**5. Summary**

712 Biomedical Science, Engineering and Technology

made using modular assembly. While effective, this method is labor intensive and requires additional expertise. The third method, referred to as OPEN (Oligomerized Pool ENgineering) is based on bacterial 2-hybrid (B2H) selection and has been proven to be a rapid platform for plant and human cells with high targeting efficiency (ranging from 1 to 50% at different loci) and less toxicity compared to modular assembly system. However, utilization of OPEN requires an archive of pre-selected zinc-finger pools and *E. coli* selection (Maeder et al., 2008). Furthermore, due to the challenge of engineering the endonucleases, orthophenanthroline (OP, a DNA cleaving molecule) was conjugated with triplex-forming oligonucleotides (TFOs, sequence-specific binding capacity) to induce targeted DSBs and stimulate mutations at the target site in approximately 10% of treated human cells (Cannata et al., 2008). TFO conjugating to OP or other DNA cleaving molecules may provide a useful tool to induce targeted gene modification because triplex-forming sequences are frequent in mammalian genes. While ZFN-driven gene targeting can be much more efficient than homologous recombination-based methods, the design and development of highly specific ZFNs remain difficult because of the lack of a simple correspondence between amino acid

Recently, several groups have shown that transcription activator–like effectors (TALEs) from the bacterial genus *Xanthomonas* contain a central domain of tandem repeats that can be readily engineered to bind virtually any DNA sequence (Boch et al., 2009; Christian et al., 2010; Morbitzer et al., 2010). The structure of the central protein domain, highly conserved in all the known TALEs, includes 17.5 tandem repeats with 34 amino acids per repeat. In each repeat monomer of a TALE, only amino acid positions 12 and 13 are hypervariable (repeat variable diresidues) (Boch et al., 2011), which can specifically recognize a single nucleotide in the target site (Boch et al., 2009; Moscou et al., 2009). Thus, the correspondence between each repeat variable diresidues and the binding nucleotide in DNA sequence opens the possibility to create novel sequence-specific DNA binding proteins by rearrangement of TALE repeats. The engineered hybrid TALE nucleases (TALNs), produced by fusion of the *FokI* endonuclease domain with the high-specificity DNA-binding domains of TALEs, can bind and create targeted DSBs in tobacco and yeast (Mahfouz et al., 2011; Li et al., 2011), showing the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific genome modification. Recently, Miller et al. (2011) reported the generation of discrete edits or small deletions within endogenous human NTF3 and CCR5 genes and the insertion of 46-bp sequence at CCR5 locus into the genome of human K562 cells using designed TALNs, demonstrating the effective application of TALNs to modify endogenous genes. While the simple DNA-binding code of TALEs enables easier design strategies as compared to ZFPs, the repetitive nature of TALE DNA-binding domains results in difficulty to efficiently synthesize new TALEs by currently used vector construction methods. To overcome this problem, Zhang et al. (2011) recently developed a new strategy to construct

Owing to the lack of ES cells in domestic animals, it is difficult to replicate strategies routinely used to create genetically modified mice. As an alternative, cell-based transgenesis via SCNT is currently used to produce genetically modified domestic animals. Recent advancements in the ability to generate induced pluripotent stem (iPS) cells may open another potential strategy to improve the efficiency of SCNT in domestic animals. Induced pluripotent stem cells in mice and human were successfully generated by reprogramming

sequence and DNA recognition sequence.

repeat domains of TALEs by hierarchical ligation.

**4.3 Induced pluripotent stem (iPS) cells** 

While the potential opportunities of transgenic domestic animals in biomedicine and agriculture are significant, current procedures, including cell-based transgenesis via SCNT, to produce genetically modified domestic animals are not without limitations. The combination of new technologies, including ZFNs/TALNs to enhance targeted genome modification and iPS cells and other strategies to improve epigenetic remodeling of SCNT embryos, represent pathways for improving the success rates of current genome manipulation strategies resulting in transgenic domestic animals. These modern approaches may have limitations of their own, such as the difficulty and high cost to design, produce and validate the target-specific ZFPs, constructing custom-designed TALEs, and maintenance of iPS cells. However, despite these limitations, we expect to see these strategies become widely utilized as a result of the potential opportunities that utilization of these strategies offers to the field of targeted genome manipulation in domestic animals.
