An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.

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Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP C , such as PrP BSE in bovine spongiform encephalopathy (BSE) in cattle and PrP CJD in Creutzfeldt-Jakob disease (CJD) in humans 1 . Disruption of PrP C expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities [2][3][4][5] . However, the impact of ablating PrP C function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP C -deficient cattle produced by a sequential gene-targeting system 6 . At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification 7 . PrP C -deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.To generate PrP C -deficient (PRNP −/− ) cattle, we transfected a male Holstein primary fetal fibroblast line 6594 with first and second knockout (KO) vectors (pBPrP(H)KOneo and pBPrP (H)KOpuro vectors) 6 to sequentially disrupt the two alleles of PRNP. PRNP −/− fetal cell lines were established at 40-60 d of gestation and three of the PRNP −/− fetal cell lines (5211, 5232 and 4296) were recloned to produce calves (Table 1 and Fig. 1a). To verify that the calves possess the PRNP −/− genotype, we collected ear biopsies and established fibroblast cell lines for genotyping. Genotyping was done by genomic PCR specific to each gene targeting event 6 (primer pairs: neoF7 × neoR7 and puroF14 × puroR14, Fig. 1b COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnology website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Fig. 1c) and confirmed the disruption of PRNP-specific mRNA expression in PRNP −/− calves. For protein expression analysis, we performed PrP-specific western blot analyses on fibroblasts (Fig. 1d), peripheral blood lymphocytes (Fig. 1e) and brain stem (Fig. 1f) from wild-type and PRNP −/− calves using the mouse anti-bovine PrP monoclonal antibody F89. We detected PrP-specific bands in the wild-type calves, whereas no reaction was observed in PRNP −/− calves and negative control mouse fibroblasts. These data clearly demonstrate that the PRNP gene is functionally inactivated in the PRNP −/− calves.PRNP −/− cattle were monitored for growth and general health status from birth to 20 months of age. Mean birth weight was 46 kg and average daily gain was 0.91 kg/d to 10 months. Both values were in the normal range for Holstein bulls. Serum chemistry was evaluated at 6 months of age and compared with published reference ranges. All the values for PRNP −/− calves (n = 12) were well within the reference range (Supplementary Table 1) and obvious abnormalities w...

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)