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Role and Gonadotrophic Regulation of X-Linked: MATERIALS AND METHODS(2)

Adenoviral LacZ, XIAP sense and antisense cDNAs, and rabbit polyclonal anti-human XIAP antibodies were generously provided by Dr. Eric LaCasse (^Jgera Therapeutics, Inc,, Ottawa ON, Canada). Rabbit polyclonal anti-XIAP antibody was raised against a glutathione S-transferase fusion protein that was expressed in Escherichia coli using pGEX vector (Amersham Pharmacia Biotech, Arlington Heights, IL) containing full-length XIAP cDNA. The antibody was affinity-purified by passing through a glutathione S-transferase-XIAP glutathione-Sepharose column. Specificity was confirmed on Western blots (using the antibody-depleted eluate from the affinity column), and cross-reactivity with other IAPs was not noted. canadian health care mall

Construction of recombinant adenovirus was carried out as described previously with some modifications. Briefly, the open reading frame of XIAP was amplified by polymerase chain reaction, cloned in the pCR2.1 vector (Invitrogen, Carlsbad, CA), and sequenced. The open reading frame was cut out and ligated into the Swa-1 site of pAdex1CAwt cosmid DNA. The vector was packaged with the Promega cosmid packaging extracts (Madison, WI) and used to infect E. coli. Colonies were picked and screened for presence of the insert in the antisense orientation relative to the chicken p-actin promoter. The CsCl-purified cosmid DNA was cotransfected with wild-type adenovirus DNA that was allowed to generate infectious adenovirus DNA only when homologous recombination with cosmid DNA occurred. The final recombinant adenovirus contained a linear, double-stranded genome of 44 820 base pairs (bp) plus the antisense XIAP insert (1500 bp). Adenoviral expression system was generated with an Ad E1 insertion vector. Virus titer was determined by the plaque assay.