Development of a DNA-labeling system for array-based comparative genomic hybridization.

Chromosomal amplifications and deletions are critical components of tumorigenesis and DNA copy-number variations also correlate with changes in mRNA expression levels. Genome-wide microarray comparative genomic hybridization (CGH) has become an important method for detecting and mapping chromosomal changes in tumors. Thus, the ability to detect twofold differences in fluorescent intensity between samples on microarrays depends on the generation of high-quality labeled probes. To enhance array-based CGH analysis, a random prime genomic DNA labeling method optimized for improved sensitivity, signal-to-noise ratios, and reproducibility has been developed. The labeling system comprises formulated random primers, nucleotide mixtures, and notably a high concentration of the double mutant exo-large fragment of DNA polymerase I (exo-Klenow). Microarray analyses indicate that the genomic DNA-labeled templates yield hybridization signals with higher fluorescent intensities and greater signal-to-noise ratios and detect more positive features than the standard random prime and conventional nick translation methods. Also, templates generated by this system have detected twofold differences in gene copy number between male and female genomic DNA and identified amplification and deletions from the BT474 breast cancer cell line in microarray hybridizations. Moreover, alterations in gene copy number were routinely detected with 0.5 microg of genomic DNA starting sample. The method is flexible and performs efficiently with different fluorescently labeled nucleotides. Application of the optimized CGH labeling system may enhance the resolution and sensitivity of array-based CGH analysis in cancer and medical genetic studies.

Reprogramming of Human Fibroblasts with Non-integrating RNA Virus on Feeder-Free or Xeno-Free Conditions.

Recent advances in generating induced pluripotent stem cells have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from somatic cells. However, a major obstacle to the use of iPSC for therapeutic applications is the potential genomic modifications resulted from viral insertion of transgenes in the cellular genome. Second, the culture of iPSCs and adult cells often requires the use of animal products, which hinder the generation of clinical-grade iPSCs. We report here the generation of iPSCs by an RNA Sendai virus vector that does not integrate transgenes into the cell's genome. In addition, reprogramming can be performed on a feeder-free or xeno-free condition without containing animal products. Generation of an integrant-free iPSCs in these conditions will facilitate the studies of iPSCs in cell-based therapies.

Profiling stem cells using quantitative PCR protein assays.

Reprogramming human somatic cells to induced pluripotent stem cells is an important avenue in biological research. Advances in the profiling of human stem cells have identified important pluripotency maintenance factors. The presence and relative expression levels of these essential markers are commonly used to define the pluripotency status and potential of reprogrammed stem cells. We reprogram human dermal fibroblasts with four transcription factors, OCT3/4, SOX2, KLF4, and cMYC delivered by viral vectors. We describe a real-time quantitative PCR methodology to quantify the levels of key protein factors and examine the kinetics during reprogramming as well as comparing protein expression in different iPS clones. This report describes three applications of TaqMan(®) Protein Assays for reprogramming studies: (1) monitoring of reprogramming proteins over the induction time course, (2) characterization of pluripotent cells by protein expression profiles, and (3) identification of potential iPSC colonies in high-throughput screening protocols. This approach is fast, simple, sensitive and generates a pluripotency scorecard for reprogrammed stem cells.

Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.

One of the major obstacles in generating induced pluripotent stem cells for research or downstream applications is the potential modifications of cellular genome as a result of using integrating viruses during reprogramming. Another major disadvantage of reprogramming cells with integrating vectors is that silencing and activation of transgenes are unpredictable, which may affect terminal differentiation potential and increase the risk of using iPSC-derived cells. Here we describe a protocol for the generation of induced pluripotent stem cells using a non-integrating RNA virus, Sendai virus, to efficiently generate transgene-free iPSCs starting with different cell types as well as in feeder-free conditions.

Generation of human-induced pluripotent stem cells (hiPSCs) using episomal vectors on defined Essential 8™ Medium conditions.

Human-induced pluripotent stem cells (iPSCs) are an important potential source of cells for regenerative medicine due to their inherent ability to differentiate into all cell types of the three germ layers. Generation of iPSCs with a non-integrating reprogramming method and in culture conditions that are completely absent of animal proteins will be ideal for such regenerative and cell therapy applications. Here we describe a method to generate non-integrating iPSCs using the Episomal iPSC Reprogramming Vectors.

Generation of human-induced pluripotent stem cells by a nonintegrating RNA Sendai virus vector in feeder-free or xeno-free conditions.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells has enabled the possibility of providing unprecedented access to patient-specific iPSC cells for drug screening, disease modeling, and cell therapy applications. However, a major obstacle to the use of iPSC for therapeutic applications is the potential of genomic modifications caused by insertion of viral transgenes in the cellular genome. A second concern is that reprogramming often requires the use of animal feeder layers and reagents that contain animal origin products, which hinder the generation of clinical-grade iPSCs. Here, we report the generation of iPSCs by an RNA Sendai virus vector that does not integrate into the cells genome, providing transgene-free iPSC line. In addition, reprogramming can be performed in feeder-free condition with StemPro hESC SFM medium and in xeno-free (XF) conditions. Generation of an integrant-free iPSCs generated in xeno-free media should facilitate the safe downstream applications of iPSC-based cell therapies.

Applications of quantitative polymerase chain reaction protein assays during reprogramming.

The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.01% to >1%, depending on the specific reprogramming factors used, the mode of delivery of the reprogramming factors, properties of the starting cells, and culture conditions. We have applied a quantitative polymerase chain reaction methodology, TaqMan Protein Assays to directly quantify the kinetics, and cellular levels of crucial transcription factors during the reprogramming process. Further, we have used the assays to ascertain the threshold levels of reprogramming protein factors required to generate iPSC colonies, to characterize the protein expression signatures of different iPSC lines, and to rapidly identify iPS versus non-iPSC colonies based on expression of pluripotency markers. These data demonstrate that TaqMan Protein Assays can be used as tools to dissect and gain greater understanding of the mechanisms guiding reprogramming and to further characterize individual established iPSC lines.

Chromatin insulator elements block transgene silencing in engineered human embryonic stem cell lines at a defined chromosome 13 locus.

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken ß actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.

Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases.

One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integrase into a pseudo attP site, and then the R4 integrase was used to place a gene of interest into specific R4 target site. The authors demonstrate the successful and rapid retargeting of a G-protein-coupled receptor (cholecystokinin receptor A, CCKAR), an ion channel (the transient receptor potential cation channel, subfamily M, member 8, TRPM8), and a GFP-c-Jun(1-79) fusion protein into the specific loci in these cell lines and show that these retargeted cell lines exhibit functional and pharmacological responses consistent with those reported in the literature.

Altering the expression kinetics of VP5 results in altered virulence and pathogenesis of herpes simplex virus type 1 in mice.

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U(L)38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.