Microchip-based forensic short tandem repeat genotyping.

Micro total analysis system (µTAS) or lab-on-a-chip (LOC) technology has advanced over decades, and the high performance for chemical and biological analysis has been well demonstrated with advantages of low sample consumption, rapid analysis time, high-throughput screening, and portability. In particular, µTAS or LOC based genetic applications have been extensively explored, and the short tandem repeat (STR) typing on a chip has garnered attention in the forensic community due to its special use for human identification in the field of mass disaster and missing person investigation, paternity testing, and perpetrator identification. The STR typing process consists of sample collection, DNA extraction, DNA quantitation, STR loci amplification, capillary electrophoretic separation, and STR profiling. Recent progress of microtechnology shows its ability to substitute the conventional analytical tools, and furthermore demonstrates total integration of the whole STR processes on a single wafer for on-site STR typing. In this review article, we highlighted some representative results for fluorescence labeling techniques, microchip-based DNA purification, on-chip polymerase chain reaction (PCR), a capillary electrophoretic microdevice, and a fully integrated microdevice for STR typing.

Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells.

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W(v) mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

H19 gene is epigenetically stable in mouse multipotent germline stem cells.

Testis-derived germline stem (GS) cells can undergo re-programming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.