| With the sequence of many genomes now accessible, we can begin to gain a deeper understanding and mastery of biological systems. Areas that were previously difficult to approach, such as gene interactions, gene and chromosome organization, and even evolution of genomes, now have the potential of being elucidated. By understanding the function of genes and gene networks, manipulating the genome in a meticulous and meaningful way becomes not only possible, but also necessary. In my thesis, I explore different methods of manipulating eukaryotic genomes precisely and efficiently. I specifically use the rate of homologous recombination (HR) as the deciding factor to choose the method for inducing targeted genome modifications in cells.; In Saccharomyces cerevisiae, an organism with high rate of HR, foreign DNA materials are incorporated into the genome quite readily, providing that there is homology with the host DNA. Taking advantage of its high rate of HR, I have developed an iterative method of designing and incorporating a long synthetic DNA fragment (around 30 kb) into yeast cells, and also developed a system of screening and analyzing a large number of transformants to ensure complete, proper and accurate integration of the synthetic fragment replacing the wild type DNA segment. My thesis work shows the feasibility of creating a synthetic yeast chromosome, using an iterative replacement strategy of 30 kb DNA segment at a time.; On the other hand, in human, an organism with very inefficient HR system, a stimulus is needed to increase the rate of HR. Zinc finger nucleases (ZFNs) are becoming powerful molecular tools to deliver such a stimulus to achieve gene correction or targeted mutagenesis in many different organisms by inducing a targeted double strand break (DSB) at a predetermined site to stimulate cell's own natural DNA repair mechanisms. However, with all the progress made in the study and applications of ZFNs, their cytotoxicity remains an issue. Here, I address the problem by designing, engineering and characterizing in vitro 4-finger ZFNs that target the human cystic fibrosis transmembrane conductance regulator (hCFTR) gene and human chemokine (C-C) motif receptor 5 (bCCR5), respectively. A pair of 4-finger ZFNs recognizing a 24 bp sequence provides greater DNA recognition specificity than 3-finger ZFNs; therefore, I expect that this would lead to lower toxicity in cells. Experiments are underway to test the efficacy and efficiency of ZFN-mediated gene targeting in human cells using these specific designer 4-finger ZFNs. |
