Reproduction in Domestic Animals

Steps to Improve Farm Animal Cloning Efficiency 411been reported in mouse, it is currently unknown ifzygotes can also improve cloning efficiency in thisspecies. Taken together, these studies unequivocallydemonstrated that zygotes retain the factors necessaryto completely reprogram embryonic and somatic genomes.These are likely to reside in the pronuclei duringinterphase, being redistributed throughout the cytoplasmduring mitosis.Improving Reprogramming In vitroA number of in vitro approaches have been devised toincrease somatic cloning success. These include treatingdonor cells with pharmacological agents to alter theirepigenetic marks (Enright et al. 2003; Shi et al. 2003),fusing transiently permeabilized cells containing artificiallycondensed chromatin (Sullivan et al. 2004) orusing serial NT (Ono et al. 2001). One of the mostpromising methods has been embryo aggregation. Inmouse, aggregation of two or three NT embryos led tonormalized gene expression and higher cloning efficiencyin some studies (Boiani et al. 2003) but not in others(Yabuuchi et al. 2002). In bovine, we aggregated threeone-cell NT embryos during in vitro culture (Obacket al. 2003; Misica-Turner et al. 2007). Aggregationaffected development of embryonic and somatic clonedembryos differently. In aggregates of embryonic clones,in vitro development was impaired, but the fewdeveloping blastocysts appeared molecularly normal.Theoretically, one would expect an n-fold increase inpost-blastocyst survival using n numbers of aggregationpartners, provided there were no positive or negativeinteractions within the composite blastocyst. Embryoclones were in agreement with this prediction, showingon average 2.5-fold higher cloning efficiency. In otherwords, there was no evidence for complementary interactionsresulting in increased survival beyond whatwould be expected as a direct numerical consequence ofaggregation. A similar increase in term survival ofaggregated embryonic clones has been described before(Peura et al. 1998). In contrast, SCNT aggregatesdeveloped normally in vitro, but the resulting blastocystsshowed reduced POU5F1 expression and no effecton vivo survival, indicating that they were in factcompromised compared with embryo clone aggregates.These differences in timing of developmental failurereveal striking biological differences between embryonicand somatic clones in response to aggregation.Measuring Reprogramming Before EmbryoTransferCloned embryos before transfer display a number ofepigenetic abnormalities such as aberrant DNA-methylation(Kang et al. 2003) and post-translational histonemodifications (Santos et al. 2003; Wang et al. 2007;Yang et al. 2007a,b). It is likely that abnormal epigenotypewill result in aberrant gene expression. At leastfor one gene, a mechanistic link between DNA-methylationand transcriptional errors has indeed been established.This gene encodes the transcription factorPOU5F1, which is essential for regulating embryoniccell pluripotency and has often been used to monitorreprogramming success after NT (Boiani et al. 2002;Munsie et al. 2002; Bortvin et al. 2003; Misica-Turneret al. 2007; Wuensch et al. 2007). In interspecies NTexperiments using Xenopus recipient oocytes and mousedonor thymocytes, selective POU5F1 promoter DNAdemethylationwas causally linked to incorrectlyre-activating POU5F1 transcription (Simonsson andGurdon 2004). Because discrepancies between RNAand protein levels exist (Tian et al. 2004), it remains tobe formally demonstrated that POU5F1 protein expressionwas also aberrant and that this subsequentlycontributed to some of the aberrant organismal phenotypesobserved in clones. Proteins ultimately carry outcellular functions, thus their expression profiling representscellular phenotype better than RNA profiling.However, the proteome with its many low-abundanceproteins, often carrying post-translational modifications,is more difficult to analyze and has consequentlynot been comprehensively profiled in clones (Fig. 3).RNA levels, on the contrary, can be amplified, makingthem easier and cheaper to quantify globally. NumerousNT ES cell-like cellsFig. 3. Genome-wide profiling ofclones at different levels. Shown isnuclear transfer (NT) embryodevelopment from one-cell to blastocyst,which can either be used togenerate ES cell-like cells or clonedoffspring. Bars indicate differentgenome-wide profiling approachesto examine different levels ofcellular information (DNA, RNA,protein, etc.) at different developmentaltime windows. Shadingwithin bars indicates to what degreeeach approach has been used for aparticular developmental interval,ranging from little use (white) toheavy use (dark shading). Arrowsbetween bars indicate the flow ofinformation from genotype tophenotype+NTrecipient donor NT 1-cell NT blastocystGenomeEpigenomeTranscriptomeProteomePhenomeNT animalÓ 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag