Construction and characteristic of 3-end enriched cDNA library from single bovine embryo

摘要

To analyze gene expression profile of the large animals and explore the species-dependent embryogenesis, here we adapted techniques to generate 3-end enriched cDNA libraries from single bovine blastocysts based on RT-PCR method. The cDNA library was tested by PCR with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3(Octmer-binding transcription factor), IFN-ι, IGF-2r (Insulin like growth factor 2 receptor) and house-keeping gene H2A and β-actin. The results showed that these genes were expressed in individual pattern presumably because of slightly difference of blastocyst growth phase and/or developmental competence. From the cDNA library, 134 clones were randomly picked for sequencing and showed that structure related elements accounted for over 33.5% transcripts and the energy and metabolism related genes were also an important part about 11.9% in the cDNA library. Approximately 14% of genes were functionally unknown in the library including over 5% of genes, which were probably novel since no significant identity in the Genbank. The frequency of structure related genes such as β-actin and ribosomal proteins in the cDNA library corresponded to other reported cDNA libraries and it suggested that the cDNA library constructed by RT-PCR might proportionally represent the mRNA population. Construction of cDNA libraries from different stage embryos will provide the powerful tool to explore the novel genes relevant to embryogenesis and determine the profiling of stage-specific gene expression.