281 EXPRESSION PROFILE AND KNOCKDOWN ANALYSIS OF A FUNCTIONALLY UNKNOWN DD2-2 GENE IN MOUSE PRE-IMPLANTATION EMBRYOS

Abstract

Zygotic gene activation (ZGA) starts at the G₂ phase at the 1-cell stage in the mouse. However, the molecular mechanism of ZGA has not been completely elucidated. We have investigated the molecular functions of many gene clusters, DD clones obtained by differential display assays for ovulated eggs at the M II stage and 1-cell stage embryos at the G² phase. As a result, we have identified a functionally unknown gene, whose sequence did not match a known transcript in the gene bank DD2-2 gene. Here, we report the expression profile and knockdown analysis of the DD2-2 gene in mouse pre-implantation embryos. Nucleotide sequence analysis of the DD2-2 cDNA revealed that the open reading frame of 1056 bp encodes a protein of 351 amino acids with a predicted molecular mass of 41.5 kDa. The deduced amino acid sequence indicated that DD2-2 protein might be a soluble protein without a signal peptide. We first investigated the expression profiles of DD2-2 in pre-implantation embryos by quantitative real-time PCR using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). To investigate the effect of knockdown of the DD2-2 gene on the development of pre-implantation embryos, we injected pβ-act/antisenseDD2-2/IRES/EGFP into male pronuclei of embryos at 7 to 9 h after insemination (hpi) and observed the development of embryos that showed EGFP expression at 24 hpi. Real-time PCR analysis of pre-implantation embryos showed that maternal DD2-2 mRNA at a low level significantly increased up to the early 2-cell stage, and significantly decreased by the 4-cell stage and later, suggesting that DD2-2 gene specifically expresses at major ZGA. In the knockdown analysis, EGFP-positive embryos with pβ-act/antisenseDD2-2/IRES/EGFP showed a lower rate of development to the 4-cell stage and later, compared with that of EGFP-positive embryos with pβ-act/luc+/IRES/EGFP [72% (94/130) vs. 54% (71/131); P < 0.05], indicating that the knockdown of DD2-2 by antisense RNA resulted in a inhibition of pre-implantation development. In conclusion, the DD2-2 gene, a functionally unknown gene, may play an important role in pre-implantation development.

This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.