bluuebunny/crossref_metadata_2025_split huggingface dataset:
Construction and use of somatic cell hybrids
Authors: Naylor Susan l. 
Published: November 2023

Abstract
Somatic cell hybrids provided the first parasexual means for studying human genes (1, 2). Interspecific hybrids were found to lose the chromosomes of one species, allowing genetic characters to be assigned to particular chromosomes by following their segregation among hybrid clones (1). Since this tenuous beginning in 1967, thousands of genes and DNA markers have been located on human chromosomes using somatic cell hybrids. The same hybrid technology is also used in the formation of hybridomas to make monoclonal antibodies (3) and to map genes in other species (4).
The use of microdissected chromosomes in genome mapping
Authors: senger, Uwe claussen. Gabriele , chuboda ilse 
Published: November 2023

Abstract
The mapping and cloning of defined regions of the genome requires a high density of DNA markers for the region of interest. Flow-sorting, as discussed in the previous chapter. provides a source of intact chromosomes. The physical dissection of banded metaphase chromosomes complements flow-sorting, making it possible to isolate small, defined parts of chromosomes in pure form.
Chromosome walking
Authors: ragoussis Jiannis , Olavesen mark g. 
Published: November 2023

Abstract
The assembly of overlapping clones to form a ‘contig’ can be done in one of two ways. Where large regions (or entire genomes) are to be covered, it is often most efficient to select clones at random from a library representing the region, to characterize them, and thereby establish which clones overlap one another; this is ·shotgun’ contig assembly (Chapter 10). However, when smaller regions are to be covered (for example, the gap between two genetic markers known to flank a locus of interest), a more systematic approach becomes necessary. One clone is chosen as a starting point or ‘founder’. and clones which partially overlap it are selected (rather than fortuitously found) from the clone library. One of these new clones is then chosen, and the process repeated. In this way, a series of partially overlapping clones is assembled progressively-a process called ·chromosome walking’ (1, 2).
In situ hybridization
Authors: Southern Shirley A , Herrington C Simon 
Published: November 2023

Abstract
Polymerase chain reaction in situ hybridization (PCR ISH) combines components of both PCR and ISH. It is important, therefore, to have some understanding of ISH before attempting its combination with PCR. In direct PCR ISH, the cell/tissue pre-treatment protocols are based on ISH and similar detection systems are used. In indirect PCR ISH (see Chapter 4), PCR is followed by conventional ISH and the procedures are much the same as those used without PCR.
Automation of in situ amplification
Authors: O’leary John J 
Published: November 2023

Abstract
In situ polymerase chain reaction (PCR) amplification has been carried out using standard thermocyclers, heating blocks, or cycling ovens, none of which was specifically designed to perform these techniques. Initially most investigators covered a standard multi-well PCR block with an aluminium foil boat into which the slide containing the tissue section or cell suspension was placed and overlaid with PCR reaction mix and mineral oil.
Total DNA isolation
Authors: Milligan Brook g. 
Published: November 2023

Abstract
Population geneticists are relying increasingly on the study of DNA variation within and among populations. However, the feasibility of such studies is often limited by one's ability to isolate DNA from many individuals. The problem of dealing with many samples is particularly acute for the study of plants and ancient or forensic samples, because often intact DNA must be isolated from a complex and usually unknown chemical mixture. In the case of plant samples, difficulties encountered include DNA degradation due to the presence of either native DNases or secondary plant compounds, and the co-isolation of polysaccharide contaminants that inhibit further enzymatic reactions. In the case of ancient or forensic samples, difficulties include contamination with modem DNA and in situ degradation under inevitably sub-optimal preservation conditions.
Detection of genetic variation by DNA conformational and denaturing gradient methods
Authors: dean Michael , Milligan brook g. 
Published: November 2023

Abstract
Single-stranded conformation polymorphism (SSCP) is one of the most widely used and practical approaches for the detection of mutations in DNA and for the detection and analysis of DNA variation (1). The principal advantage of the method is that it is very rapid to perform and can be carried out using equipment available in most molecular biology laboratories. If properly opti mized the method is highly sensitive, and different mutations within a frag ment can often be distinguished on the same gel (2). A number of variations of SSCP have been developed that allow analysis of RNA or for the assay to be carried out non-radioactively, as reviewed in ref. 3. SSCP is often combined with heteroduplex analysis to increase further the sensitivity of the method. Despite the denaturation of the sample prior to loading the SSCP gel, there is often the formation of a significant fraction of double-stranded DNA. This is visible at the bottom of the SSCP gel. Since heteroduplexes can occasionally be resolved from homoduplexes, the appearance of heteroduplexes on the SSCP gel can give additional information on the presence of variants.
Gene therapy applications of growth factors
Authors: Svensjo Tor , Yao Feng , Pomahac Bohdan , Eriksson Elof 
Published: November 2023

