Identification of differentially expressed genes in murine embryos at the blastocyst stage using annealing control primer system.
The identification of embryo-specific genes would provide insights into early embryonic development. However, the current methods employed to identify the genes that are expressed at a specific developmental stage are labor intensive and suffer from high rates of false positives. Here we employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method that involves annealing control primers (ACPs) to identify the genes that are specifically or prominently expressed in mouse blastocysts compared to 4-cell stage embryos. Using 120 ACPs, we identified and sequenced 74 of these differentially expressed genes (DEGs). Basic Local Alignment Search Tool (BLAST) searches revealed that 53 were known genes, 9 encoded ribosomal proteins, and 12 were unknown genes. Of the known genes, 14 were selected and further characterized using real-time quantitative PCR to assess their stage-specific expression in mouse embryos. This analysis suggests that the ACP system is a very good method for the identification of stage-specific genes in small numbers of mouse embryos. Further analysis of the differentially expressed blastocyst genes we have identified will provide insights into the molecular basis of preimplantation development.
Cui
Xiang-Shun
XS
Department of Animal Science, Chungbuk National University, Gaesin-dong, Cheongju, Chungbuk, 361-763, South Korea.
Shin
Mi-Ra
MR
Lee
Kyung-Ah
KA
Kim
Nam-Hyung
NH
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D019295:Computational Biology; D017931:DNA Primers; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005796:Genes / Q000235:genetics*; D016014:Linear Models; D051379:Mice; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D017931:DNA Primers