Reproduction in Domestic Animals

Reprod Dom Anim 43 (Suppl. 2), 407–416 (2008); doi: 10.1111/j.1439-0531.2008.01192.xISSN 0936-6768Climbing Mount Efficiency – Small Steps, Not Giant Leaps Towards Higher CloningSuccess in Farm AnimalsBjo¨rn ObackAgResearch Ltd., Ruakura Research Centre, Hamilton, New ZealandContentsDespite more than a decade of research efforts, farm animalcloning by somatic cell nuclear transfer (SCNT) is stillfrustratingly inefficient. Inefficiency manifests itself at differentlevels, which are currently not well integrated. At the molecularlevel, it leads to widespread genetic, epigenetic andtranscriptional aberrations in cloned embryos. At the organismallevel, these genome-wide abnormalities compromisedevelopment of cloned foetuses and offspring. Specific moleculardefects need to be causally linked to specific clonedphenotypes, in order to design specific treatments to correctthem. Cloning efficiency depends on the ability of the nucleardonor cell to be fully reprogrammed into an embryonic stateand the ability of the enucleated recipient cell to carry out thereprogramming reactions. It has been postulated that reprogrammabilityof the somatic donor cell epigenome is influencedby its differentiation status. However, directcomparisons between cells of divergent differentiation statuswithin several somatic lineages have found no conclusiveevidence for this. Choosing somatic stem cells as donors hasnot improved cloning efficiency, indicating that donor cell typemay be less critical for cloning success. Different recipient cells,on the otherhand, vary in their reprogramming ability. Inbovine, using zygotes instead of oocytes has increased cloningsuccess. Other improvements in livestock cloning efficiencyinclude better coordinating donor cell type with cell cycle stageand aggregating cloned embryos. In the future, it will beimportant to demonstrate if these small increases at every stepare cumulative, adding up to an integrated cloning protocolwith greatly improved efficiency.The Importance of Farm Animal CloningIn more than a decade since the birth of Dolly the sheep,cloned offspring have been produced by somatic cellnuclear transfer (SCNT) in 18 mammalian species.Despite this ever growing list, SCNT remains veryinefficient compared with other assisted reproductivetechnologies such as in vitro fertilization (IVF) orartificial insemination. Typically, cloning efficiency,quantified as the proportion of all embryos transferredinto surrogate mothers that develop into viable offspring,is about 1%–5% (Oback and Wells 2007a). Overthree-quarters of all cloning laboratories are working onfarm animals (cattle, pig, goat, sheep, buffalo and deer),illustrating that the main objective behind SCNT is stillcommercially driven – namely to multiply elite animalswith desired phenotypic traits and to produce geneticallymodified animals (Oback and Wells 2007a). As aconsequence of increased research effort and fundingin the area, the total number of cloning publications hasincreased by an order of magnitude in the past decade,and still continues to grow (Fig. 1). Cattle SCNT haslong dominated the NT publication record, accountingfor an annual average of about 25% of PubMed-listedpapers since 1994. Pig is the second most importantcloned farm animal by this measure (13% of NTpublications), followed by goat, sheep, buffalo and reddeer (altogether 6%). Overall, farm animal cloning thusaccounts for 44% of cloning publications, laboratoryanimals (mouse, rabbit, monkey and rat) for 22%, otherspecies (including human) for 16% and general reviewarticles, which are not species-specific, for the remaining18%. Based solely on past research investment andoutput, i.e. the number of labs involved and theirpublications, cattle is still the most important clonedlivestock species (Oback and Wells 2007a).Nuclear ReprogrammingAfter NT of a fully differentiated donor cell into acytoplast, the resulting reconstruct can develop into anembryo and even a viable animal. The logical alternative,i.e. that the NT reconstruct cleaves into fullydifferentiated donor cells, has never been observed.Erasing transcriptional programme and epigenetic identityof the donor cell is referred to as nuclear reprogramming.The molecular dominance of the oocyte overany somatic cell type tested may simply be due to itbeing a 1000-fold larger in volume and thus containing a1000-fold excess of oocyte-specific factors, in which casethe reprogramming dominance should disappear oncecell size differences are experimentally adjusted. This issupported by the observation that nuclear reprogrammingalso occurs in differentiated cells fused to nondividingmultinucleate heterokaryons (Blau et al. 1983;Terranova et al. 2006) with the direction of reprogrammingbeing dictated by the ratio of the nuclei derivedfrom each cell type (Pavlath and Blau 1986). Thecapacity to reverse stable heritable epigenetic modifications,such as DNA-methylation, is not particular tooocytes, but also occurs in embryonic stem (ES) cells(Tada et al. 2003) and even fully differentiated skeletalmuscle cells (Zhang et al. 2007). However, so far onlyoocytes have been capable of reprogramming somaticcells to the extent of giving rise to a completely newcloned organism. Clone survival into adulthood is thusthe most informative and meaningful measure of extensivedonor cell reprogramming. Reprogramming efficiencyafter NT critically depends on two processes: theability of the nuclear donor cell to be fully reprogrammedand the ability of the oocyte to carry out thereprogramming reactions. As it is currently unclearwhich process is more important for reprogrammingsuccess, both will be discussed in this review.Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag