title: Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts.
abstract: During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4, Otx2, Ifitm3, GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (beta-actin, beta-tubulin and GAPDH) in 21 NT blastocysts with that in genetically half-identical in vitro produced (IVP, n=19) and in vivo (n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts. Ifitm3 expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NT and IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP or in vivo embryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer. Oct4 levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed that in vivo embryos resembled each other much more than did NT and IVP blastocysts. Furthermore, in vivo embryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivo versus in vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.
authors.affiliation: AgResearch, Ruakura Campus, East Street, Hamilton, New Zealand.
authors.forename: Craig
authors.initials: C
authors.lastname: Smith
authors.forename: Debbie
authors.initials: D
authors.lastname: Berg
authors.forename: Sue
authors.initials: S
authors.lastname: Beaumont
authors.forename: Neil T
authors.initials: NT
authors.lastname: Standley
authors.forename: David N
authors.initials: DN
authors.lastname: Wells
authors.forename: Peter L
authors.initials: PL
authors.lastname: Pfeffer
mesh_terms: D000818:Animals; D001755:Blastocyst; D002417:Cattle; D004622:Embryo, Mammalian / Q000502:physiology*; D047108:Embryonic Development; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D053652:Nuclear Transfer Techniques; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction