Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method.
An efficient method for constructing a recombinant adenovirus (Ad) vector, based on an in vitro ligation, has been developed. To insert the foreign gene into an adenoviral DNA, we introduced three unique restriction sites, I-CeuI, SwaI, and PI-SceI, into the E1 deletion site of the vector plasmid, which contains a complete E1, E3-deleted adenovirus type 5 genome. I-CeuI and PI-SceI are intron-encoded endonucleases with a sequence specificity of at least 9-10 and 11 bp, respectively. A shuttle plasmid, pHM3, containing multiple cloning sites between the I-CeuI and PI-SceI sites, was constructed. After the gene of interest was inserted into this shuttle plasmid, the plasmid for E1-deleted adenovirus vector could be easily prepared by in vitro ligation using the I-CeuI and PI-SceI sites. SwaI digestion of the ligation products prevented the production of a plasmid containing a parental adenovirus genome (null vector). After transformation into E. coli, more than 90% of the transformants had the correct insert. To make the vector, a PacI-digested, linearized plasmid was transfected into 293 cells, resulting in a homogeneous population of recombinant virus. The large number and strategic location of the unique restriction sites will not only increase the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vector DNA backbone.
Mizuguchi
H
H
Department of Pediatrics, Stanford University School of Medicine, CA 94305, USA.
Kay
M A
MA
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017384:Sequence Deletion; D000515:alpha 1-Antitrypsin / Q000235:genetics
D017931:DNA Primers; D000515:alpha 1-Antitrypsin