Detect Marker Genes Research Articles

Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions

The use of strong promoter systems for recombinant protein production generates high product yields, but also overburdens the host cell metabolism and compromises production. Escherichia coli has highly developed regulatory pathways that are immediately responsive to adverse conditions. To gain insight into stress response mechanisms and to detect marker genes and proteins for stress specific monitoring time course analysis of controlled chemostat cultivations was performed using E. coli total microarray and difference gel electrophoresis (Ettan™ DIGE). In order to detect differences and consistencies of stress response as well as the impact of the inducer isopropyl-β- d-thiogalactopyranosid on cells, expression of two recombinant proteins (hSOD and GFPmut3.1) was investigated. Genes involved in aerobic metabolism under control of the ArcB/ArcA two component system were found to be down-regulated, and the interplay of the psp operon, ArcA system and guanosine tetraphosphate is suggested to be involved in stress regulatory mechanisms. A distinct impact of the two recombinant proteins was observed, particularly on levels of known stress regulatory genes and proteins, as well as on the response associated with ArcA and psp. Altogether, 62 genes as well as seven proteins showed consistent expression levels due to recombinant gene expression, and are therefore suggested to be appropriate monitoring targets.

Application of suspension array assay to detect marker genes expression of circulating tumor cells for early prediction of breast cancer metastasis

To develop a suspension array assay to detect the expression of multiple genes in the circulating breast cancer cells simultaneously so as to identify the marker genes for human breast cancer metastasis. Peripheral blood samples were obtained from 73 breast cancer patients, including 31 breast cancer metastasis patients, 30 patients with benign breast diseases, and 40 healthy women, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted and cDNA was synthesized. PCR was used to amplify 8 breast cancer-related genes: hMAM, HER2, CK19, SBEM, EPG2, hTERT, beta-HGG, and B305D. Suspension array of the PCR products was developed and underwent Luminex 100 laser Flow-type analysis to read the fluorescence signal. COX proportional hazard model was used to find the independent prognostic predictors of breast cancer metastasis. hMAM expression was detected in 57.5%, HER2 in 57.5%, CK19 in 53.4%, SBEM in 52.1%, EPG2 in 31.5%, hTERT in 26.1%, beta-HCG in 21.9%, and B305D in 15.1% of the blood samples respectively. Compared with serum CA15-3 detection, the multigene detection has higher sensitivity (P < 0.05). The expression of SBEM-mRNA in the peripheral blood was correlated with the stage of breast cancer (P < 0.05); and hMAM, SBEM, HER2, and ER could be considered as the independent prognostic predictors of breast cancer metastasis (all P < 0.05). The suspension array assay thus developed is practical in diagnosis of the prognosis of breast cancer. The expression of hMAM, SBEM, and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.

Global Gene Expression Profiling of Circulating Endothelial Cells in Patients with Metastatic Carcinomas

Increased numbers of endothelial cells are observed in peripheral blood of cancer patients. These circulating endothelial cells (CECs) may contribute to the formation of blood vessels in the tumor or reflect vascular damage caused by treatment or tumor growth. Characterization of these cells may aid in the understanding of the angiogenic process and may provide biomarkers for treatment efficacy of angiogenesis inhibitors. To identify markers typical for CECs in cancer patients, we assessed global gene expression profiles of CD146 immunomagnetically enriched CECs from healthy donors and patients with metastatic breast, colorectal, prostate, lung, and renal cancer. From the generated gene profiles, a list of 61 marker genes for CEC detection was generated, and their expression was measured by real-time quantitative PCR in blood samples from 81 metastatic cancer patients and 55 healthy donors that were immunomagnetically enriched for CECs. A set of 34 genes, among which novel CEC-associated genes, such as THBD, BST1, TIE1, POSTN1, SELE, SORT1, and DTR, were identified that were expressed at higher levels in cancer patients compared with healthy donors. Expression of the VWF, DTR, CDH5, TIE, and IGFBP7 genes were found to discriminate between cancer patients and "healthy" donors with a receiver operating characteristic curve accuracy of 0.93. Assessment of the expression of these genes may provide biomarkers to evaluate treatment efficacy.

