Gene targeting is a genetic technique that utilizes homologous recombination between an engineered exogenous DNA fragments with the endogenous genome of an organism. In domestic animal, gene targeting has provided an important tool for producing Knock-out pig for GGTA1 gene to use xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse ES cells. So the application of gene targeting in somatic cells has been limited by its low efficiency. Recently, knock-out rat and mouse was generated by introducing nonhomologous end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knock-out and knock-in techniques in domestic animal is very important in biomedical research. In this present study, we investigated whether homologous recombination events occurs at cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. CMAH-targeted ZFN DNA and mRNA were purchased from SIGMA-Aldrich. CMAH neo targeting vector consists of the neomycin resistance gene as a positive selectable marker gene, 789 bp 5’ arm and 763 bp 3’ arm from Exon 8 of CMAH gene. For transfection, the targeting vector and ZFN DNA or mRNA were introduced into ear fibroblasts cells of Chicago miniature pig by electroporation. After selection of G-418, PCR analysis was performed using 213 colonies transfected with ZFN DNA or mRNA. As a result, 39 positive colonies were identified in colonies transfected with ZFN DNA or mRNA. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in the production of CMAH knock-out for xenotransplantation.

Plant breeding is a multidisciplinary science of changing the genetic makeup of plants in order to generate desired traits or characteristics, and thus it can be accomplished through many different techniques ranging from simply selecting plants with desirable traits for propagation to more complex molecular techniques. Both conventional and genetically modified (GM) plant breeding alter or modify the genes of a plant so that a better variety is developed. Breeding using GM tools is achieved for the same reasons as conventional breeding. One prominent distinction is that instead of randomly mixing genes in conventional breeding, which occurs as a result of a sexual cross, a specific gene is directly transferred or selectively inactivated in the new plant variety through GM plant breeding. Site-specific mutagenesis and selection of gene knockout mutants are readily carried out in model plant species, such as Arabidopsis. However, targeted mutation of a specific gene is technically impractical, if not impossible, in most cases. As an alternative approach, RNA interference (RNAi), which is mediated by small interfering RNA (siRNA) and microRNA (miRNA), is routinely employed for targeted silencing of genes in academic and biotechnological studies. Recently, engineered nuclease-based genome editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been developed to induce site-specific genome modifications in both animals and plants. ZFNs are chimeric DNA restriction enzymes that consist of the nuclease domain of the Fok1 restriction enzyme, which triggers double strand breaks in genomic DNAs, and a custom-designed ZF DNA-binding domain, which guides the ZFNs to specific sequences within genomic DNAs. The double-strand breaks are rejoined by cellular DNA repair machinery, resulting in targeted mutagenesis or targeted gene replacement. In this work, we employed the ZFN tool to specifically inactivate two flowering genes, such as FCA and GI that also mediate high temperature responses and clock output signaling, respectively, in a bioenergy grass crop, Brachypodium distachyon. We designed extensive sets of ZF recognition sequences that recognize target sequences within the FCA and GI genes. The potential ZFN cassettes were transformed into Brachypodium ecotype Bd21-3. The transformants will be screened to identify those carrying targeted gene mutations. We will also discuss the extension of the ZFN tool to other plant species, including crops.