CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS).

Manual Feeding Device Experiences of People With a Neurodisability.

Objective: Neurological bilateral upper limb weakness can result in self-feeding difficulties and reliance on care providers. Mealtimes become time consuming and frustrating. In this exploratory inquiry, we examined the experiences of users of a feeding device.

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN GS ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells.

Evaluation of homogeneity and genetic stability of REOLYSIN (pelareorep) by complete genome sequencing of reovirus after large scale production.

REOLYSIN (pelareorep) is a proprietary isolate of the reovirus T3D (Type 3 Dearing) strain which is currently being tested in clinical trials as an anticancer therapeutic agent. Reovirus genomes are composed of ten segments of double-stranded ribonucleic acid (RNA) characterized by genome size: large (L1, L2, and L3), medium (M1, M2, and M3), and small (S1, S2, S3, and S4). The objective of this work was to evaluate the homogeneity and genetic stability of REOLYSIN.

Overview of emerging technologies to detect adventitious agents.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA).

The fecal viral flora of California sea lions.

California sea lions are one of the major marine mammal species along the Pacific coast of North America. Sea lions are susceptible to a wide variety of viruses, some of which can be transmitted to or from terrestrial mammals. Using an unbiased viral metagenomic approach, we surveyed the fecal virome in California sea lions of different ages and health statuses.

Ensuring the safety of vaccine cell substrates by massively parallel sequencing of the transcriptome.

Massively parallel, deep, sequencing of the transcriptome coupled with algorithmic analysis to identify adventitious agents (MP-Seq™) is an important adjunct in ensuring the safety of cells used in vaccine production. Such cells may harbour novel viruses whose sequences are unknown or latent viruses that are only expressed following stress to the cells. MP-Seq is an unbiased and comprehensive method to identify such viruses and other adventitious agents without prior knowledge of the nature of those agents.

Massively parallel sequencing: a new tool in virus discovery and vaccine safety.

The introduction of new sequencing technologies is revolutionizing virus discovery and providing a new means to demonstrate the safety of vaccines. Since these methods do not depend on prior assumptions of the types of viruses that may be present, they have detected viruses missed by other methods like degenerate or, family specific, PCRs. We have used massively parallel sequencing (MP-Seq) to detect new viruses in bovine serum and in the faeces of animals.

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine.

Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA.

Massively parallel sequencing, a new method for detecting adventitious agents.

There has been an upsurge of interest in developing new veterinary and human vaccines and, in turn, this has involved the development of new mammalian and insect cell substrates. Excluding adventitious agents from these cells can be problematic, particularly for cells derived from species with limited virological investigation. Massively parallel sequencing is a powerful new method for the identification of viruses and other adventitious agents, without prior knowledge of the nature of the agent.

A vector expressing feline mature IL-18 fused to IL-1beta antagonist protein signal sequence is an effective adjuvant to a DNA vaccine for feline leukaemia virus.

DNA vaccination using vectors expressing the gag/pol and env genes of feline leukaemia virus (FeLV) and plasmids encoding feline interleukin-12 (IL-12) and IL-18 completely protected cats from viraemia following challenge [Hanlon L, Argyle D, Bain D, Nicolson L, Dunham S, Golder MC, et al. Feline leukaemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors. J Virol 2001;75:8424-33].

Characterization of germline porcine endogenous retroviruses from Large White pig.

Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses.

Absence of replication-competent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine.

The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission.

Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature Swine.

The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.

Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria.

E3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants. This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus. Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs.

In vivo analysis of porcine endogenous retrovirus expression in transgenic pigs.

Xenotransplantation offers a potential solution to the shortage of donor organs for allotransplantation. In vitro studies that demonstrate the transmission of porcine endogenous retroviruses (PERV) from porcine cells to human cells and cell lines have raised concerns regarding the potential transmission of PERV to both xenograft recipients and their contacts (1-4). While no evidence of infection has been detected in any patients who have been treated with a variety of different porcine tissues (5-8), two studies have shown that severe combined immunodeficient (SCID) mice can be infected by PERV after the transplantation of porcine islets (9-10).

Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems.

Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species.

Evaluating the oncogenic risk of residual DNA in vaccines: the potential role of transgenic mice.

There is a general consensus that residual DNA from immortalised cells is very unlikely to pose a safety risk for biotechnology products but there is little experimental data to support this contention. Transgenic mice primed with one or more oncogenes or tumour suppressor deletions offer a potentially sensitive and quantitative method for determining the presence of oncogenic DNA. Separate lines of mice transgenic for myc and Cbfa1 may provide a means of detecting oncogenic DNA containing genes belonging to different complementation groups.

Mapping full-length porcine endogenous retroviruses in a large white pig.

Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus.

Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors.

The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-gamma).

Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography.

Characterization of the feline thyroglobulin promoter.

The feline thyroglobulin promoter was identified by a combination of standard polymerase chain reaction (PCR) techniques, using primers designed according to regions of homology in published sequences from other species, then adaptor ligated PCR. A 310 bp fragment of the feline thyroglobulin promoter was generated, including 8 nucleotides of adaptor sequence at the 5' end and, based on the putative transcription start site, 36 nucleotides of the thyroglobulin mRNA (untranslated portion). The homology between the feline promoter sequence (from 193 bp upstream to the putative cap site) and canine, bovine and human sequences was 89%, 81% and 78%, respectively.

Xenotransplantation: an overview of microbiological risks and potentials for risk management.

Xenotransplantation is the use of animal organs, tissues or cells for transplantion into humans to treat a variety of medical conditions. If proven efficacious, the technique could be used as one means of alleviating the disparity between the growing demand for transplantable organs, tissues and cells, and the availability of human-origin transplants world-wide. Just as the practicality and efficacy of the technology need to be investigated, so too does the potential for associated infectious disease risk.

Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice.

Animal donors such as pigs could provide an alternative source of organs for transplantation. However, the promise of xenotransplantation is offset by the possible public health risk of a cross-species infection. All pigs contain several copies of porcine endogenous retroviruses (PERV), and at least three variants of PERV can infect human cell lines in vitro in co-culture, infectivity and pseudotyping experiments.