Prospects for using genetic transformation for improved SIT and new biocontrol methods.

The genetic manipulation of non-drosophilid insect species is possible by the creation of recombinant DNA constructs that can be integrated into host genomes by several transposon-based vector systems. This technology will allow the development and testing of a variety of systems that can improve existing biological control methods, and the development of new highly efficient methods. For programs such as sterile insect technique (SIT), transgenic strains may include fluorescent protein marker genes for detection of released insects, and conditional gene expression systems that will result in male sterility and female lethality for genetic sexing. Conditional expression systems include the yeast GAL4 system and the bacterial Tet-off and Tet-on systems that can, respectively, negatively or positively regulate expression of genes for lethality or sterility depending on a dietary source of tetracycline. Importantly, strains for male sterility must also incorporate an effective system for genetic sexing, since typically, surviving females would remain fertile. Models for the use of these expression systems and associated genetic material come from studies in Drosophila and, while many of these systems should be transferable to other insects, continued research will be necessary in insects of interest to clone genes, optimize germ-line transformation, and perform vector stability studies and risk assessment for their release as transgenic strains.

Protein Transduction Domain of HIV-1 Tat Protein Promotes Efficient Delivery of DNA into Mammalian Cells

The plasma membrane of mammalian cells is one of the tight barriers against gene transfer by synthetic delivery systems. Various agents have been used to facilitate gene transfer by destabilizing the endosomal membrane under acidic conditions, but their utility is limited, especially for gene transfer in vivo. In this article, we report that the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) greatly facilitates gene transfer via membrane destabilization. We constructed recombinant lambda phage particles displaying Tat peptide on their surfaces and carrying mammalian marker genes as part of their genomes (Tat-phage). We demonstrate that, when animal cells are briefly exposed to Tat-phage, significant expression of phage marker genes is induced with no harmful effects to the cells. In contrast, recombinant phage displaying other functional peptides, such as the integrin-binding domain or a nuclear localization signal, could not induce detectable marker gene expression. The expression of marker genes induced by Tat-phage is not affected by endosomotropic agents but is partially impaired by inhibitors of caveolae formation. These data suggest that Tat peptide will become a useful component of synthetic delivery vehicles that promote gene transfer independently of the classical endocytic pathway.

Utilization of polymerase chain reaction technology in the detection of solid tumors.

Most cancer detection tests currently performed are based on either antibody assays to a marker protein with altered expression in cancer patients or on imaging studies to identify characteristic lesions. Generally, for a positive result, these detection assays require that a tumor have a significant volume of cancer cells. Advances in diagnostic techniques and technology may allow for cancer detection at earlier stages, when the tumor burden is smaller and potentially more curable. The molecular techniques of polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) are highly sensitive methods for detecting a small number of cancer cells. Over the past few years, numerous clinical studies have used PCR techniques to detect physical alterations of genes, such as mutations, deletions, translocations and amplification, the presence of oncogenic viruses, and the expression of genes specific to tissue, cancer, and metastasis. The current status of PCR as a method for detecting marker genes in the management of solid tumors is reviewed. A review of the literature on the clinical utility of PCR and RT-PCR in the detection of solid tumor micrometastasis was conducted. Amplification by PCR is a highly sensitive method to determine gene expression. A single cell expressing a tumor marker among 10-100 million lymphocytes can be detected by the PCR assay. This approach has been used to detect tumor cells in approximately 18 different solid tumor types, with melanoma and carcinoma of the breast and prostate the most widely investigated to date. PCR-based assays have been used to detect cancer cells in biopsies of solid tissue, lymph nodes, bone marrow, peripheral blood, and other body fluids. Several studies have reported a high specificity and sensitivity of tumor marker detection and a high correlation between PCR results and the presence of metastatic disease. However, in a few studies, PCR assays have not consistently demonstrated a higher sensitivity and specificity of detection than traditional modalities for many types of cancer. There has been a wide range in sensitivity and specificity among the studies, which may be partly attributed to the lack of uniformity among the PCR protocols used in different studies. PCR can detect tumor marker-expressing cells that are otherwise undetectable by other means in patients with localized or metastatic cancer. Reports from various study groups have lacked uniformity in their protocols, and this has prevented adequate comparison. The clinical utility of this assay as a tool for the prognosis and management of cancer patients remains and area of active investigation. PCR is a powerful tool in the study of the biology of cancer metastasis and will likely serve as a useful adjunct to clinical decision-making in the future.

