EFFICIENT CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR BY AN IMPROVED IN-VITRO LIGATION METHOD

An efficient method for constructing a recombinant adenovirus (Ad) vec tor, based on an in vitro ligation, has been developed. To insert the foreign gene into an adenoviral DNA, we introduced three unique restri ction sites, I-CeuI, SwaI, and PI-SceI, into the El deletion site of t he vector plasmid, which contains a complete El, E3-deleted adenovirus type 5 genome. I-CeuI and PI-SceI are intron-encoded endonucleases wi th a sequence specificity of at least 9-10 and 11 bp, respectively. A shuttle plasmid, pHM3, containing multiple cloning sites between the I -CenI and PI-SceI sites, was constructed. After the gene of interest w as inserted into this shuttle plasmid, the plasmid for El-deleted aden ovirus vector could be easily prepared by ill vitro ligation using the I-CeuI and PI-SceI sites. SwaI digestion of the ligation products pre vented the production of a plasmid containing a parental adenovirus ge nome (null vector). After transformation into E. coil, more than 90% o f the transformants had the correct insert. To make the vector, a PacI -digested, linearized plasmid was transfected into 293 cells, resultin g in a homogeneous population of recombinant virus. The large number a nd strategic location of the unique restriction sites will not only in crease the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vec tor DNA backbone.