JoVE General
Adam M. McCoy, Michelle L. Collins, Luis A. Ugozzoli
Gene Expression Division, Bio-Rad Laboratories, Inc.
Denna procedur visar hur du använder Gene Pulser MXcell elektroporation systemet för att snabbt och enkelt identifiera de bästa elektroporation förutsättningarna för mus embryonala fibroblaster (MEFs) eller andra primära celler. Överväganden för felsökning diskuteras också i den tillhörande video.
JoVE General
Adam M. McCoy, Claudia Litterst, Michelle L. Collins, Luis A. Ugozzoli
Gene Expression Division, Bio-Rad Laboratories
Denna procedur beskriver ett snabbt och enkelt arbetsflöde för att införa siRNA in svårt att transfektera cellinjer och följa genuttrycket realtids-PCR. Användning av en automatiserad celltalsräknare, med flera bra elektroporation platta och automatiserad elektrofores stationen ger snabba och tillförlitliga resultat utan behov av dyra robotiserade hantering.
Proceedings of the National Academy of Sciences of the United States of America. Sep, 2010 | Pubmed ID: 20736348
With malaria parasites (Plasmodium spp.), Toxoplasma, and many other species of medical and veterinary importance its iconic representatives, the protistan phylum Apicomplexa has long been defined as a group composed entirely of parasites and pathogens. We present here a report of a beneficial apicomplexan: the mutualistic marine endosymbiont Nephromyces. For more than a century, the peculiar structural and developmental features of Nephromyces, and its unusual habitat, have thwarted characterization of the phylogenetic affinities of this eukaryotic microbe. Using short-subunit ribosomal DNA (SSU rDNA) sequences as key evidence, with sequence identity confirmed by fluorescence in situ hybridization (FISH), we show that Nephromyces, originally classified as a chytrid fungus, is actually an apicomplexan. Inferences from rDNA data are further supported by the several apicomplexan-like structural features in Nephromyces, including especially the strong resemblance of Nephromyces infective stages to apicomplexan sporozoites. The striking emergence of the mutualistic Nephromyces from a quintessentially parasitic clade accentuates the promise of this organism, and the three-partner symbiosis of which it is a part, as a model for probing the factors underlying the evolution of mutualism, pathogenicity, and infectious disease.