Abstract
Gene therapy (GTh) involves transfer of DNA to somatic cells with the purpose of getting those cells to express certain proteins or antisense RNA. Currently all protocols for GTh are experimental, but with the ultimate goal of curing patients. Remarkable progress has marked the field of GTh in the past decade. Since the first Recombinant DNA Advisory Committee (RAC) approved treatment that employed gene transfer (GTr) to study and treat human disease (1) the number of clinical trials involving GTh has increased exponentially, and as of March 1996 there were 145 approved human GTh protocols in the USA (2). Diseases that have been targeted include several congenital disorders (e.g. adenosine deaminase (ADA) deficiency, cystic fibrosis, and familial hypercholesterolaemia) and acquired immunodeficiency syndrome (AIDS), but more commonly the therapies are targeted at cancer (3).
Characterization of liposoms
Authors: Zuidam Nicolaas Jan , Vrueh Remco de , Crommelin Daan J A 
Published: November 2023

Abstract
The chemical and physical characteristics of liposomes determine their in vivo and in vitro behaviour (1, 2). Thus, the availability of analytical methods to establish these characteristics is essential. Extensive work was performed over the last thirty years on characterization of liposomes and up until now FDA approved four parenteral liposome drug products. But, no formal guidelines for liposome characterization have been defined yet (for reviews see refs 3 and 4, and references listed therein). Quality control of liposomal dispersions is complicated by the supermolecular nature of the systems to be analysed. The various components are held together by relatively weak interactions to form lipid vesicles of the desired size and lamellarity. Moreover, all chemical analyses require dissolution, solubilization, or extraction of the liposomal components, which must be complete, non-selective, and enable analysis and quantification of each of the components. Table 1 lists various quality control characteristics of a liposomal formulation that should be assessed during the different phases of its development from research product to commercial product.
Encapsulation of Weakly-Basic Drugs, Antisense Oligonucleotides, and Plasmid DNA within Large Unilamellar vesicles for Drug Delivery Applications
Authors: Fenske David B , Maurer Norbert , Cullis Pieter R 
Published: November 2023

Abstract
Liposomes are microscopic spheres consisting of one or more lipid bilayers arranged concentrically about a central aqueous core. Liposomes were first described over thirty-five years ago (1), and it was not long after that their usefulness as models of biological membranes and their potential as systems for the systemic delivery of drugs was recognized (2). Development of this potential required techniques for the generation of unilamellar vesicles and encapsulation of drugs and macromolecules within them. Although a wide variety of methods were developed for the formation of liposomes (3, 4), many of them were technically demanding, time-consuming, and did not generate liposomes of optimal size and polydispersity. Likewise, early attempts at encapsulating drugs within liposomes relied on passive entrapment methods, which resulted in low encapsulation levels (&lt;30%) and poor retention of drugs (5). Nevertheless, early in vivo experiments on liposomal drug systems were encouraging enough to fuel further development (see ref. 5 and references therein).
Liposomes in Biological Systems
Authors: Kamps Jan A A M , Scherphof Gerrit L 
Published: November 2023

Abstract
The interaction of liposomes with biological systems is very complex but has bearing on most topics in liposomal drug delivery. In this chapter we will describe the various interactions occurring in biological systems and the factors that determine these interactions. Biological systems in liposome research include relatively simple cell culture set-ups, organ perfusion systems, and the intact organism. Although we will touch on these different systems, emphasis will be on liposome-cell interaction and whole body distribution of liposomes, including the means to determine the interaction processes involved. The first part of this chapter deals with in vitro liposome-cell interactions. The different kinds of interaction that can occur at the level of liposome-cell contact will be discussed, including practical methods to distinguish them. The consequences of these interactions for the different liposomal components, i.e. bilayer lipids and the liposomal contents, and various options to analyse these will be reviewed. Most of the above aspects will also be dealt with in the second section of this con-tribution, which covers biodistribution of liposomes in animal models. Considerable attention will be given to the role of the liver as the main site of uptake from the blood and to the contribution of the different liver cell types to the in vivo uptake of liposomes. The experimental protocols given are a selection of protocols available in this research area but they are all practical and generally do not require exceptional laboratory equipment.
Introduction
Authors: Forrest Sue 
Published: November 2023

Abstract
Mutation detection is becoming vital to the whole of biology, let alone medicine. In medical research it is fundamental to disease gene isolation, mutation spectrum studies, and diagnosis. It is most important in the area of cancer and cancer research where increasing numbers of cancer genes have been isolated over recent years. In biology, commercially important genes can be identified by the mutations they contain. Strains of organisms can be characterized because they differ by a number of mutations. It is not surprising then that there is a rising interest in mutations and mutation detection.
Single-stranded conformation polymorphism and heteroduplex analysis
Authors: Gerrard Bernard , Dean Michael 
Published: November 2023

Abstract
Single-stranded conformation polymorphism (SSCP) alone or in combination with heteroduplex analysis (HA) is one of the most widely used and practical approaches for mutation detection (1). The principal advantage of this method is that it is rapid to perform and can be carried out using equipment available in most molecular biology laboratories. When properly optimized, the method is highly sensitive, and many different mutations within a DNA fragment can often be distinguished on the same gel (2, 3). SSCP and HA can be performed on the same gel since, after the denaturation of the sample prior to loading, there is often the re-formation of a significant amount of double stranded DNA which will appear in a lower position from the single-stranded products on the SSCP gel. Since heteroduplexes can often be resolved from homoduplexes, the appearance of heteroduplexes on the SSCP gel can give additional information on the presence of variants.
Histomorphometry
Authors: Reynolds A J 
Published: November 2023

