Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system.

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.

3.6-KB mouse cyclin C promoter fragment is predominantly active in the testis.

Cyclin C is a highly conserved protein that regulates cell-cycle, messenger RNA transcription and cell adhesion. Recently published studies demonstrate that this protein is an essential player during early embryonic development of multicellular eukaryotes as well. In order to understand better its complex function at the level of tissues or organs, spatial expression characteristics of cyclin C and regulatory components of its expression are needed to be determined. In vitro studies on human cells suggested that approximately the first 3 kilobases of the cyclin C promoter might contain all the regulatory elements that might mimic transcription of cyclin C. To test the hypothesis, we generated reporter transgenic lines where the first 3.6-kilobase region of mouse cyclin C promoter fragment drives the transcription of a marker gene. Messenger RNA levels of the marker gene and cyclin C isoforms were measured in nine organs with reverse transcription coupled quantitative realtime polymerase chain reaction and their expression patterns were compared. The marker gene is predominantly transcribed in testes and does not follow the transcriptional regulation of the examined cyclin C isoforms. Thus, the isolated promoter fragment alone is not sufficient for the complete physiological modulation of cyclin C RNA levels, however, it is capable of enhancing testicular transcription which can be exploited in future applications.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.