Description of AdEasy™ Kit Reagents

pShuttle

pShuttle is an Ad transfer vector containing a multiple cloning site in which an expression cassette can be inserted. The cloning capacity of pShuttle is 7.5 kb. Upon homologous recombination in bacteria with a plasmid containing the E1-E3 Ad5 genome, a new plasmid is generated in which the expression cassette is inserted into the original E1 region of the adenovirus genome. This resulting plasmid is then transfected into QBI-293A cells to generate a recombinant adenovirus expressing the gene product.

pShuttle-CMV

pShuttle-CMV is an Ad transfer vector containing a multiple cloning site in which a cloned gene is under the control of the human CMV promoter for constitutive recombinant expression in a wide variety of cell lines. The cloning capacity of pShuttle-CMV is 6.6 kb. Upon homologous recombination in bacteria with a plasmid containing the E1-E3 Ad5 genome, a new plasmid is generated in which the expression cassette is inserted into the original E1 region of the Ad genome. The resulting plasmid is then transfected into QBI-293A cells to generate a recombinant adenovirus expressing the gene of interest under the CMV promoter.

BJ5183 EC

BJ5183 EC are electrocompetent (EC) Escherichia coli cells used in the AdEasy™ system for homologous recombination. Genotype: endA sbcBC recBC galK met thi-1 bioT hsdR (Strr).

QBI-293A CELLS

The QBI-293A cell line is a permanent line of primary human embryonic kidney cells transformed by sheared human Ad5 DNA. The QBI-293A cell line contains the E1A and E1B Ad5 viral genes, which complement the deletion of this essential region in the recombinant adenovirus. We have selected a superior sub-clone of 293A that adheres strongly to plastic dishes and thus performs very well in plaque assay and transfection experiments.

pAdEasy-1

pAdEasy-1 is a 33.4kb plasmid containing the adenovirus serotype 5 (Ad5) genome with deletions in the E1-E3 regions.

DH5 EC

DH5 EC are electrocompetent (EC) Escherichia coli cells used in the AdEasy™ system for amplification of recombinant plasmid. Genotype: F- deoR recA1 endA1 hsdR17(rk-,mk ) supE44 l- thi-1 gyrA96 relA1.

QBI-INFECT

The Ad5.CMV-LacZE1/E3 viral particles can be used to optimize adenovirus culture techniques before production of your recombinant virus. QBI-Infect can also be used to prepare control plaques for people inexperienced with virology techniques. Once amplified, QBI-Infect particles can be used to test the infectivity of cell lines or tissues other than QBI-293A cells or commonly used eukaryotic cells. The success of DNA transfer can be monitored with a substrate for -galactosidase.

TRANSFECTION REAGENTS

CaCl₂ 2M, 0.1x TE and 2X HBS. All reagents have been tested to give optimal calcium phosphate transfection in QBI-293A cells.