Isolation of epiblast-specific cDNA clones by differential hybridization with polymerase chain reaction-amplified probes derived from single embryos.
A mouse day 7.5 embryonic ectoderm cDNA library containing 2 x 10(6) clones was screened by differential hybridization with polymerase chain reaction (PCR)-amplified probes derived from a single embryo. Day 7.5 ectoplacental cone and embryonic ectoderm served as the source of mRNA to make minus and plus probes, respectively. In a limited screen of fewer than 2,000 clones, 23 up-regulated clones were identified by the difference in hybridization signal with the two probes. DNA sequence analysis revealed the nature of some, but not all, of the clones. Northern blot and in situ hybridization with a subset of the clones confirmed the utility of the approach, since the differential signal was also observed in these experiments. This approach may prove useful for identifying genes that play a role during development.
Varmuza
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Department of Zoology, University of Toronto, Ontario, Canada.
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D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes; D004277:DNA, Single-Stranded; D005775:Gastrula / Q000166:cytology / Q000378:metabolism*; D015698:Genomic Library; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D004277:DNA, Single-Stranded