In this study, we successfully produced a somatically cloned transgenic piglet using recombinant cells obtained after gene transfer of a transgene (carrying both EGFP and pac expression units) and subsequent in vitro selection with a low concentration (2 ^g/ml) of puromycin. In contrast, no piglets were born when the conventional method, in which a period of 7 days is used for the selection of transfected cells and a further additional 7 days for the growth of cells before nuclear transfer, was applied in an attempt to produce transgenic cloned piglets (Table 1). In fact, during culture, the growth ability of cultured cells decreased and the morphology of the cells changed in this study (data not shown). Thus, we attempted to shorten the period of time used to culture isolated fetal porcine cells as much as possible to eliminate possible changes in cell potentiality. These attempts included two main strategies: i) gene delivery immediately after isolation of fetal somatic cells and ii) short-term selection (within 7 days of transfection) of transfected cells in the presence of 2 ^g/ml of puromycin. To our knowledge, the method we have described here appears to be the first report using another selection drug other than G418 for the production of transgenic somatically cloned animals. buy zyrtec online

A long-term culture of primary cultured cells has been thought to cause cellular aging that leads to a reduction in the proliferative activity of cells. Nuclei of aged cells have been associated with reductions in developmental potentiality in somatic cell cloning systems. In this context, our present strategy, employing direct gene delivery to freshly isolated cells and short-term selection of transfected cells in the presence of puromycin, would clearly overcome these earlier problems. To our knowledge, there are very few reports concerning the successful gene delivery to cells freshly isolated from fetal tissue. This may be due to the widely accepted concept for a transfection protocol that exogenous DNA is easily introduced to rapidly dividing cells. However, we observed a highly efficient gene transfer rate (1/50) into isolated fetal cells using an electroporation method. The method of electroporation-mediated gene transfer to freshly isolated (but not cultured) cells reduces time and cost and appears to be a powerful tool in various fields related to gene delivery to cells. Furthermore, employment of puromycin as a means of drug selection appears to be of importance for the systematic production of transgenic somatically cloned animals. This is solely because puromycin is effective at very low concentrations (>2 ^g/ml) (see Fig. 2), even in slowly dividing cells such as cells from newborn piglets. If puromycin is added to more actively dividing cells, which synthesize proteins actively, it appears to block the growth of cells at concentrations lower than 2 ^g/ml. The culture period required for obtaining stable transfectants after gene transfer and selection with puromycin is only 7 days. This appears also to be important in shortening the culture period because selection of G418, which is most widely used as a selection drug, requires at least 10 days to obtain stable transfectants. Because the selection period of somatic cells influences success rate in the somatic cloning system , we believe that puromycin is one of the best selection drugs to be reported thus far. Puromycin and G418 each work in an independent manner in mice ES cells. If this is also true in pigs, then it follows that puromycin and G418 may be used as selection drugs to obtain doubleknockout somatic cells in which a certain target locus is disrupted by insertion of the neocontaining gene construct and the corresponding allele by the pac-containing gene construct. Double-knockout cells would allow us to produce null-mutant pigs at founder level using the somatic cell cloning technique. Recently, Sharma et al. reported a method for the successful production of double-knockout somatic cells. Briefly, they disrupted both alleles of the alpha 1,3-galactosyltransferase gene (1,3-GTase) in cultured somatic cells using a conventional gene targeting technique and produced somatic cell-derived cloned fetuses using the targeted cells. After the resulting fetuses were dissected out, primary-cultured fibroblastic cells were propagated in vitro and treated with anti-GTase monoclonal antibody to eliminate cells expressing GTase and concentrate doubleknockout recombinant cells. However, the efficiency of this method (which is called mitotic recombination) to obtain double-knockout cells was very low. We believe that simultaneous use of both G418 and puromycin may greatly enhance the production rate of double-knockout somatic cells. Interestingly, Taniguchi et al. demonstrated that puromycin is useful as a dominant marker for Cre recombinase expression in ES cells in the conditional gene targeting system. This indicates the potential usefulness of puromycin within the conditional gene targeting (and probably other fields related to gene engineering), as well as in somatic cloning system.

In the present study, we observed that most of our newborn piglets died postnatally. The reason for this high mortality is still unclear because histological and anatomical abnormality could not be found in these dead piglets. At present, there is little knowledge about the effect of puro-mycin N-acethyltransferase to early development of somatic cell clones in pigs. However, we believe that pac gene expression or puromycin selection itself is not deleterious to cell function because the EGFPac transfectants exhibited normal cell morphology, which was indistinguishable from that of untransfected cells, and exhibited EGFP without change in fluorescent strength. There is, however, a possibility that EGFP protein expressed in fetal piglets after nuclear transfer may be deleterious to postnatal development. Arat et al. reported detrimental effects of the GFP protein during the somatic cloning procedure in cattle. These authors report that donor cells expressing GFP protein had reduced developmental ability when compared with nontransfected ones after nuclear transfer. These results indicated the harm that GFP protein may cause during early embryo development. Alternatively, abnormal genomic imprinting or epigenetic alteration in host chromosomal DNA, which probably generates during the nuclear transfer procedure, may result in the postnatal death of cloned piglets. Further research will be needed to make this point clear.

We possess a living transgenic cloned piglet (L15-112), aged 10 mo; he has no behavioral or metabolic abnormality at the present time. We are planning to obtain F1 offspring from L15-112, and using these offspring, we plan to address those issues mentioned above in more detail.

In conclusion, puromycin is useful as an effective drug for the selection of recombinant cells that will be used as donor cells in the production of somatically cloned gene engineered animals. Furthermore, we demonstrated that the direct gene transfer to freshly isolated porcine fetal cells is possible using electroporation. This method does not require short-term cultivation of cells which is always needed for the standard gene transfer protocol targeted to primary-cultured cells, and would accelerate production speed for transgenic somatic cell clone piglets by nuclear transfer. At present, however, this method is neither rapid nor convenient but does provide a novel means of producing transgenic farm animals using cloning technology. Moreover, the present study provides the foundation for the development of further methods.