Reproduction in Domestic Animals

412 B Obackstudies have shown that the spatiotemporal regulationof gene expression is abnormal in cloned pre-implantationembryos. This applies to both termination of thesomatic and initiation of the embryonic gene expressionprogramme. Analysis of transcriptional reprogramminghas focused on two developmental stages: early-cleavagestages at the time of embryonic genome activation andblastocysts.Repressing donor genesTransient and reversible changes in donor cell-specificgene expression can occur simply as an artefact oftrypsinization (Green et al. 2007), emphasizing theimportance of analyzing cells in the state they are inwhen used for NT. However, even if the initial silencingof some genes reflects their manipulation for NT, manydonor cell-specific mRNAs remain repressed until theblastocyst stage. Microarray data have shown that therecipient ooplasm, which is transcriptionally inactive atthe time of NT (Latham et al. 1992), establishes atranscriptionally repressive state within the donor cellgenome of the early NT reconstruct (Vassena et al.2007). As a result, the transcriptome of cloned murineone-cell embryos was very similar to that of controls.Only 259 (=1.6%) of total mRNAs were differentiallyexpressed between SCNT and fertilized embryos at theone-cell stage, with 53 (=20%) of those being newtranscripts and 206 (=80%) being maternally suppliedmRNAs, which were either stabilized or precociouslydegraded in clones. The few newly transcribed genesthat were overexpressed in SCNT embryos, comparedwith both fertilized and parthenogenetic controls,encoded mostly transcription factors that were alsohighly expressed in the donor cells.Reprogramming is a process that occurs over aprotracted period of time, and the next developmentalstage that was analyzed in great detail is the blastocyst.Expression of several donor-specific candidate genesfrom different lineages, e.g. cumulus (Bortvin et al.2003; Inoue et al. 2006) and haematopoietic in mouse(Inoue et al. 2006), skeletal muscle in bovine (Greenet al. 2007) and antler vs adipocyte in cervine NT (Berget al. 2007) was extinguished by that stage. Likewise,global gene expression comparisons demonstrated thatthe majority of genes was differentially expressedbetween donors and their NT blastocyst derivatives,indicating extensive donor transcriptome reprogramming(Smith et al. 2005). Even two female adult ear skinfibroblast lines that differed in the expression of over3000 transcripts prior to NT resulted in indistinguishablegene expression profiles at the blastocyst stage(Smith et al. 2005). Despite their similar degree of geneexpression reprogramming, these two lines still showedsignificantly different cloning efficiency, suggesting thatglobal transcriptional profiling may be of limited valuewhen screening for ‘good’ vs ‘bad’ donor lines. However,not every gene was properly repressed, leaving asmall set of differentially expressed genes that maintainedtheir expression levels in both donors and theircorresponding blastocysts. These fibroblast genes wereinvolved in various biological functions and it would beinformative to determine if the same genes, or theirassociated chromatin modifications, are generally resistantto silencing, independent of cell type. Other genesthat maintain epigenetic donor cell memory have beendescribed in endoderm and neuroectoderm donors infrog (Ng and Gurdon 2005) and myogenic donors aftermouse (Gao et al. 2003) and frog NT (Ng and Gurdon2008). Apart from altered metabolic demands (Gaoet al. 2003), functional consequences of continued donorcell gene expression in cloned embryos are not known.In summary, the donor cell genome was largelyrepressed by the late one-cell stage, but still detectablewith a small array of transcripts at both early and latepreimplantation stages. This raises the question ifinduced donor genome silencing would improve development.Activating embryo genesFollowing fertilization, the embryo undergoes drasticmorphological changes that culminate in the differentiationof two separate lineages, trophectoderm andinner cell mass. During this process, failure to correctlyactivate embryonic genes at the right time, place andabundance will have detrimental effects on development,as illustrated by a large number of single genemutations that interfere with normal embryogenesis inthe mouse. Cloned embryos are clearly defective inrecapitulating this correct stage-specific gene expression.In mouse, already the first transiently inducedgenes (TIG) transcribed from the embryonic genomewere absent or greatly reduced in cloned two-cellembryos, especially if they were also absent from thedonor cell (Vassena et al. 2007, 2008). Even expressionof those few TIG transcripts already present in thedonor cell was reduced in clones, probably becausethey became silenced after NT and only partially reactivatedat the two-cell stage. Other studies, usingdifferent sets of early markers, also observed suppressionof embryonically activated mRNAs in clonedtwo-cells; however, the donor cell expression was notanalyzed (Suzuki et al. 2006; Inoue et al. 2007). Microarrayanalysis confirmed substantial differences betweenthe transcriptomes of NT vs fertilized or artificiallyactivated embryos at the one-cell and, at an order ofmagnitude greater, the two-cell stages (Vassena et al.2007). At the one-cell stage, 53 (=0.3% of total) newtranscripts were at least twofold differentially expressed(8 down- and 45 upregulated), at the two-cell stage thatnumber increased to 1539 (452 down- and 1087upregulated) of different transcripts relative to controlembryos. In addition, a large number of maternalmRNAs underwent either precocious or delayed degradation.The combined effects of aberrant transcriptionand mRNA handling prominently affected genesinvolved in transcriptional regulation. SCNT embryosmay be predisposed to aberrantly express genes encodingproteins that are responsible for establishing andmaintaining a stable donor cell differentiation status.Their widespread dysregulation likely caused a ‘rippleeffect’ that alters the transcriptome of many otherfunctions including oxidative phosphorylation, membranetransport, and mRNA transport and processing(Vassena et al. 2007).Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag