Supplementary materials to Chen WV, Delrow J, Corrin PD, Frazier JP and Soriano P (2004).
Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis.
Nature Genetics, 36, 304-312
Abstract
We developed a versatile,
high-throughput genetic screening strategy by coupling gene mutagenesis and
expression profiling technologies. Using a retroviral gene-trap vector
optimized for efficient mutagenesis and cloning, we randomly disrupted genes in
mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that
transcriptional target genes of platelet-derived growth factor (PDGF) can be
efficiently and reliably identified in physiologically relevant cells and are
immediately accessible to genetic studies to determine their in vivo roles and relative contributions
to PDGF-regulated developmental processes.
The same platform can be used to search for genes of specific biological
relevance in a broad array of experimental settings, providing a fast track
from gene identification to functional validation.
This web page provides the complete microarray data sets as well as the oligo sequences used in the publication.
A. Testis versus brain
expression profiling (Figure 4)
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MIAME checklist:
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Gene expression data table: |
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Image analysis output files: |
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B. PDGF
transcriptional response profiling (Figure 5)
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MIAME checklist:
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Gene expression data table: |
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Image analysis output files: |
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C. Oligo sequences
3’ RACE:
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Anchoring oligo (QT) |
CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC(T)17 |
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Anchoring primer 1 (QA) |
CCAGTGAGCAGAGTGACGAGGAC |
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Anchoring primer 2 (QB) |
GACGAGGACTCGAGCTCAAGC |
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Hygromycin-specific primer (HYGF) |
ACTCGTCCGAGGGCAAAGGAATAGG |
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SD exon-specific primer (SDEXF) |
GCTAGCGCGTTCGTCCTCACTCT |
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SD intron-specific oligo (SDINF) |
GTGAGTACTCCCTCTCAAAAGCGGGCATGACTTC |
5’ genomic anchoring PCR:
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Adapter oligo 1(PDA-L) |
AGCAGCGAACTCAGTACAACAACTCTCCGACCTCTCACCGAGT |
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Adapter oligo 2 (PDA-S) |
ACTCGGTGA |
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Distal adapter primer (DAP) |
AGCAGCGAACTCAGTACAACA |
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SA-specific primer (SASP) |
GAAAGACCGCGAAGAGTTTG |
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U5-specific primer (U5SP) |
CTGTTCCTTGGGAGGGTCTC |
ROSA71 genotyping:
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Forward gene-specific primer (R71F) |
GCCTTTCTACCCACACAACTACA |
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Reverse gene-specific primer (R71R) |
CTGGAAAACCGTTGTTTTGACTG |
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Beta-gal-specific primer (BGALR) |
CGGGCCTCTTCGCTATTACGC |
Strap RT-PCR:
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Forward primer (5’ of insertion site) |
ATCACGCCTTACGGCTACTTT |
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Reverse primer (3’ of insertion site) |
GTGTGAAATCCACAGTCTTGACA |
For further information, contact genetrap@fhcrc.org.