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.

Thomas Millard - Captain's Appointment 1814

Document appointing Thomas Millard, Esquire to as "Captain of a Troop in the North Somerset Regiment Cavalry". The appointment is signed and declared "By the Right Honourable John Earl Poulett, Viscount, and Baron of Hinton Saint George of the most Ancient Order of the Thistle and Lord Lieutenant of the County of Somerset". Dated 1814

Pen and sunlight sketches of scenery reached by the Grand Trunk Railway and connections : including Niagara Falls, Thousand Islands, Rapids of the Saint Lawrence, Montreal, Quebec, and the mountains of New England.--

At head of title: "The tourist route of America".

BP BLOGGER: DESCRIPTIVE STUDY OF ELECTRONIC KNOWLEDGE DISSEMINATION IN LONG TERM CARE

Little is known of the uptake and use of knowledge disseminated in electronic formats, especially in Long Term Care (LTC) settings. The purpose of this descriptive study was to examine the dissemination of the BP Bloggers, a series of brief, evidence summaries designed to meet the knowledge needs of LTC staff. Guided by Rogers’ (2003) Diffusion of Innovations theory, the study documents dissemination of the BP Blogger and examines factors affecting dissemination, awareness, perceptions and its use. The survey of BP Blogger recipients was conducted electronically (n=114) online (n=10), by telephone (n=55), and print (n=144). Managers usually received the newsletter electronically while staff in LTC were more likely to receive printed copies. Participants disseminated the newsletter through paper, email, or posting in the workplace. Most participants rated the content, format, and usefulness of the BP Blogger as good or excellent. Time and lack of email access were barriers to dissemination.