Identification of the rat homologue of the mouse capsulin gene by cDNA representational difference analysis1
Introduction
To identify the factors that regulate gut epithelial stem cell commitment into particular lineages, it will be necessary first to understand when tissue-specific genes are expressed in the developing gut, and to identify factors that may regulate this process in the embryo. In this study, we have used the powerful technique of representational difference analysis (RDA) to detect embryonic gut-specific genes. We undertook a pilot study to evaluate the sensitivity of the technique in our hands and to test whether cDNA RDA, being a PCR-based strategy has any bias towards smaller DpnII-generated gene fragments, a bias that would mitigate against its usefulness.
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Materials and methods
RDA was performed as previously described (Hubank and Schatz, 1994) except that commercially synthesized oligonucleotides for adaptors were cartridge-purified (Life Technologies, USA), and following digestion excess adaptors were removed by Chromaspin 100 columns (Clontech, Palo Alto, CA), and half the recommended cDNA concentrations were used for all subtractive hybridizations. All subsequent enzyme and reagent concentrations and reaction volumes were altered accordingly. The RNase Protection
Results and discussion
In the first round of cDNA RDA, we used rat embryonic day-14 brain cDNA as both driver (20 μg) and tester (from 0.2 μg to 50 pg). pBluescript (Stratagene, La Jolla, CA), containing a fragment of the rat GAPDH gene, was subjected to Sau3AI digestion to generate fragments that were used to spike the tester cDNA. The following approximate sized fragments were selected: 260 bp (0.04 pg), 340 bp (4 pg), 560 bp (40 pg) and 740 bp (0.04 pg), spiked at the amounts indicated in parentheses. Note that, in order to
Conclusions
- 1.
cDNA RDA is a powerful, sensitive and reproducible technique that does not have any PCR bias towards smaller gene fragments.
- 2.
This technique has been used successfully to identify the rat homologue of the mouse capsulin gene.
Acknowledgements
The authors wish to thank Dr D. Newgreen for critical reading of the manuscript. A.S.G. is a recipient of a National Health and Medical Research Council Grant.
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Accession No. AF061752.