nuclear genome integration with elements for eukaryotic gene expression; and a plastid
vector, with elements that allow prokaryotic gene expression. Using such vector design fea-
tures, the risk of gene flow and product contamination is reduced, since chloroplasts are
maternally inherited in crop species and pollen produced by these plants would contain
only half of the protein-coding fragment.
Innovations in vector construction and plant transformation technology can influence the
character of the resulting transgenic crop. Gene silencing and transgene containment are
two important issues in plant biotechnology, the impact of which can be reduced by geneti-
cally engineering the plastid genome rather than the nuclear genome. This is because,
unlike the nuclear genome, gene silencing does not occur in the plastid genome and, in
most agronomically important plant species, plastids are maternally inherited, preventing
pollen-mediated outcrossing. Vectors for chloroplast transformation are designed and
Figure 7.23.Site-specific integration is achieved by two homologous recombination events, one on
either side of the DNA fragment to be integrated. During insertion, the targeted region of the vector
replaces the targeted region of the plastid genome, and the vector backbone is lost. The inserted
DNA fragment contains a selectable marker (here, theaadAgene encoding aminoglycoside 3^0 -
adenylyltransferase, providing spectinomycin resistance) and can contain either a single gene
flanked by independent 5^0 and 3^0 regulatory regions, including a promoter; a 5^0 UTR and a 3^0
UTR; or, as is the case here, multiple genes with a single promoter that regulates the expression of
the operon with individual ribosome binding sites (RBS), upstream of each ORF. In this example,
theBacillus thuringiensis(Bt)cry2Aa2 operon is inserted in the plastid genome generating insectici-
dal proteins in plant cells. The 16Sribosomal RNA (rRNA) promoter (Prrn) drives the expression of
theaadAgene and the three genes of thecry2Aa2 operon. The terminator is thepsbA 30 region of a
gene encoding the photosystem II reaction center component of the tobacco chloroplast genome.
7.6. SAFETY FEATURES IN VECTOR DESIGN 187