Researchers at the University of California have developed an efficient way to genetically modify human embryonic stem cells. The study aims to substitute Bacterial Artificial Chromosomes (BAC) in place of defective copies to genes. Xu and co-authors Hoseok Song and Sun-Ku Chung, both postdoctoral fellows in Xu’s research group, California described their technique in the January 8 issue of the journal Cell Stem Cell.
BACs are synthesized circles of human DNA, which bacteria will replicate just like their own native chromosomes. Commercially available BACs can be modified within bacterial cells to insert altered copies of specific genes. Once the modified BACs are introduced into human cells, they will sometimes pair up with a matching segment of a human chromosome and swap segments of DNA, a process called homologous recombination. The advantage of using BACS to alter the genetic code in human cells comes from the long flanking sequences on either side of the modified gene, which increases the chance that the BAC with line up with native DNA in position for a swap.
The researcher team successfully transferred a defective copy of the gene p53, which suppresses cancer, into a human embryonic stem cell line. By repeating the process in a second round, they developed a line of cells in which both copies of the genes were disrupted. They also report success with a different gene, ATM, which when mutated in humans causes Ataxia-telangiectasia, a disease characterized by a host of systemic defects including increased cancer risk, degeneration of specific types of brain cells and degraded telomeres, the protective caps at the end of each chromosome. Genetically engineered mice with two bad copies of the ATM gene share some of these traits with human patients, but not all. Neurons don’t degenerate in ATM mice, for example, and the telomeres are long. “If you want to study accelerated shortening of telomeres, you can’t do it in the mouse. You can only do it in human cells,” Xu said. Those differences propelled Xu’s group to develop human cell lines instead, with the hope that some of the processes that go wrong in human patients could be studied in the lab.
In the initial stage, Xu’s group used a cell line that easily forms new colonies from single cells, but they also repeated the procedure in a cell line called H9, which has proved difficult to manipulate.Because H9 was among the few cells lines approved for use by researchers funded by the federal government, many researchers already have considerable experience with coaxing the cells into differentiating into specific types of tissues, for example, which would make the ability to genetically modify them particularly valuable.
The California Institute for Regenerative Medicine, the state’s stem cell research agency, funded this project.