Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein.

Fifth series of historical documents : [All relating to the War of 1812].

Published under the auspices of "The Literary and Historical Society of Quebec".

Why are we still at war? : or, The American question considered ; in a series of essays rejected by the journalists as unpopular ; recommended to a candid perusal /

In the Pamphleteer, vol. 4, p. 551-576. London, 1814.

Why are we still at war? : or, The American question considered ; in a series of essays rejected by the journalists as unpopular ; recommended to a candid perusal

In the Pamphleteer, vol. 4, p. 551-576. London, 1814

Niagara River and Falls from Lake Erie to Lake Ontario : a series of one hundred and fifty-three original etchings (1886)

A portfolio of 50 folio and 103 vignette etchings, printed on fine china paper, which has been pressed into a sturdy hand-made paper. The plates were printed by J.H. Daniels of Boston in black, brown, sepia or blue ink. All prints are dated, numbered, and signed or initialed in the plate by Sangster, and the folio prints have pencil signatures. Vol. 1. Vignettes (nos. 1-53) -- vol. 2. Vignettes (nos. 54-103) -- vol. 3. Plates (vol. 1 and 2, nos. 1-50).

Niagara River and Falls from Lake Erie to Lake Ontario : a series of one hundred and fifty-three original etchings (1886) Vol.2

A portfolio of 50 folio and 103 vignette etchings, printed on fine china paper, which has been pressed into a sturdy hand-made paper. The plates were printed by J.H. Daniels of Boston in black, brown, sepia or blue ink. All prints are dated, numbered, and signed or initialed in the plate by Sangster, and the folio prints have pencil signatures. Vol. 1. Vignettes (nos. 1-53) -- vol. 2. Vignettes (nos. 54-103) -- vol. 3. Plates (vol. 1 and 2, nos. 1-50).

Niagara River and Falls from Lake Erie to Lake Ontario : a series of one hundred and fifty-three original etchings (1886) Vol. 3

A portfolio of 50 folio and 103 vignette etchings, printed on fine china paper, which has been pressed into a sturdy hand-made paper. The plates were printed by J.H. Daniels of Boston in black, brown, sepia or blue ink. All prints are dated, numbered, and signed or initialed in the plate by Sangster, and the folio prints have pencil signatures. Vol. 1. Vignettes (nos. 1-53) -- vol. 2. Vignettes (nos. 54-103) -- vol. 3. Plates (vol. 1 and 2, nos. 1-50).