animals. Site-specific integration of DNA will likely be used in the next generation of agri-
cultural products. The recombinase-mediated integration process is more efficient for deli-
vering precise single-copy DNA into a host genome. More importantly, it has the potential
to cluster the introduced DNA at a single locus in the genome. Clustering the transgenic
traits provides an important benefit in the breeding process. Over time, more and more
genetic improvements will be added or “stacked” to a transgenic plant or animal.
Currently, this gene stacking is conducted by combining different transgenes from indepen-
dent transformation events (integration sites). For example, the coassortment of 4 indepen-
dent loci is 1 in 16 progeny, which can be found with ease among the typically hundreds of
plant progeny (although it is already beyond the litter size limits of many farm animals). For
10 independent loci, however, this would be 1 out of 1024; and here we are not even con-
sidering the nontransgenic traits that also must be combined into the genome of the desired
commercial product, or the possibility of linkage drag that makes the selective assortment of
some of the genetic loci more difficult. If the transgenic traits were clustered, however, then
there would be fewer genetic loci to assemble in a breeding program.
16.3 Zinc-Finger Nucleases
In the high-flying world of human gene therapy, a technology that has captured the imagin-
ation of researchers is calledzinc-finger nucleases(ZFNs). In 2005 researchers corrected a
mutation in severe combined immune deficiency (SCID) in cultured cells (Urnov et al.
2005). ZFNs are similar to restriction enzymesin their action of digesting DNA at
certain sequences, but, unlike restriction endonucleases, they can be made to be specific
to single locations in a genome. These designer proteins are created from zinc finger and
nuclease domains.Zinc-finger domainsbind DNA at specific sequences, with each zinc
finger recognizing primarily a three-nucleotide sequence (Porteus and Carroll 2005).
Hence, a domain composed of three zinc fingers would recognize a specific 9-bp sequence.
The theory behind this approach is that with the addition of a zinc-finger domain to a DNA
endonuclease, the endonuclease would be directed to cut the DNA near the site where the
zinc-finger peptide binds. The damaged DNA would then activate the host DNA repair
Figure 16.3.Recombination sites that flank the entire transgenic locus permits removal of transgenic
DNA on induced expression of a recombinase gene. For instance, if the recombinase gene is placed
under the control of sperm-specific or fruit-specific promoters, the excision of transgenic DNA may
help reduce the outcross of transgenes, or minimize the production of transgene-encoded proteins
needed elsewhere in the plant but not in the edible portions of food.
16.3. ZINC-FINGER NUCLEASES 363