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Context: Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry. Methods: In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption. Key results: Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance. Conclusions: Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress. Implications: The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions. With warming climates, grazing cattle are becoming increasingly heat stressed. Embryo-mediated genome editing provides a strategy to rapidly adapt cattle. It was successfully applied to introduce a mutation in the PMEL gene, known to lighten coat colour, which reduced thermal energy absorption and might help to reduce heat stress impacts. Photograph by G. Laible. [ABSTRACT FROM AUTHOR]

We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from nuclear transfer of an identical nuclear genetic background (mural granulosa donor cells including surrounding cytoplasm) into enucleated oocytes with either Bos indicus or B. taurus mtDNA. Here we report the first cases of coexisting mtDNAs of two closely related subspecies following nuclear transfer. Heteroplasmy (0.6-2.8%) was found in 4 out of 11 cross-subspecies cloned cattle. Quantitation was performed using 'amplification refractory mutation system (ARMS) allele-specific real-time PCR.' We determined that the ratio of donor cell to recipient cytoplast mtDNA copy number was 0.9% before nuclear transfer. Therefore, we concluded that the percentage of donor cell mtDNA in the heteroplasmic intersubspecific cloned animals is in accordance with neutral transmission of donor mtDNA. We determined an amino acid sequence divergence of up to 1.3% for the two subspecies-specific mtDNA haplotypes. In addition, intrasubspecific B. indicus heteroplasmy of ~1% (but up to 7.3 and 12.7% in muscle and follicular cells of one animal) was detected in 7 out of the 9 B. indicus intrasubspecific cloned cattle.

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost‐effective, flexible, large‐scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti‐cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10 g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat‐produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody‐dependent cell‐dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large‐scale production of therapeutic mAbs. [ABSTRACT FROM AUTHOR]

Pervoe svidanie (First Meeting) is Belyi's 1921 long poem, which has often been praised as one of his most successful and accessible works in verse. Two particular elements- the Wagnerian concept of leitmotiv and the ideas of Rudolf Steiner are examined, and they discuss the symbolic structure of the poem and an analysis of how they combine with Belyi's thematic aims further, is also done.

Somatic cell nuclear transfer (SCNT), commonly referred to as cloning, results in the generation of offspring that, except for mitochondrial DNA, are genetically identical to the nuclear donor. We previously used a genetically modified bovine cell line as the donor for SCNT and obtained a calf, named Daisy, that was born without a tail. To determine whether the missing tail was a result of the genetic modification, we performed recloning experiments by using either cells from a sacrificed pregnancy of a second clone (Daisy's 'twin' clone) or cells from tailless Daisy as donors for SCNT. Cloned fetuses from aborted pregnancies and a cloned live calf that died shortly after birth were examined and confirmed to all possess tails. Hence, the observed phenotype of Daisy's lacking tail is not due to the introduced transgene or a mutation present in the cell that was used for her production. Rather, the missing tail has most likely arisen from an epigenetic reprogramming error during development. [ABSTRACT FROM AUTHOR]

A Companion to the History of the Book Simon Eliot and Jonathan Rose (eds) Malden MA Wiley-Blackwell 2009 599p ISBN 9781405192781 (pbk) AUD$64.95 The 'history of the book' as currently [...]

Transferred illusions: digital technology and the forms of print Marilyn Deegan and Kathryn Sutherland Farnham Surrey Ashgate 2009 xi 204pp ISBN 9780754670162 55.00 [pounds sterling] By now it is not [...]

Public perception of somatic cell nuclear transfer (SCNT) in the production of agricultural animals is surrounded by fear, which is exacerbated by the inability to differentiate animals generated by SCNT from those generated by natural mating or artificial insemination (AI). Unfortunately, the DNA sequence of animals produced by SCNT is indistinguishable from those generated by fertilization. With the current banning of all SCNT animal products from entering the food supply in some countries, the lack of a diagnostic test to identify SCNT animals may jeopardize market access for producers. The aim of this research was to exploit differences in epigenetic reprogramming that occur during SCNT and fertilization in the early embryo. The resulting differences in epigenetic signatures that persist to adulthood are proposed as the basis for a diagnostic test to identify animals generated by SCNT. Here we describe differences in DNA methylation at eight CpG sites in the retrotransposon-like 1 ( Rtl1) promoter region in cattle blood and test whether these differences could be used as a diagnostic tool. For a definitive diagnosis, it is critical that no overlap in DNA methylation levels is observed between individuals produced by SCNT and fertilization. This was the case for the cohort of SCNT animals studied, their female half-siblings generated by AI, and a collection of unrelated cows also generated by AI. Further rigorous testing is required to determine what effects donor cell type, age, sex, genetic background, SCNT methods, and the environment have on the DNA methylation across this region, but the Rtl1 promoter is currently a promising candidate for the identification of SCNT generated cattle. [ABSTRACT FROM AUTHOR]

