Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts

The successful production of embryos by nuclear transfer (NT) employing cul tured somatic donor cells depends upon a variety of factors. The objective of the present study was to investigate the effects 1) of two different act ivation protocols, 2) the use of quiescent or nonquiescent donor cells (G(0 ) or G(1) of the cell cycle), and 3) passage number of donor cells on the r elative abundance (RA) of eight specific mRNAs (DNA methyltransferase, DNMT ; mammalian achaete-scute homologue, Mash2; glucose transporter-1, Glut-1; heat shock protein 70.1, Hsp; desmocollin II, Dc II; E-cadherin, E-cad; int erferon tau, IF; insulin-like growth factor 2 receptor, Igf2r) in single bl astocysts employing a semiquantitative reverse transcription-polymerase cha in reaction assay. The results were compared with those for their in vitro (IVP)- and in vivo-generated noncloned counterparts. In experiment 1, emplo ying either FBA (fusion before activation) or AFS (fusion and activation si multaneously) to generate NT blastocysts, Hsp mRNAs were not found in NT em bryos from either protocol, whereas Hsp transcripts were detectable in IVP embryos. The relative abundance (RA) of IF transcripts was significantly in creased in the AFS and IVP groups compared to the FBA treatment. In experim ent 2, the use of either G(1) or G(0) donor cells to produce cloned embryos both significantly reduced the relative amount of DNMT transcripts and sig nificantly increased the RA of Mash2 compared to the IVP embryos. In additi on, IF transcript levels were significantly elevated in NT blastocysts empl oying G(1) donor cells for NT compared to IVP embryos and those generated u sing G(0) cells. In experiment 3, donor cells, either from passsage 5/6 or 8, were employed for NT. DNMT transcripts were significantly decreased, whe reas Mash2 transcripts were significantly increased in both NT groups compa red to their IVP counterparts. The amount of IF mRNA was significantly high er in P8-derived than in P5/6 and IVP embryos. In experiment 4, the RA of D NMT transcripts was decreased in in vivo-derived blastocysts compared to th ose produced in vitro. Mash2 expression was increased in in vivo embryos an d those IVP embryos produced in medium containing Sigma BSA. The RA of Hsp was higher in IVP embryos produced in serum containing medium than in those produced in Sigma BSA or in vivo. In vivo embryos and those produced in Li fe Technologies BSA had the lowest expression of IF transcripts. Expression of all other genes was not affected by variation in NT methodology or IVP culture systems throughout experiments 1-4. In conclusion, depending on ste ps of the cloning procedure NT-derived embryos display marked differences f rom their IVP- and in vivo-derived counterparts. An aberrant expression pat tern in NT embryos was found with respect to genes thought to be involved i n stress adaptation, trophoblastic function, and DNA methylation during pre implantation development.