Improving the long-term storage of a mammalian biosensor cell line via genetic engineering.

The unique properties of mammalian cells make them valuable for a variety of applications in medicine, industry, and diagnostics. However, the utility of such cells is restricted due to the difficulty in storing them non-frozen for an extended time and still maintaining their stability and responsiveness. In order to extend the active life span of a mammalian biosensor cell line at room and refrigerated temperatures, we have over expressed genes that are reported to provide protection from apoptosis, stress, or oxidation. We demonstrated that over expression of genes from the extremophile, Artemia franciscana, as well as GADD45beta, extends room-temperature storage of fully active cells 3.5-fold, while over production of several anti-apoptotic proteins extended 4 degrees C storage 2- to 3-fold. Methodologies like these that improve the stability of mammalian-cell-based technologies in the absence of freezers may enable widespread use of these tools in applications that have been considered impractical based solely on limited storage characteristics.

UV irradiation and desiccation modulate the three-dimensional extracellular matrix of Nostoc commune (Cyanobacteria).

Cyanobacterium Nostoc commune can tolerate the simultaneous stresses of desiccation, UV irradiation, and oxidation. Acidic WspA, of approximately 33.6 kDa, is secreted to the three-dimensional extracellular matrix and accounts for greater than 70% of the total soluble protein. The wspA gene of N. commune strain DRH1 was cloned and found in a single genomic copy, in a monocistronic operon. Transcription of wspA and sodF (superoxide dismutase), and synthesis and secretion of WspA, were induced upon desiccation or UV-A/B irradiation of cells. Recombinant WspA binds the UV-A/B absorbing pigment scytonemin through non-covalent interactions. WspA peptide polymorphism, and heterogeneity of multiple wspA sequences within cells of a single colony, account for distinct WspA isoforms. WspA has no similarity to entries in the sequence databases and wspA, a possible xenolog, is restricted to a subset of strains in the "form species" N. commune characterized through group I intron phylogeny. We hypothesize that WspA plays a central role in the global stress response of N. commune through modulation of the structure and function of the three-dimensional extracellular matrix, particularly the transport, distribution, and/or macromolecular architecture of mycosporine and scytonemin UV-A/B absorbing pigment complexes.

Genomic DNA of Nostoc commune (Cyanobacteria) becomes covalently modified during long-term (decades) desiccation but is protected from oxidative damage and degradation.

Genomic DNA of Nostoc commune (Cyanobacteria) became covalently modified during decades of desiccation. Amplification of gene loci from desiccated cells required pretreatment of DNA with N-phenacylthiazolium bromide, a reagent that cleaves DNA- and protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modified DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of amplification products were obtained with DNA from desiccated/rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible fingerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5'-GWCWATCGCC-3') based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection efficiency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N.commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation.