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1.  Rapid site-directed domain scanning mutagenesis of enteropathogenic Escherichia coli espD 
We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 - 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5-10 ng/μl and the final concentration of template to 1-2 ng/μl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest.
PMCID: PMC2211572  PMID: 18213361
Mutagenesis, Site-Directed; Polymerase Chain Reaction; Plasmids; Sequence Deletion
2.  Development of human gene reporter cell lines using rAAV mediated homologous recombination 
Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used for selection of correctly targeted alleles by taking advantage of known regulatory conditions that activate transcription of specific genes. Using this method, we describe the generation of primary human fibroblasts that express EGFP-Luciferase under the control of the c-Myc oncogene.
PMCID: PMC2374725  PMID: 18464937
Dependovirus; Proto-Oncogene proteins c-myc
3.  Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations 
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.
PMCID: PMC2275047  PMID: 18385809
receptors, antigen, b-cell; immunomagnetic separation
4.  Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine 
Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.
PMCID: PMC2274965  PMID: 18385803
electrophoresis, gel, two-dimensional; oxidation-reduction; protein processing, post-translational
5.  An in vitro method to study the effects of hematopoietic regulators during immune and blood cell development 
In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1Î and the neuropeptide, substance P (SP). SDF-1Î production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1Î levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34+ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.
PMCID: PMC2266633  PMID: 18335004
cell culture techniques; hematopoiesis; cytokines; hematopoietic stem cells
6.  Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (Sparus aurata L.) 
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.
PMCID: PMC2211574  PMID: 18213363
Sea Bream; Flow Cytometry; Respiratory Burst
7.  Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array 
We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.
PMCID: PMC2211573  PMID: 18213362
Genotype; Microspheres; Polymorphism, Genetic
8.  Generation of shRNAs from randomized oligonucleotides 
Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can be used for various functional assays, such as target validation when a suitable screening or selection method is available.
PMCID: PMC2211575  PMID: 18213360
RNA Interference; RNA, Small Interfering; Oligonucleotides
9.  A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections 
We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of β-galactosidase (βgal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of βgal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.
PMCID: PMC1821346  PMID: 17364022
Mice, Transgenic; Brain Chemistry; beta-Galactosidase

Results 1-9 (9)