Zinc finger nucleases and TALENs belong to a class of DNA- binding proteins or artificial restriction enzymes that facilitate targeted editing of the genome by creating double-strand breaks

529 The creation of new genetically modified animals has increased exponentially over the last ten years. this is due, in large part, to the continued development and advancement in the technology involved in creating genetically modified animals. Although the continued development of new technologies to create genetically modified animals is useful in creating more sophisticated experimental animal models, it also is important to look at and develop new technologies that will help us implement the 3Rs. transgenic laboratories often are at the cutting edge of new technology when it comes to creating genetically modified animals. Since this is the case, they also should be among the first groups to look at the technology available that allows them to modify and refine their procedures in the implementation of the 3Rs. the 3Rs transgenic laboratories can have the biggest impact when working either to reduce their animal numbers or refine their procedures to become less invasive. Although a large number of developing technologies may be implemented, this discussion will address only five of the newest ones available, with a brief discussion of their advantages and disadvantages. The five technologies I would like to discuss are: zinc finger nucleases and transcription activator-like effector nucleases (tAleNs) to create gene targeted rodents, NSet – non surgical embryo transfer, deathless transgenesis, Internet database searches, and genotyping genetically engineered animals. Zinc finger nucleases and TALENs belong to a class of DNAbinding proteins or artificial restriction enzymes that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specific locations. There are several advantages to using this technology. The first is unlimited species possibilities for creating genetically modified animals. Until now, only animals that had established eS cell methods and cell lines could be targeted to create disease-representative animal models. However, zinc finger nucleases and TALENs do not depend on creating new eS stem cell lines but instead use the host genome information for targeting, and once the sequence information for a species is known it can then be targeted. the mutation rate is also very robust. Mutation rates at around 1015% have been reported, while traditional eS cell technology often runs only 1-4%. This improved efficiency in mutation targeting rates will help reduce animal numbers. lastly and most importantly, this technology has been demonstrated to be heritable through the germ line. Once targeted animals have been created and identified, they can transmit the desired mutation with standard Mendelian genetics to their offspring. Some disadvantages must be taken into consideration, however. First, this is a new technology that is still in development. Second, future developers likely will be faced with patent and governmental regulation issues. NSet is a simple method of transferring embryos non-surgically to the uterine horns of the mouse (Fath-Goodin et al., 2006). Briefly, embryos are loaded using a modified capillary tube and transferred through the cervix into the uterine horn of an unanesthetized mouse. The advantages to this technique are several, including, elimination of the pain and distress of surgery, thus completely removing the need for anesthetics and analgesics. It also eliminates the need for post-surgical monitoring and reduces the regulatory burden on laboratories and institutes. this is a valuable savings in staff time, which translates to a decrease in costs. This is a significant savings, since staff salaries are often are cited as the most costly expense to laboratory institutes. the only drawback to this technology is that the technology itself is expensive, since each individual unit costs $ 25. Hopefully, as this technique is established in a larger number of labs the increased demand could reduce the costs to purchase this product. Deathless transgenics is a common name for a new spermatogonia-mediated in vivo approach for the generation of transgenic mice (Majumdar et al., 2008). It is an alternative method to generating transgenic animals. the technique inRefinement of Genetic Engineering Procedures