Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

Kuroiwa, Yoshimi; Kasinathan, Poothappillai; Matsushita, Haruo; Sathiyaselan, Janaki; Sullivan, Eddie; Kakitani, Makoto; Tomizuka, Kazuma; Ishida, Isao; Robl, James M.

doi:10.1038/ng1373

Cited by 162 publications

(124 citation statements)

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“…Wild-type calves served as controls. For mRNA expression analysis, we performed RT-PCR 6 (primer pairs: PrPmF3 × PrPmR3, Fig. 1c) and confirmed the disruption of PRNP-specific mRNA expression in PRNP −/− calves.…”

mentioning

confidence: 72%

“…Cloned fetuses and calves were produced using the chromatin transfer procedure as described 6 . In vitro embryo development rate is provided in Supplementary Table 5.…”

Section: Embryonic Cloningmentioning

confidence: 99%

“…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 …”

Section: Introductionmentioning

confidence: 99%

“…Negative PCR analysis 6 was also carried out to confirm the absence of wild-type PRNP alleles (primer pairs: BPrPex3F × BPrPex3R, Fig. 1b).…”

mentioning

confidence: 99%

“…The cerebellar symptoms in these Prnp −/− mouse strains were suggested to be caused by ectopic expression of Dpl protein in brain 28 . In the PRNP −/− cattle described here, only the ORF of the bovine PRNP gene was disrupted by insertion of the neo and puro cassettes without any deletion of PRNP genomic sequences, so that any splicing donor/acceptor sites remain intact 6 . It remains to be determined whether or not the bovine PRND locus might be affected by the disruption of PRNP, but if it occurs, its effect would appear to be minimal because obvious abnormalities, such as ataxia and Purkinje cell loss, were not found in PRNP −/− cattle up to 20 months of age.…”

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confidence: 99%

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Production of cattle lacking prion protein

et al. 2006

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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...

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confidence: 72%

“…Cloned fetuses and calves were produced using the chromatin transfer procedure as described 6 . In vitro embryo development rate is provided in Supplementary Table 5.…”

Section: Embryonic Cloningmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Negative PCR analysis 6 was also carried out to confirm the absence of wild-type PRNP alleles (primer pairs: BPrPex3F × BPrPex3R, Fig. 1b).…”

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confidence: 99%

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Gene Targeting by Homologous Recombination

Reh

Vásquez

2014

Encyclopedia of Life Sciences

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Gene targeting by homologous recombination involves the exchange of genetic information between genomic and exogenous deoxyribonucleic acid (DNA) molecules via crossover events. These exchanges are guided by homologous sequences acted on by enzymatic machinery of the cell. Homologous recombination provides a mechanism for targeting defined modifications into genes of interest, making gene‐targeting technologies valuable tools to explore gene function and to develop human, genetic disease models. Gene targeting, however, is inefficient, making the process challenging. Advances in technology now allow us to direct repair enzymes to the targeted site by inducing site‐specific DNA damage using zinc‐finger nucleases, transcription activator‐like effector nucleases, clustered regularly interspaced short palindromic repeats and triplex‐forming oligonucleotides. Further, antirecombinogenic pathways can now be transiently suppressed using ribonucleic acid (RNA) interference (RNAi) and other small molecule approaches. These and other techniques can greatly enhance gene‐targeting efficiency. The coincident development of human stem cell technology brings forth the potential of gene‐targeting strategies for therapeutic application. Key Concepts: Homology‐directed gene targeting utilises homologous recombination to introduce defined modifications into sequences of interest in mammalian genomes. Gene‐targeted knockout and knock‐in models are instrumental in elucidating gene function and studying human genetic diseases. Combining stem cell and gene‐targeting technologies opens potential avenues for gene therapy. Recent technological advances have greatly enhanced gene‐targeting efficiencies. Due to technological advances, most genes in a variety of mammalian species can now be manipulated by homology‐directed gene targeting.

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Monoclonal Antibodies, Human, Engineered

Helguera

Daniels

Rodríguez

et al. 2009

Encyclopedia of Industrial Biotechnology

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Antibodies are unique proteins of the immune system that exhibit high affinity and specificity, which have emerged as powerful molecular tools with a broad range of applications in research, diagnosis, and treatment of diseases. Since the 1970s, the hybridoma technology allowed the large‐scale generation of rodent monoclonal antibodies with selectivity for a single antigen. However, the therapeutic efficacy of these antibodies was disappointing due to inefficient activation of the human effector functions, short half‐life, and elevated immunogenicity that in some cases resulted in severe allergic reactions. The recombinant DNA technology allowed the engineering of a new generation of therapeutic antibodies by replacement of the murine components of antibodies with their human counterparts. This process started with the generation of mouse/human chimeric antibodies, followed by humanized antibodies, and finally fully human antibodies, each step significantly diminishing the immunogenicity of the antibodies. Importantly, the presence of the human fragment crystallizable (Fc) region in these antibodies makes them more effective in patients. These improvements resulted in a class of drugs that exhibit high specificity and functionality with less, if any, immunogenicity and toxicity, emerging as the fastest growing class of biological therapeutics. A rapidly growing number of human‐engineered antibodies are being used as therapeutic agents alone or in combination with other therapeutic interventions for the treatment of a wide variety of diseases that include cancer, transplant rejection, allergy, and autoimmune disorders. Further, modifications in the structure of antibodies that modulate or add new biological properties are broadening the range of their clinical applications.

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Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

Cited by 162 publications

References 26 publications

Production of cattle lacking prion protein

Production of cattle lacking prion protein

Gene Targeting by Homologous Recombination

Monoclonal Antibodies, Human, Engineered

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