The Philosophy of Information editor Ken Herold Urbana-Champaign Ill University of Illinois Graduate School of Library and Information Science 2004 (Special Issue of Library Trends vol 45 no3 pp373-670) This [...]

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies. [ABSTRACT FROM AUTHOR]

One of the features of the early Russian Symbolist movement in the 189os is its appropriation of literary models previously championed by the civic tradition which preceded it, and to which it was both philosophically and aesthetically opposed. One example can be found in treatments of the English poet Percy Bysshe Shelley. This article compares civic and Symbolist translations of Shelley's sonnet 'Ozymandias', showing that the same material could be used to support radically different views, and that the literary world of the period was a particularly fluid space in which multiple overlapping trends competed for the attention of readers. [ABSTRACT FROM AUTHOR]

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low. [ABSTRACT FROM AUTHOR]

Milk from dairy cows contains the protein ß-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA-me-diated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits. [ABSTRACT FROM AUTHOR]

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine b-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences. [ABSTRACT FROM AUTHOR]

The cloning of animals by somatic cell nuclear transfer (SCNT) has the potential to allow rapid dissemination of desirable traits from elite animals. However, concern has been expressed that aberrant epigenetic marks in SCNT-derived animals may be passed onto the next generation, even though the offspring of clones appear to be mainly normal. Here, we compared the DNA methylation patterns at 10 genomic regions in sperm from SCNT bulls with that from normal, naturally conceived bulls and with the nuclear donor somatic cells. Eight of the 10 genomic regions were differentially methylated in sperm compared with the donor cell DNA. All three satellite sequences examined here were less methylated in sperm than in the donor cells, contradicting the belief that the sperm genome is always highly methylated. The DNA methylation patterns at all 10 regions were almost identical between SCNT and control sperm, with only one out of the 175 CpG sites/groups of sites examined showing significant difference. These results provide the first molecular evidence that the donor cell genome is correctly reprogrammed upon passage through the germ line in males, and that any epigenetic aberrations harbored by SCNT bulls are unlikely to be passed onto their offspring. [ABSTRACT FROM AUTHOR]

The article explores the engagement of Australian poet A. D. Hope with Russian literature and argues for its centrality in Hope's thinking in the last three decades of his poetry. The author discusses Hope's essays, epistolary correspondences, and the notebooks archived at the National Library of Australia to trace Hope's familiarity with the Russian language, his translation projects, and the Russian essays Hope includes in his book "The New Cratylus: Notes on the Craft of Poetry."

The article discusses the development of Russian symbolism in literature with a focus on the influence of poet Dmitrii Merezhkovsky. The poet's book of verse "Simvoly" or "Symbols" which was published in 1892 is discussed. Topics include a mystical or religious slant on idealism in literary symbols and the continuity of thematic motifs in translations. Merezhkovsky's poetic technique in three collections of verse, "Simvoly," Stikhotvoreniia," and "Novye Stikhotvoreniia," is compared. His lectures "O prichinakh upadka i o novykh techeniiakh v sovremennoi russkoi literature" or "The Reasons for the Decline and the New Currents in Contemporary Russian Literature" are mentioned.

This article discusses the symbolic role played by food and drink in the Russian discovery of Japan. The Russian discovery of Japanese food culture in the eighteenth, nineteenth and early twentieth centuries illustrates the various points of conflict and convergence in two distinct cultural systems. An analysis of material drawn from memoirs written by Russians who visited Japan between the eighteenth century and the First World War is presented. The major themes that emerge from the Russian narratives in relation to food and hospitality can be broadly designated economic, political, cultural and aesthetic.

Examines the developments in the transgenic experimentation of cattle. Use of transgenesis in livestock to aid sustainable agriculture; Biomedical applications of transgenic farm animals; Details of performing cell-mediated transgenesis through nuclear transfer.