tetracycline and Teratoma

The use of human embryonic stem cells (hESCs) as a research and therapeutic tool will be facilitated by conditional gene expression. Here, we report drug-induced transgene expression, both in vitro and in vivo, from a tet-on hESC line with >95% purity. Using green fluorescent protein as an indicator, we demonstrated that the tet-on system allowed a tight control of the gene expression in both undifferentiated hESCs and differentiated cells of the three germ layers. More importantly, after the cells were transplanted into animals, the gene expression remained to be regulated by an orally administered drug. These results provide a technical basis for regulation of gene expression in hESCs and derivatives in vitro and in vivo.

Topics: Administration, Oral; Animals; Brain Tissue Transplantation; Cell Differentiation; Cell Line; Doxycycline; Embryonic Stem Cells; Gene Expression; Green Fluorescent Proteins; Humans; In Vitro Techniques; Insulin; Mice; Mice, SCID; Myocytes, Cardiac; Neurons; Recombinant Proteins; Teratoma; Tetracycline; Transfection

Pancreatic development in mammals is controlled in part by the expression and function of numerous genes encoding transcription factors. Yet, how these regulate each other and their target genes is incompletely understood. Embryonic stem (ES) cells have recently been shown to be capable of differentiating into pancreatic progenitor cells and insulin-producing cells, representing a useful in vitro model system for studying pancreatic and islet development. To generate tools to study the relationships of transcription factors in pancreatic development we have established seven unique mouse ES cell lines with tetracycline-inducible expression of either Hnf4alpha, Hnf6, Nkx2.2, Nkx6.1, Pax4, Pdx1, and Ptf1a cDNAs. Each of the cell lines was characterized for induction of transgene expression after exposure to doxycycline (DOX) by quantitative real-time PCR and immunofluorescence microscopy. Transgene expression in the presence of DOX was at least 97-fold that seen in untreated cells. Immunofluorescent staining of DOX-treated cultures showed efficient (>95% of cells) transgene protein expression while showing <5% positive staining in uninduced cells. Each of the ES cell lines maintained their pluripotency as measured by teratoma formation. Furthermore, transgene expression can be efficiently achieved in vivo through DOX administration to mice. The establishment of ES cell lines with temporally controllable induction of critical pancreatic transcription factor genes provides a new set of tools that could be used to interrogate gene regulatory networks in pancreatic development and potentially generate greater numbers of beta cells from ES cells.

Topics: Animals; Base Sequence; Cell Line; DNA Primers; DNA, Complementary; Doxycycline; Embryonic Stem Cells; Homeobox Protein Nkx-2.2; Mice; Molecular Sequence Data; Pancreas; Teratoma; Tetracycline; Transcription Factors

Metabolic changes in human spermatozoa related to capacitation were investigated. Glycolysis, motility, oxygen uptake, survival time, and c hanges in tetracycline-binding capacity were studied in spermatozoa expo sed to the following possible capacitating agents: 1) human cervical mucus, 2) human serum, 3) hydroxalpinx fluid, and 4) follicular cyst fluid. Mucus and serum did not produce marked changes. Hydrosalpinx fluid produced an increase in oxygen uptake in the presence of citrate and succinate while follicular cyst fluid produced modifications that could be related to capacitation such as increase in oxygen uptake, increase in motility, and release of bound tetracycline. Similar changes were seen with cyclic adenosine monophosphate and adenosine triphosphate treatment but not with other related nucleotides. The parameters studied in this work are suggestive of functional capacitation.

Topics: Adenosine Triphosphate; Cell Movement; Cervix Mucus; Cyclic AMP; Exudates and Transudates; Female; Glycolysis; Humans; In Vitro Techniques; Male; Ovarian Cysts; Oxygen Consumption; Parovarian Cyst; Sperm Capacitation; Spermatozoa; Stimulation, Chemical; Teratoma; Tetracycline; Tritium