| 1. |
- Ahmad, Faiyaz, et al.
(författare)
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Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B
- 2005
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. ; 307, s. 93-107
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.
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| 2. |
- Allen, Marie, et al.
(författare)
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Mitochondrial D-loop and coding sequence analysis using pyrosequencing
- 2005
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Ingår i: Methods in Molecular Biology. - New Jersey : Humana Press. - 1064-3745 .- 1940-6029. ; 297, s. 179-196
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Tidskriftsartikel (refereegranskat)abstract
- In forensic casework analysis, mitochondrial deoxyribonucleic acid (DNA) often is used when the evidence material contains scarce amounts of DNA. Here, a mitochondrial DNA typing system for D-loop and coding region analysis based on pyrosequencing is described. Pyrosequencing is a real-time, single-tube sequencing-by-synthesis method, in which a cascade of enzymatic reactions yields detectable light. This pyrosequencing system has a higher resolution than the D-loop analysis performed routinely today as it also covers informative positions in the mitochondrial coding region. The system is composed of 16 polymerase chain reaction (PCR) fragments and 24 pyrosequencing reactions with a turn around time for a 96-well plate of less than 3 h after PCR.
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| 3. |
- Allen, Marie, et al.
(författare)
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Universal tag arrays in forensic SNP analysis.
- 2005
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 297, s. 141-154
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Tidskriftsartikel (refereegranskat)abstract
- Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.
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| 4. |
- Altai, Mohamed, et al.
(författare)
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Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting.
- 2020
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2105, s. 283-304, s. 283-304
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins.
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| 5. |
- Alvarez-Castro, Jose, et al.
(författare)
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Estimation and interpretation of genetic effects with epistasis using the NOIA model.
- 2012
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 871, s. 191-204
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Tidskriftsartikel (refereegranskat)abstract
- We introduce this communication with a brief outline of the historical landmarks in genetic modeling, especially concerning epistasis. Then, we present methods for the use of genetic modeling in QTL analyses. In particular, we summarize the essential expressions of the natural and orthogonal interactions (NOIA) model of genetic effects. Our motivation for reviewing that theory here is twofold. First, this review presents a digest of the expressions for the application of the NOIA model, which are often mixed with intermediate and additional formulae in the original articles. Second, we make the required theory handy for the reader to relate the genetic concepts to the particular mathematical expressions underlying them. We illustrate those relations by providing graphical interpretations and a diagram summarizing the key features for applying genetic modeling with epistasis in comprehensive QTL analyses. Finally, we briefly review some examples of the application of NOIA to real data and the way it improves the interpretability of the results.
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| 6. |
- Andersson, Jan O.
(författare)
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Horizontal gene transfer between microbial eukaryotes.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 532:4, s. 473-487
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Tidskriftsartikel (refereegranskat)abstract
- Comparative genomics have identified two loosely defined classes of genes: widely distributed core genes that encode proteins for central functions in the cell and accessory genes that are patchily distributed across lineages and encode taxa-specific functions. Studies of microbial eukaryotes show that both categories undergo horizontal gene transfer (HGT) from prokaryotes, but also between eukaryotic organisms. Intra-domain gene transfers of most core genes seem to be relatively infrequent and therefore comparatively easy to detect using phylogenetic methods. In contrast, phylogenies of accessory genes often have complex topologies with little or no resemblance of organismal relationships typically with eukaryotes and prokaryotes intermingled, making detailed evolutionary histories difficult to interpret. Nevertheless, this suggests significant rates of gene transfer between and among the three domains of life for many of these genes, affecting a considerably diversity of eukaryotic microbes, although the current depth of taxonomic sampling usually is insufficient to pin down individual transfer events. The occurrence of intra-domain transfer among microbial eukaryotes has important implications for studies of organismal phylogeny as well as eukaryote genome evolution in general.
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| 7. |
- Andreasson, Erik, et al.
(författare)
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Isolation of Apoplast
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1511, s. 233-240
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Tidskriftsartikel (refereegranskat)abstract
- The apoplast can be described as the soluble fraction of the extracellular space of plant tissue, and it plays an important role in signaling, nutrient transport, and plant-pathogen interactions. In this protocol, we describe a method where leaves are infiltrated with phosphate buffer under vacuum. The apoplast can then be extracted by centrifugation and simultaneously collected in a protease inhibitor solution. Using this protocol, typically 3 mu g of apoplastic proteins can be obtained in a volume of 300 mu L from five potato leaflets, with minimal contamination by non-apoplastic proteins.
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| 8. |
- Archer, Amena, et al.
(författare)
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Expression Profiles of Estrogen-Regulated MicroRNAs in Cancer Cells.
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 313-343
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs play critical roles through their impact on posttranscriptional gene regulation. In cancer, they can act as oncogenes or tumor suppressors and can also function as biomarkers. Here, we describe a method for robust characterization of estrogen-regulated microRNA profiles. The activity of estrogen is mediated by two nuclear receptors, estrogen receptor alpha and estrogen receptor beta, and a transmembrane G-protein coupled estrogen receptor 1. This chapter details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, different microRNA profiling approaches, and subsequent confirmations.
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| 9. |
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| 10. |
- Aslıyüce, Sevgi, et al.
(författare)
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Preparation of Staphylococcal Protein A Imprinted Supermacroporous Cryogel Beads
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2466, s. 261-273
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Bokkapitel (refereegranskat)abstract
- Protein A is the most commonly used ligand in IgG purification due to its specific binding to the Fc receptor of most immunoglobulins, making it commercially important. Molecular imprinting is a method based on the selective recognition of various molecules. Molecular imprinted polymers are materials that are easy to prepare, durable, cheap and have molecular recognition capability. Cryogels are prepared by radical polymerization in a partially frozen environment. The unique structure of cryogels combined with osmotic, chemical and mechanical stability make them attractive chromatography matrices for a variety of biological compounds/specimens (plasmids, pathogens, cells). In this protocol, protein A imprinted supermacroporous poly(2-hydroxyethyl methacrylate) cryogels were prepared in spherical form for protein A purification. The characterization of the prepared cryogels were made by swelling test, scanning electron microscopy (SEM), Fourier transform infrared spectrophotometer (FTIR), and Brunauer–Emmett–Teller (BET) surface area analysis. After characterization, optimum conditions for protein A adsorption were determined in the batch system. The maximum protein A adsorption capacity was determined after optimization of the imprinted cryogels. Protein A relative selectivity coefficients of imprinted cryogels were examined for both Fc and protein G. Protein A was isolated from the bacterial cell wall using fast performance liquid chromatography (FPLC). The separated protein A was determined by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). In the last stage, the reusability of the cryogel was examined.
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