Production of Prnp −/− goats by gene targeting in adult fibroblasts

Abstract

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines

“…It is also supported by their growth pattern (Fig. 2), and thus these cells can be potentially used for gene-targeting in goats (Keefer et al 2001;Zhu et al 2009). As seen in Fig.…”

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

“…Moreover, long-range chromosomal rearrangements, including deletions, inversions and translocations can be accomplished by nuclease-induced DSBs. The introduction of SCNT opened the possibility of conventional HR-based gene targeting in somatic cells of livestock [194,195], an approach that has been inapplicable before because of the lack of germline competent stem cells in these species [196][197][198][199]. However, the extremely low rate of HR in somatic cells (10-to 100-fold lower than that in murine ES cells) [200][201][202], along with the inherent inefficiency of the SCNT technique [203] made this approach cumbersome and tedious.…”

Five Questions on Prion Diseases

“…In 2007, viable PRNP knockout cattle were obtained by sequential gene targeting in somatic cells and nuclear transfer. Targeted disruption of PRNP in goats, which frequently suffer from the prototypic prion disease, scrapie, was accomplished through a similar strategy [28], [29].…”