Efficient exogenous DNA-free reprogramming with suicide gene vectors

Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications. Stem cells: Cleaner generation by selective killing Using genetic engineering to convert cells into stem cells without leaving unwanted foreign DNA behind is made easier and more effective by a method that allows cells acquiring unwanted DNA to be selectively killed. Janghwan Kim, Mi-Young Son and colleagues at the Korea Research Institute of Bioscience and Biotechnology, Daejeon, included a cell “suicide” gene in the DNA carrier that is used to transfer desired genes into cells. The suicide gene codes for an enzyme that converts an inactive drug into its active form. If undesired DNA from the carrier integrates into the cell DNA, as occasionally occurs, the action of the suicide gene selectively kills the affected cells. The method will improve the supply of stem cells that are used to study disease, test new drugs, and carry out research on correcting genetic diseases.