Methods

Homologous Recombination

By flanking the genetic cassette that needs to be inserted into the genome, for example, bacterial replicon and selection markers, with homologous arms, a F-plasmid can be created and can be co-transfected with native viral genome into mammalian cells or insect cells. Recombination of viral genome and the plasmid takes place naturally, inserting the cassette into the viral genome at random sites. Homology arms can be acquired by PCR amplifying the homology sequences in the isolated viral genome. F-plasmid can be acquired through the PCR amplification of other Bacterial Artificial Chromosomes (BACs) and then self-ligation into a circular plasmid. Cells infected with HSV and transfected with the plasmid will be cultured and passaged. Once cells are sufficiently infected, the supernatant will be extracted and used to infect more cells. Next, supernatant from the cell culture will be collected, the viral DNA extracted through phenol treatment and ethanol precipitation. The resulting recombinant viral BAC will be transformed in bacteria and sequenced for the correct recombinant genome.⁴

Blue-White Selection

The Lac Z gene provides a transcript for the eventual translation of an enzyme (beta-galactosidase) that leads the formation of a blue precipitate. When foreign DNA is inserted into the Lac Z site (either replacing part of the Lac Z sequence or removing it completely), the enzyme can no longer be translated. Therefore, the colony will not be blue (will be white).

Suppose that we want the plasmid to uptake a certain DNA sequence . We can insert it to the Lac Z site of a plasmid (i.e. via homologous recombination) and place the plasmid into the bacteria via transformation. We can then select for white colonies. These colonies are unable to express the blue enzyme (beta-galactosidase), meaning that their Lac Z site must be disrupted. This implies that our desired DNA sequence is added to the E.coli.