RESUMO
Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.
Assuntos
Culicidae/virologia , Densovirinae/fisiologia , Proteínas não Estruturais Virais/fisiologia , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Núcleo Celular/química , Citoplasma/química , Densovirinae/genética , Deleção de Genes , Viabilidade Microbiana/genética , Microscopia de Fluorescência , Fatores de Tempo , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Replicação Viral/genéticaRESUMO
Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80 degrees C while attached to the substratum of 25 cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.
RESUMO
Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K), we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.
