Reproduction in Domestic Animals

356 CBA Whitelaw, SG Lillico and T Kingpromoter element, the vector is otherwise transcriptionallysilent. This is achieved by introducing mutationsinto the viral genome transcription control sequences(the long-terminal repeats) to generate a self-inactivatingvector (Pfeifer 2004). This distinguishes the lentivirusvector from other retrovirus vectors, which rely on viraltranscriptional elements for transgene activity (Pfeifer2004).With regard to transgenesis, lentivirus vectors haveseveral advantages (Pfeifer 2004; Whitelaw 2004).A distinguishing property of lentivirus vectors is theirability to infect both dividing and non-dividing cells. Itis this property that has promoted their development asgene delivery vectors since they can be delivered to eitherthe egg or zygote. Second, delivery of the vector,although still requiring the use of microinjection equipment,is less damaging to the egg ⁄ zygote when comparedwith pronuclear microinjection resulting in aconsiderable proportion of injected embryos developingto term. Exploiting the natural efficiency of infectionafforded to virus by evolution, the vector fuses with thecell (egg or zygote) and is internalized. This means thatthe lentivirus vector can be introduced (Fig. 1), byinjection into the perivitelline space of the zygote (Loiset al. 2002; Ritchie et al. 2007) or by co-culture with azona-free (or laser microdrilled) zygote (Ewerling et al.2006). This delivery method results in a very highproportion of injected embryos surviving. Recent datasuggests that many embryo manipulation methods causeDNA damage and thus limited embryo development(Yamauchi et al. 2007). Lentivirus vector-mediatedtransgene delivery appears not to suffer from thisproblem, perhaps because of the ‘natural’ route ofinfection that they use to deliver the transgene.A further and significant property of lentivirus vectorsis the spectacular efficiency of transgenesis that can beachieved. Transgenic mice (Lois et al. 2002; Pfeifer et al.LTRPromoter - transgeneFig. 1. Diagram of lentiviral vector delivery methods. A prototypicalvector is shown long-terminal repeats encompassing the promotertransgeneof choice. Packaged viral vectors can then be either coculturedwith zona denuded (or microdrilled) zygotes, which requiresindividual culturing of each embryo or injected into the perivitellinespace of an oocyte or zygote. Both methods have been demonstrated toproduce live transgenic animalsLTR2002), rats (Lois et al. 2002; Dann 2007; Michalkiewiczet al. 2007), poultry (McGrew et al. 2004; Lillico et al.2007), pigs (Hofmann et al. 2003; Whitelaw et al. 2004),cattle (Hofmann et al. 2004), sheep (Whitelaw, unpublished)and recently rabbits (Zsuzsa Bosze, personalcommunication) have been made using this method. Formany of these species, transgenesis rates of up to 100%of injected embryos carrying an integrated transgenehave been achieved. This had to be compared with thestandard pronuclear injection method, where 5% ofmouse zygotes and 1% of livestock zygotes result in atransgenic founder animal. Furthermore, unlike pronuclearinjection, most of these integration events carrytranscriptionally active transgenes. Taken together, theadvantageous properties of lentivirus vectors make thema very attractive transgene delivery route, especially forspecies, where the more standard methods are verycostly in terms of animal numbers and in the financialsupport required.Unfortunately like all good things, lentivirus vectorshave properties, which can constrain their application(Pfeifer 2004; Whitelaw et al. 2004). Perhaps, the mostsignificant concern is the reduced ‘cargo’ capacity. Thesevectors can only carry small transgenes (up to 8 kb),although in practice, transgenes bigger than 6 kb oftendisplay poor packaging efficiency and low vector titres.Second, the vector integrates as a single copy butmultiple integrations can occur. This leads to segregationissues in subsequent generations. Yet, since founderanimals carry different copies of the transgene, this canbe an advantage for founder population studies, offeringa dose–response to transgene activity.Integration is random, as in the pronuclear injectionmethod. This can result in position-effect phenomenonthat is often associated with pronuclear injection.Position-effects can result in aberrant or ectropic transgeneactivity, and poorly expressing or silent transgeneloci (Wilson et al. 1990). In our experience, the authorshave rarely seen these inappropriate transgene expressionprofiles, although others have (Hofmann et al.2006). The authors have seen expression in 4-year-oldpigs at the same level they displayed as a young pig; insheep, pigs and chickens the vast majority of transgenicanimals that they produced using lentiviral vectorsexpress the transgene; although the authors haveobserved transgenerational reduction in expression(after the 6th generation) in a few lines of transgenemice (the majority of lines do not show silencing), theauthors have yet to see any transgenerational silencingin chickens and pigs. Random integration also raises thepossibility of functional impairment of the host genomeat the site of integration. Lentiviral transgene integrationsites have yet to be mapped in animals but do lieclose to genes in studies on human cells and, although atvery low frequency, can cause gene activation (Themiset al. 2005; Montini et al. 2006). To overcome thislimitation, there is a considerable effort being directed todesign lentivirus vectors that can target transgeneintegration to specific sequences within the genome(Balciunas et al. 2006; Philippe et al. 2006; Clark et al.2007; Lombardo et al. 2007; Shinohara et al. 2007).Finally, it must be remembered that VSV-G pseudotyped(Pfeifer 2004) lentivirus vectors have the potentialÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag