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...

α-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca 2⍣ -dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca 2⍣ -dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca 2⍣ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca 2⍣ and is blocked by drugs depleting Ca 2⍣ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca 2⍣ -independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca 2⍣ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTXstimulated exocytosis depends upon the co-operative action of external and intracellular Ca 2⍣ involving G proteins and PLC, whereas the Ca 2⍣ -independent release is largely non-vesicular.

Calcitonin gene-related peptide (CGRP) is thought to be a prominent neuropeptide in cardiovascular regulation and neuroimmune modulation. There are two isoforms of CGRP (␣CGRP and ␤CGRP), and the main CGRP receptors are probably composed of a calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP)1. However, the physiological functions of CGRP that are mediated through the CLR/RAMP1 receptors remain to be clarified. For an improved understanding of the functions, we generated mice deficient in RAMP1, a specific subunit of CGRP receptors, by a conditional gene-targeting technique. The RAMP1-deficient mice (RAMP1 ؊/؊ ) exhibited high blood pressure, with no changes in heart rate. ␣CGRP was found to have a potent vascular relaxant activity compared with ␤CGRP in the artery of the WT (RAMP1 ؉/؉ ) mice. The activities of both CGRP isoforms were remarkably suppressed in the arteries of the RAMP1 ؊/؊ mice. The LPS-induced inflammatory responses of the RAMP1 ؊/؊ mice revealed a transient and significant increase in the serum CGRP levels and high serum levels of proinflammatory cytokines compared with the RAMP1 ؉/؉ mice. ␣CGRP and ␤CGRP equally suppressed the production of TNF-␣ and IL-12 in bone marrow-derived dendritic cells stimulated with lipopolysaccharide. Their inhibitory effects were not observed in the bone marrow-derived dendritic cells of the RAMP1 ؊/؊ mice. These results indicate that CGRP signaling through CLR/RAMP1 receptors plays a crucial role in the regulation of both blood pressure by vascular relaxation and proinflammatory cytokine production from dendritic cells.gene-disrupted mice ͉ calcitonin gene-related peptide ͉ adrenomedullin ͉ neuropeptide ͉ dendritic cells C alcitonin gene-related peptide (CGRP) is a 37-aa neuropeptide that is produced in the neural body of dorsal root ganglion cells and released from sensory nerve endings. There are two isoforms of CGRP: ␣ and ␤ in rats and mice and I and II in humans. These differ in their peptide sequences by 1 aa (rats) and 3 aa (mice and humans) of the 37 aa (1). ␣CGRP is produced mainly in the nervous system by the tissue-specific alternative splicing of the primary RNA transcript of the calcitonin/CGRP gene. On the other hand, ␤CGRP is produced not only in the neuronal tissues but also in the enteric nerves of the intestine (2) and in immune cells such as T cells (3). Pharmacologically, ␣CGRP is known to have the most potent vasodilatory activity (4). Most blood vessels are surrounded by a dense perivascular CGRPergic neural network, suggesting the physiological importance of CGRP in vasodilatory regulation (5). ␣CGRP also contributes to local neurogenic inflammation and nociception (6). Moreover, the functions of immune cells such as macrophages (7,8), Langerhans cells (8), and T cells (9, 10) are modulated by ␣CGRP. However, the precise functional differences between ␣CGRP and ␤CGRP remain unclear.Historically, CGRP receptors have been classified into two classes: CGRP 1 and CGRP 2 receptors. The CGRP 1 receptors are m...

Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.

Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-µ (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.