare cDNA.
2) Short Hybridization – supress common
abundant cDNA.
Materials and Methods
Subtractive Hybridization CONTINUED
3) PCR
4) Hybrids were removed by extractions using
Streptavidin
5) While residual driver fragments were
removed by digestion with EcoRI
Enrichment of Up-Regulated Genes
Enrichment of Down-Regulated Genes
Results
After 3 Cycles of Subtractive Enrichment:
1) The enriched cDNA fragments were
amplified with 30 cycles of PCR.
2) They were cleaved with EcoRI and
ligated to a vector for transformation.
3) 3000 colonies of each cDNA library were
screened.
Comparison of differentially expressed genes
Results
1) Alltogether they found 30 non-crosshybridizing cDNA fragments
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5 from (-) group, down-regulated
25 from (+) group, up-regulated
2)They perfomed Nothern blots to check for
redundancy of the cDNA
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Found 6 (+) and 1 (-) were redundant
3) Thus, they isolated 16 up-regulated genes
and 4 down regulated genes.
Conclusion
A Gene Expression Screen
• Poisson distribution was used to predict how
many total genes were involved using statistics.
Limitations
1)Gene expression screen will not detect:
• genes that lack a poly(A) tail
• Genes that are small enough to escape
restriction enzyme digestion
• Genes that can not be amplified by PCR
2)PCR also provides its own bias
• PCR does not amplify everything uniformly so
biases in frequency may result.
In Case You Want More . . .
• The thyroid hormone-induced tail resorption program during
Xenopus laevis metamorphosis
Donald D. Brown et al. 1995
• Application of Suppression Subtractive Hybridization (SSH) to
Cloning Differentially Expressed cDNA in Dunaliella salina
(Chlorophyta) Under Hyperosmotic Shock
Xiao-Ning Zhang et al. 2002
• Thyroid Hormone-Dependent Gene Expression in Differentiated
Embryonic Stem Cells and Embryonal Carcinoma Cells:
Identification of Novel Thyroid Hormone Target Genes by
Deoxyribonucleic Acid Microarray Analysis
Y.-Y. Liu et al. 2005