BAC mutagenesis techniques. (a) generation of a deletion mutant via homologous recombination. (1) Amplification of the ka expression cassette by PCR using a primer pair adding 40-bp (or longer) homologies flanking ORFX. (2) Viral BAC DNA is introduced into DY380 by electroporation. (3) Homologous recombination between upstream and downstream homologies of Gene X replaces Gene X with a selectable marker (the ka cassette), creating the Gene X-deletion viral BAC. (4) The recombinants are selected based upon their ability to grown on LB agar plates containing kanamycin. (5) The viral BAC DNA is isolated, and the deletion of Gene X is confirmed by PCR analysis. The integrity of the viral genome (after homologous recombination) is examined by restriction enzyme digestion. (6) Purified BAC DNA is transfected into a human cell line. (7) Viral proteins are expressed, and a functional virus is created. B. Generation of Rescue Virus. 1. Gene X is amplified by PCR from the wild-type BAC DNA. (2) Gene X is cloned into a bacterial plasmid. (3) Gene X and a selectable maker (zeocin) are amplified via PCR using a primer pair that adds at least 40 bp of nucleotide sequence that is homologous to viral genomic sequence flanking Gene X. (4) The PCR product was transformed via electroporation into DY380, now carrying the Gene X deletion mutant. (5) and (6) Gene X (with a zeocin marker) is inserted back into the BAC by homologous recombination (7) The Ze vector sequence is removed (by cotransfecting a Cre recombinase-expressing plasmid with the prepared viral BAC DNA). Rescue virus DNA is ready to be purified and transfected into human cells. (c) GalK-based Mutagenesis (1). Insert galK sequence flanked by a sequence homologous to the viral BAC sequence flanking Gene X. (2) Gene X is replaced by the galK gene via homologous recombination. (3) Replace the galK gene with PCR product containing desired mutation in Gene X (referred to as Gene Y). (4) Transfect viral BAC into mammalian cells to produce infectious mutant virus. (d) Transposon Mediated Mutagenesis. (1) and (2) A temperature-sensitive plasmid donor containing transposon (Tn) is inserted into E. coli cells already containing viral BAC that was inserted via electroporation). Once the donor plasmid is inside the cell, the transposon will be inserted into the BAC genome. (3) An increase in temperature will remove the donor plasmid. The transposon mutant is now ready to be purified and transfected into human cells. PCR primers pre-engineered into the transposon insertion site can be used to sequence the insertion region of any transposon mutants with interesting phenotypes.
