Ectodomain Cleavage of ErbB-4: Characterization of the Cleavage Site and M80 Fragment

The Journal of Biological Chemistry. Oct, 2003  |  Pubmed ID: 12869563

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646-657 was incubated with recombinant tumor necrosis factor-alpha-converting enzyme, fragments representing residues 646-651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.

Enhanced Sensitivity RNA Gel Loading Buffer That Enables Efficient RNA Separation on Native Gels

BioTechniques. Feb, 2004  |  Pubmed ID: 14989099

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.

A Method for Cross-species Gene Expression Analysis with High-density Oligonucleotide Arrays

Nucleic Acids Research. 2004  |  Pubmed ID: 15247326

DNA microarrays have been widely used in gene expression analysis of biological processes. Due to a lack of sequence information, the applications have been largely restricted to humans and a few model organisms. Presented within this study are results of the cross-species hybridization with Affymetrix human high-density oligonucleotide arrays or GeneChip using distantly related mammalian species; cattle, pig and dog. Based on the unique feature of the Affymetrix GeneChip where every gene is represented by multiple probes, we hypothesized that sequence conservation within mammals is high enough to generate sufficient signals from some of the probes for expression analysis. We demonstrated that while overall hybridization signals are low for cross-species hybridization, a few probes of most genes still generated signals equivalent to the same-species hybridization. By masking the poorly hybridized probes electronically, the remaining probes provided reliable data for gene expression analysis. We developed an algorithm to select the reliable probes for analysis utilizing the match/mismatch feature of GeneChip. When comparing gene expression between two tissues using the selected probes, we found a linear correlation between the cross-species and same-species hybridization. In addition, we validated cross-species hybridization results by quantitative PCR using randomly selected genes. The method shown herein could be applied to both plant and animal research.

A Method for Gene Expression Analysis by Oligonucleotide Arrays from Minute Biological Materials

Analytical Biochemistry. Aug, 2004  |  Pubmed ID: 15265739

Gene expression profiling has been widely used in identifying differentially expressed genes. One of the most popular formats is oligonucleotide array. A limitation of oligonucleotide arrays is the requirement of relatively large amounts of biological starting materials for gene expression analysis. We have developed a simple method for gene expression profiling from very small amounts of biological material by combining exponential (PCR) and linear (T7 RNA polymerase) amplification. By modifying the widely used SMART protocol, we combined T7 promoter ligation and PCR amplification in one step and generated around 0.5 microg of PCRcDNA from 30 ng of total RNA in a single PCR. The PCRcDNA was in vitro transcribed by T7 RNA polymerase to generate complementary RNA (cRNA), which then was used to hybridize Affymetrix GeneChips. Our results demonstrated a linear correlation between the PCR amplification and the conventional linear amplification in gene expression ratios of individual transcript species between two different RNA preparations. The method was further validated by TaqMan reactions. Moreover, both linear and PCR methods showed some inherent bias as to which transcripts were detected, suggesting that using both in parallel may provide a more comprehensive coverage of the transcriptome present in a given sample.

A Method for Determining Zygosity of Transgenic Zebrafish by TaqMan Real-time PCR

Analytical Biochemistry. Sep, 2005  |  Pubmed ID: 16061191

When producing a genetically modified organism, intended genes are often integrated into a target genome by random insertions. Subsequently, it is often desirable to know the gene copy number of the transgenic organism and the zygosity of its offspring. Because of the random insertions, the estimation can be made only by quantitative measurement of the genes. Even though TaqMan real-time PCR has been used in gene expression analysis, it is routinely used to quantify differences larger than twofold or more than one PCR cycle. In this study, we employed TaqMan quantitative PCR to determine zygosity of transgenic fluorescent zebrafish in which a homozygote and a hemizygote differ by only twofold. We measured relative quantities of the transgene by taking the threshold cycle (Ct) of both the transgene and an internal control zebrafish genomic DNA. Using scatterplots and statistical inference, we demonstrated that homozygotes and hemizygotes could be differentiated unambiguously when multiple measurements were taken. We discuss the relationship between the repetitive measurements and TaqMan precision with a statistical model. The result illustrates that the method can be extended to some areas that require even higher precision such as determining the polyploidy of an organism.

Amplification of Nanogram Amounts of Total RNA by the SMART-based PCR Method for High-density Oligonucleotide Microarrays

Clinical Chemistry. Dec, 2005  |  Pubmed ID: 16306094

Linkage Analysis and Gene Expression Profile of Pancreatic Acinar Atrophy in the German Shepherd Dog

Mammalian Genome : Official Journal of the International Mammalian Genome Society. Dec, 2005  |  Pubmed ID: 16341675

Pancreatic acinar atrophy (PAA) is a degenerative disease of the exocrine pancreas and is the most common cause of exocrine pancreatic insufficiency in the German Shepherd Dog. Analyses of inheritance have shown that a single gene segregating in an autosomal recessive fashion is causative for PAA. To date the gene and causative mutation have not been determined. To identify a region of interest and/or candidate genes, we conducted linkage and gene expression studies. Analysis of 384 microsatellite markers resulted in a maximum two-point LOD score of 2.5 for FH2107 on CFA03. We used an oligonucleotide array to generate gene expression profiles for normal and affected pancreata. It revealed 244 genes with greater than two-fold difference in expression levels. Five genes of interest were further assessed by TaqMan quantitative real-time RT-PCR that confirmed trends observed using the microarray. One gene, gp25L, located on CFA03, was found to be downregulated by more than 500-fold in affected pancreata and was further investigated as a candidate gene. Sequence data did not reveal a mutation in the coding sequence that segregates with PAA.

Global Gene Expression Analysis of Bovine Blastocysts Produced by Multiple Methods

Molecular Reproduction and Development. May, 2008  |  Pubmed ID: 17886272

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.

Aggregation of Bovine Cloned Embryos at the Four-cell Stage Stimulated Gene Expression and in Vitro Embryo Development

Molecular Reproduction and Development. Aug, 2008  |  Pubmed ID: 18196553

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.

Transcript Levels of Several Epigenome Regulatory Genes in Bovine Somatic Donor Cells Are Not Correlated with Their Cloning Efficiency

Cloning and Stem Cells. Sep, 2009  |  Pubmed ID: 19751110

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

[Application of Bio-chip in Lung Cancer Research]

Zhongguo Fei Ai Za Zhi = Chinese Journal of Lung Cancer. May, 2011  |  Pubmed ID: 21569652

Lung cancer is a complicated disease involving many genes and important biologic processes. Genetic variation or aberrant expression of some oncogenes, tumor suppressor genes and DNA repair genes might be related to its incidence and development. How to find out the pathogenic factors of lung cancer and its molecular mechanism of multistage carcinogenesis from massive data is a key question that researchers and clinicians must answer. The bio-chip technology provides a high throughput and efficient method for cancer research. The latest bio-chip progress in lung cancer research is summarized in this minireview.