The Expression of Prostate-Specific Membrane Antigen in Peripheral Blood Leukocytes

Purpose Prostate-specific membrane antigen (PSM) and prostate-specific antigen (PSA) have been used as marker genes for detection of cancer cells in circulation of prostate cancer patients. However, PSA was recently found to be expressed in non-prostate cell lines and normal blood. To evaluate this phenomenon for PSM, we studied its mRNA expression in non-prostatic cells and cell lines, in blood from healthy donors and patients with prostate cancer. Materials and Methods We studied PSM expression by a highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) in peripheral blood of 24 patients with cancer of the prostate (CAP) and 13 healthy young male and female donors, in four non-prostatic cell lines and in isolated blood cells. The identity of the RT-PCR product was confirmed by sequencing. Contamination of the samples with cDNA or prostatic RNA was rigorously excluded by subjecting each sample to PCR reaction without RT-enzyme and by RT-PCR with PSA primers, respectively. Results We found PSM mRNA expression in blood from 18 of 24 CAP patients and 12 of 13 healthy donors and in the leukocyte fraction of normal blood cells. PSM expression could not be detected in erythroblasts, platelets, K-562, U-937, HL-60 or Jurkat cell lines. Conclusions Our results indicate that sensitive nested RT-PCR method detects PSM mRNA in the leukocyte fraction of normal blood. This “background” expression is probably caused by a leaky promoter of PSM. We conclude that it is necessary to develop quantitative RT-PCR assays to differentiate PSM mRNA expression derived from circulating cancer cells from background expression in blood cells.

Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida

In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE-ColA protein was expressed in E. coli under conditions of phosphate starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.

Intrachromosomal homologous recombination in whole plants.

A system to assay intrachromosomal homologous recombination during the complete life-cycle of a whole higher eukaryote was set up. Arabidopsis thaliana plants were transformed with a recombination substrate carrying a non-selectable and quantitatively detectable marker gene. The recombination substrates contain two overlapping, non-functional deletion mutants of a chimeric beta-glucuronidase (uidA) gene. Upon recombination, as proven by Southern blot analysis, a functional gene is restored and its product can be detected by histochemical staining. Therefore, cells in which recombination events occurred, and their progeny, can be precisely localized in the whole plant. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Meristematic recombination events revealed cell lineage patterns. Overall recombination frequencies typically were in the range 10(-6)-10(-7) events/genome. Recombination frequencies were found to differ in different organs of particular transgenic lines.

Inheritance of isozyme variants in leaf tissues of hinoki, Chamaecyparis obtusa, and allozyme diversity of two natural forests.

Isozyme variation found in leaf tissues of hinoki, Chamaecyparis obtusa, were investigated using polyacrylamide vertical slab gel electrophoresis. Twelve enzyme systems were examined in selfed and crossed families to detect marker genes, and electrophoresis banding variants for 10 of the 12 enzyme systems were observed. Judging from the segregation data, 14 Ioci were identified. The genetic variation of three populations of two natural forests was surveyed using these isozyme loci. The average proportion of polymorphic loci (95 % criterion) was 56.4%, the mean number of alleles per locus was 2.00, and the mean expected heterozygosity was .228. The results of the gene diversity analysis and genetic distance indicated these populations are differentiated little from each other.

Genetic Control of Isozyme Variation in Needle Tissues of Cryptomeria japonica

Using polyacrylamide vertical slab gel electrophoresis, we developed methods for detecting allozyme variants of Cryptomeria japonica from needle tissues. We examined 18 enzyme systems to detect marker genes