Single cell gene expression analysis of pluripotent stem cells.

Analyzing gene expression profiles from cells en masse provides an average profile for the population which may obscure differences in individual cells. Using an optimized workflow for qRT-PCR, gene expression profiles of undifferentiated pluripotent stem cells reveal distinct gene expression profiles for individual cells, and a large expression level range of almost every gene. Importantly, this technique allows for the identification and characterization of small subpopulations.

Aggregation of bovine cloned embryos at the four-cell stage stimulated gene expression and in vitro embryo development.

Pre-implantation embryos produced by somatic cell nuclear transfer (SCNT) have varied developmental potentials. The majority of SCNT blastocysts do not develop to term, and the mechanisms inhibiting development are still largely unknown. Aggregation of cloned embryos has been attempted to compensate for the developmental deficiency of individual cloned embryos. In this report, we investigated the impact of aggregation of bovine cloned embryos at the four-cell stage on in vitro development and gene expression of the embryos. Cell numbers and development rate of aggregated (NTagg) and non-aggregated (NT) blastocysts were characterized and compared. The blastocyst formation after aggregation was modeled using the binominal distribution. The results indicate that aggregation enhances the blastocyst formation but does not increase the overall blastocyst rate. Additionally, utilizing microarray gene chip analysis 8.8% of 8,059 genes analyzed were differentially expressed between NTagg and NT blastocysts, with more than 80% of the differentially expressed genes up-regulated in the NTagg blastocysts. Up-regulated genes include those involved in transcription, biosynthesis and signaling such as TDGF1, HNFA, CAV1, GLU5, and CD81. Our results indicate that aggregation of bovine cloned embryos at an early stage promotes the in vitro development of the resulting pre-implantation embryos.

Global gene expression analysis of bovine blastocysts produced by multiple methods.

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.

Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs.

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.

"Blue heart": characterization of a mifepristone-dependent system for conditional gene expression in genetically modified animals.

A detailed characterization of a cardiac muscle-specific, ligand-regulated gene expression system was performed in transgenic mice using the inducing ligand mifepristone (MFP). Several lines of double transgenic mice were created that expressed a bacterial lacZ reporter gene in the heart, under the control of a MFP-activated transcription factor constitutively expressed in cardiac muscle. The transgenic mice, which were administered MFP at a dose of 1 micromol/l in the drinking water, responded to the ligand within 24 h. Induction of beta-galactosidase enzyme activity in the heart continued for up to 21 days and resulted in an average 17-fold increase in enzyme activity. The highest individual animal response measured was a 94-fold increase in enzyme activity. The EC(50) for MFP induction of beta-galactosidase activity in the heart was 0.7 micromol/l when MFP was administered in the drinking water. Pharmacokinetic analysis of MFP dosing in wild-type FVB/N mice showed that absorption was very rapid (T(max) 1-10 min), bioavailability was modest ( approximately 10%) and the t(1/2) of MFP in mouse plasma was determined to be approximately 5 h. Thus, the system functions effectively in transgenic mouse heart where induction of gene expression is sensitive and can be accomplished by a simple and broadly applicable drinking water protocol.