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Updated: Jul 15, 2026
Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos
Published on: April 29, 2011
[Difference of gene expression between 8-cell early embryos and 8-cell compacted embryos in mice]
1Insitute of Reproductive and Stem Cell Engineering, Central South University, Changsha, China.
Objective:
To investigate the differences of gene expression before and after the compaction to search for the control mechanism of compaction.
Methods:
8-cell embryos (626 mice) and 8-cell compacted embryos (437 mice) were collected from Kunmingbai mice, respectively. The cDNA of early 8-cell embryos was used as a tester, and the cDNA of 8-cell compacted embryos as a driver. Differences of gene expression were investigated between early 8-cell embryos and 8-cell compacted embryos using combining SMART (switching mechanism at 5o end of the RNA transcript) -PCR and suppression subtractive hybridization (SSH).
Results:
Four hundreds and seventy eight positive clones were obtained, of which 384 clones with a range of 200~1000 bp and low redundancy were selected for further analysis. Eighty-three ESTs (expressed sequence tags) of the genes expressed differently were gained between early 8-cell embryos and 8-cell compacted embryos. Bioinformatic analysis showed that among the screened 83 putative positive ESTs, 51 ESTs matched 36 known genes, 27 ESTs matched 7 hypothetical genes, and 5 ESTs were new. Genes, which were up-regulated during the compaction, included cytoskeleton(4.8%); enzymes (9.6%); transcriptional factor/regulatory factor (12%); binding protein, protein-protein interactions and transport(13.3%); structural constituent of ribosome (14.5%); pluripotency associated gene(27.7%), and molecular function unknown(18%). Novel ESTs and 17 ESTs which matched the hypothetical genes were put into GenBank with accession numbers.
Conclusion:
Combining SMART-PCR and suppression subtractive hybridization is an efficient method to investigate the gene expression difference in a few samples. During the compaction, the genes maintaining cell pluripotentiality express actively, which may be related to maintaining the embryonic cell totipotence in the differentation environment. All the 17 ESTs might be novel genes related to the compaction in the compacted embryos.
