MATRIZ CONCEPTUAL LECURA: Sociedades Americanas (1842)

In the last years the Chair for Molecular Animal Breeding and Biotechnology was able to generate different transgenic pigs using several types of porcine primary cells (PKCs, PFFs, and PEFs) transfected with the nucleofector technology.

Transfection for additive gene transfer or homologous recombination has been done with conventional DNA plasmids that were linearized to remove the backbone which was important for propagation in bacteria. In BAC vectors the risk to achieve undesirable DNA molecules generated by random linearization within the BAC construct prior to the integration is reduced (Giraldo and Montoliu 2001).

In additive gene transfer experiments, litter rates between 41% and 50% were achieved using transgenic PKCs and PFFs, respectively, after the first transfection as nuclear donor cells. In opposite, Hyun et al. (2003) achieved litter rates between 6.7% and 7.7% using untransfected and genetically modified PFFs for SCNT. The cells of several cloned transgenic animals were used for a second transfection. Here, the litter rates were highly different between PKC (52.6%) and PEF (20%) as donor cells, taking into account that only one transfected PEF line was used for SCNT and ET, whereas five different PKC populations were used. As expected, the obtained efficiency after first and second transfection of PKCs as donor cells was similar. In recloning experiments, a similar trend was observed in obtained litter rates using PKCs (45.2%), PFFs (33.3%) and PEFs (26.7%) as donor cells, which is comparable to the in vitro development competence of embryos using several donor cells. In addition, also a low efficiency was obtained by cloning of untransfected PFFs with 21%, whereas no litter was achieved after two ETs using PSF. Lai and Prather (2003) got similar cloning success rates between 20-30% using untransfected PFFs. Certainly, there were differences in achieved litter rates using transfected (50%), recloned (33.3%) and cloned PFFs (21%). This could belong to different stages of development and quality of fetuses. In recloning and cloning experiments, cells of animals were used which were already generated by SCNT.

DISCUSSION

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Classical HR is a widely used method for site-directed modifications in the mammalian genome, certainly, the principal limitation of this classical strategy is the low targeting efficiency (reviewed in Vasquez et al. 2001). In mice HR in somatic cells is less efficient than in ES cells (Arbones et al. 1994; reviewed in Wang and Zhou 2003). Due to the fact, that no true ES cells are available in pigs, gene targeting of somatic cells results in generation of a high number of cell clones and is laborious and costly.

Rogers et al. (2008b) verified that homologous recombination depends on donor cells. They targeted fibroblasts from several fetuses obtained from the same uterus at the same time. After targeting which was done by the same people and with same reagents, they achieved targeting frequencies between 0.07 to 10.93% (Rogers et al. 2008b). Using PFFs, we were not able to knock-out a gene on cell culture level, probably, due to their reduced growth potential, resulting in low number of cell clones after transfection and selection. On the other side, four different loci could be targeted using PKCs. SCNT with these successful targeted PKC cell clones resulted in cloning efficiencies from 37-50%.

Overall, these data prove that primary PKC as well as PFFs are highly sufficient for generation of genetically modified pigs by additive gene transfer. PEFs as donor cells were unsuitable for SCNT, because of low efficiency of obtained litter rates. Only with PKCs site-directed mutagenesis experiments were successfully.

5.5

Outlook

During this thesis, several donor cell sources were characterized and compared for generation of transgenic pigs using SCNT. This is a very important point, but merely one way to increase the efficiency of generating transgenic piglets. In future projects, the focus could be on manipulation of the reprogramming processes of epigenetic modifications, because this is a crucial part for dedifferentiation of donor nuclei during SCNT. It is presumed that the low cloning efficiency is largely attributable to the incomplete and faulty reprogramming of epigenetic modifications (Bourc'his et al. 2001; Xue et al. 2002; Enright et al. 2003). There are several ways to influence epigenetic modifications, for instance the chemical treatment of donor cells, oocytes as well as embryos for more efficient epigenetic reprogramming. In pig several

DISCUSSION

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studies showed higher blastocyst rate after treatment with histone deacetylase inhibitor Trichostatin A of in vitro cultivated porcine embryos and in SCNT embryos using PFFs as donor cells (Jeseta et al. 2008; Zhao et al. 2009; Himaki et al. 2010). Moreover, Kong et al. (2011) demonstrated the reactivation of silenced transgene after treatment with DNA methyltransferase inhibitor 5-Aza-2´-deoxycytidine and/or with Trichostatin A in PFFs.

Another possibility would be the improvement of the selection system for side-directed mutagenesis. The used BAC-vectors which were applied for site- directed mutagenesis contain a positive selection cassette such as blasticidin or geneticin resistance gene. The combination of a positive-negative-selection (PNS) strategy could increase the efficiency of obtained targeted clones. Cells are selected on both integration of the targeting vector due to positive selection and on the HR event as well as the resulting loss of the negative selection cassette. In murine ES cells 79% targeting efficiency was achieved with PNS strategy (Mansour et al. 1988) and 30% targeting efficiency was obtained in a rat fibroblasts (Hanson and Sedivy 1995).

SUMMARY

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6

S

Genetic modification of primary cells for the generation of