Infection efficiency is the key issue for gene delivery using adenovirus vector and usually unsatisfactory. In this study, recombinant adenoviruses encoding recombinant human EPO were prepared using the Adeasy system, and injected into the mammary gland of goats via the teat canal. BAPTA was used to treat the mammary gland to facilitate adenoviruses infection compared with EGTA. Milk serum was collected from the infected mammary gland and characterized by ELISAs and Western blotting. Expression level of rhEPO from the group treated by BAPTA was higher than that treated by EGTA.
[Show abstract][Hide abstract] ABSTRACT: Heterogenous expression of recombinant proteins in milk of livestock at a large scale is very labour-intensive to be achieved with current transgenic animals, and usually seen as time-consuming, expensive and technically most challenging. Here we describe a convenient system for transient production of recombinant human growth hormone and its extensive use in recombinant protein production for therapeutic purposes. In this study, an adenoviral vector containing the GFP gene and hGH gene was constructed for direct infusion into the epithelium of mammary glands of goats via the teat canal during the period of natural lactation. Western-blot analysis of milk samples obtained from all of the viral-treated founders indicated that the recombinant hGH (rhGH) was secreted into the milk of the goats. The concentrations of rhGH in milk ranged from 0.6 to 2.4 mg/ml and lasted for more than 10 days during lactation. These data suggest that it is possible to produce larger amounts of recombinant human growth hormone in the milk of livestock animals by using replication-defective adenoviruses.
[Show abstract][Hide abstract] ABSTRACT: This review provides an update of the genetic content, phylogeny and evolution of the family Adenoviridae. An appraisal of the condition of adenovirus genomics highlights the need to ensure that public sequence information is interpreted accurately. To this end, all complete genome sequences available have been reannotated. Adenoviruses fall into four recognized genera, plus possibly a fifth, which have apparently evolved with their vertebrate hosts, but have also engaged in a number of interspecies transmission events. Genes inherited by all modern adenoviruses from their common ancestor are located centrally in the genome and are involved in replication and packaging of viral DNA and formation and structure of the virion. Additional niche-specific genes have accumulated in each lineage, mostly near the genome termini. Capture and duplication of genes in the setting of a 'leader-exon structure', which results from widespread use of splicing, appear to have been central to adenovirus evolution. The antiquity of the pre-vertebrate lineages that ultimately gave rise to the Adenoviridae is illustrated by morphological similarities between adenoviruses and bacteriophages, and by use of a protein-primed DNA replication strategy by adenoviruses, certain bacteria and bacteriophages, and linear plasmids of fungi and plants.
Journal of General Virology 12/2003; 84(Pt 11):2895-908. DOI:10.1099/vir.0.19497-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.
Human Gene Therapy 04/2001; 12(5):455-67. DOI:10.1089/104303401300042348 · 3.76 Impact Factor
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