Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

Social Organization on the Web: How Sex workers Rights Organizations Engage in Activism and Advocacy Work Online

The purpose of this study was to examine how sex workers rights organizations use their websites as a site of activist and advocacy work and ask (i) how do various organizations conceptualize sex work on their websites, and to what extent do they incorporate an intersectional feminist perspective? (ii) what communication strategies are used by the four organizations to target audiences in the viewing public? (iii) what audiences do the four websites target? (iv) how do the four organizations discuss successes and challenges on their websites? (v) in what ways do sex worker right organizations use websites to further their goals? The websites of Maggie’s, POWER, and Stella attempt to embrace an intersectional feminist perspective of sex work, while PACE does not. The four organizations strategically use their websites to target audiences with diverse needs, specifically through advocacy efforts in educating the general public about the legitimacy of sexual labour. Additionally, to increase the use of the websites by sex workers, using social media platforms, such as Facebook and Twitter augment the untapped potential for creating action, mobilization, interaction, and dialogue on the websites.

Letter - E.C. Schmon to Arthur A. Schmon, 6 February 1917

The letter reads:" The sixth of February. Artie dear, I am going to be a real good girl and not make you read a long letter, when your thoughts are so completely occupied with "war". Newark is also greatly excited. All arrangements for the mobilization of the militia forces have been made, and in case of any local uprisings, two hundred policemen are to be added to the force. Of course everyone is thinking the worst at present, but I cannot help but believe that in a few months, people will feel like kicking themselves, for having done an unnecessary amount of worrying. (Let us hope so). If nothing unforeseen occurs, I am going to take the "Congressional limited" to Washington, Wednesday the twenty first. Saw in the paper that the public ceremonies of inauguration are to be eliminated, or I would go down the following week. Don't think to steadily about the war. It's bad for the head. Affectionately, Lessie."

Fair Copyright for Canada: Lessons for Online Social Movements from the First Canadian Facebook Uprising

Despite their growing importance, the political effectiveness of social media remains understudied. Drawing on and updating resource mobilization theory and political process theory, this article considers how social media make “political engagement more probable,” and the determinants of success for online social movements. It does so by examining the mainstreaming of the Canadian “user rights” copyright movement, focusing on the Fair Copyright for Canada Facebook page, created in December 2007. This decentralized, grassroots, social media-focused action – the first successful campaign of its kind in Canada and one of the first in the world – changed the terms of the Canadian copyright debate and legitimized Canadian user rights. As this case demonstrates, social media have changed the type and amount of resources needed to create and sustain social movements, creating openings for new groups and interests. Their success, however, remains dependent on the political context within which they operate.