Hemizygous prion protein gene (PRNP) knockout in cattle fibroblasts

Bovine spongiform encephalopathy (BSE) represents a real threat for human health, as has been demonstrated by the causal link with the variant form of Creutzfeldt-Jakob disease. The aim of our project is to create a bovine strain knockout for the prion protein gene (PRNP) that should be resistant to BSE infection. We combined the use of homologous recombination by PRNP targeting vectors in bovine fibroblasts with the subsequent use of nuclear transfer (NT). We transfected fetal (male) and adult (female) bovine fibroblasts by nucleofection, using targeting vectors disrupting the PRNP by means of loxP flanked cassettes. They expressed resistance to different drugs driven by a PGK or TK promoter and the thymidine kinase gene as a negative selection marker. We screened, by PCR, 907 drug-resistant colonies, from which we identified 8 Neo-resistant colonies with a recombined PRNP allele (overall efficiency 3.2%; 7/108 from fetal, 1/145 from adult; P < 0.5). Fibroblasts PRNP+/– Neo were used to produce NT blastocysts from which neural precursors cell lines were established (Lazzari et al. 2006 Stem Cells 24, 2514–2521). These lines were capable of extensive proliferation (over 120 doublings during 4 months of culture) and provided unlimited material for Southern blot analysis to confirm PCR findings. Three clones (2 from fetal and 1 from adult) were further analyzed and confirmed PRNP+/– by Southern blot and were subsequently used for NT to generate blastocysts for transfer to recipient heifers. On Day +40 of gestation, the pregnancy rate was 33.3% (9/30) for the fetal line and 50% (2/4) for the adult line. One of the fetuses originating from fetal fibroblasts was removed on Day +45 to establish a rejuvenated fibroblast cell line used for a second round of gene targeting to obtain a PRNP –/– clone. We nucleofected these fibroblasts with Puro, Hygro, and promoterless Hygro cassette-carrying targeting vectors. We screened 625 drug-resistant colonies by PCR but none tested positive for the second targeting. In conclusion, we have obtained heterozygous PRNP+/– fibroblasts with the Neo vector both in fetal and adult fibroblasts, but failed with other vectors. In the first targeting, the efficiency was 10 times greater in fetal v. adult fibroblasts. The derivation of neural precursor cell lines from cloned blastocysts is a useful procedure to have sufficient material for molecular analysis without the need of rejuvenating the cell through the production of a fetus. None of the vectors used for the targeting of the second allele was successful.