Abstract
The analysis of histological material has traditionally been by comparative morphological analysis. A section of tissue, suitably processed and stained, is examined under a light microscope by a skilled microscopist and a diagnosis made by observing the morphology of the tissue and cells. Whilst this approach is suitable for the majority of analyses conducted, there is a growing need for a more quantitative methodology. Visual assessment of histological preparations is poorly reproducible and measurement has several advantages such as: • objectivity • reproducibility • the ability to detect changes not immediately apparent to the naked eye (1) A problem encountered in interpreting a microscopic image is that one sees a flat image of a section cut from a three-dimensional object and the viewer is required to visualize the spatial context of the object. We are used to seeing objects as a three-dimensional projection of that object and we have a natural tendency to interpret flat sections as projections. An example is that a circular profile will generally be interpreted as being derived from a sphere but, it could also have come from a cylinder. Problems can become compounded if we want to obtain information about the actual size of spatial objects from a study of sections cut from the bulk.
Near-field optical microscopy
Authors: Van Hulst Niek F 
Published: November 2023

Abstract
This is a chapter on an emerging field in optical microscopy: near-field optics, i.e. the optics of sub wavelength-sized structures. It may seem surprising to find any aspect unexplored within the mature and well-established domain of optical microscopy. Indeed optical microscopy has reached an outstandingly high level of perfection through centuries and established itself as the main tool in biological studies through its flexibility, applicability, and multitude of optical contrast methods. Yet, a natural limitation had always to be faced: the diffraction limit. It is fundamentally impossible to focus light to an area smaller than the size of the wavelength. Consequently the spatial resolution in lens-based optical microscopy is limited to about half the wavelength. This barrier, already recognized in the previous century by Abbe, has been a stimulus to develop alternative techniques like electron and ion microscopy. Indeed these alternatives have pushed the resolution to the atomic level (in transmission electron microscopy), however generally with the loss of the ‘optical’ advantages, such as: non-invasiveness, non-destructiveness, operation in native environment (in vivo), high resolution spectroscopic contrast, polarization contrast, and time resolution.
Nonselectable modifications and removal of selection marker genes
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
One of the most straightforward purposes for using Cre/loxP is to remove a loxP-flanked (floxed) selectable marker after homologous recombinants have been identified in a gene targeting experiment. This is certainly important for any experiment designed to modify, but not abrogate, gene function by preventing possible interference of the selectable gene (and its own regulatory elements) on gene function and/or regulation (20, 21, 87). The range of modifications which can be introduced into the target gene is virtually unlimited ranging from alterations in the coding region, like point mutations or exon insertion (or deletion), to changes in regulatory elements such as the promoter/ enhancer region of a gene. Thus, the design of a targeting vector will vary depending on the nature of your specific question. In some instances the genomic sequences which are to be deleted may simply be replaced by the selectable marker gene in the targeting vector; elements to be inserted can be directly linked to the selection marker creating a single region of nonhomology in the vector. In many cases, however, it will be more appropriate to insert the selection marker (without replacing genomic sequence) into a different position than the desired modification in the targeting vector to avoid the mutation being directly neighbored by the loxP site, which remains in the genome after Cre-mediated deletion of the floxed marker gene. This is an obvious requirement if the coding region of a gene should be altered but might be equally important for the modification of regulatory regions.
Gene replacement
Authors: Replacement Gene 
Published: November 2023

Abstract
The design of targeting vectors-for gene replacement combines features of conventional replacement vectors with the strategy to generate large deletions (see above and Figure 6). In general, the aim of such experiments is to replace endogenous wildtype sequence by a heterologous sequence (e.g. the human for murine homologue). The advantage of this strategy compared to simple insertions by conventional replacement vectors lies in the option to remove regions of the original gene, together with the selection marker used in the first targeting step, by Cre-mediated deletion. In this manner, a gene replacement vector encloses a long region of nonhomology (e.g. consisting of the intron/exon or promoter region of the gene to be introduced) and a selection marker cassette with one loxP site (e.g. loxP-neo or loxP-neo-tk). A second loxP site is placed into the homology region of the vector such that Cre-mediated deletion in homologous recombinant ES cell clones generates the desired deletion of the endogenous sequence together with the selection marker cassette (Figure6).
Conditional gene modification
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
Following one of the three strategies described in this section, from a single targeting construct, ES clones can be generated which harbor either a loxP-flanked (floxed) allele suitable for conditional gene targeting or which contain the final genetic modification resulting from ere-mediated deletion or inversion. For the floxed allele subsequent ere-mediated recombination will result in gene inactivation or modification, depending on loxP positioning and orientation. The mice derived from ES cells with a floxed allele can be crossed to ere transgenic mice for conditional gene modification or inactivation in specific cell types (Chapter 11). Mice harboring the product of ere-mediated deletion or inversion of the floxed allele in all cells throughout development, like conventional mutants, can be generated simultaneously from the second type of ES clones. Alternatively, such mice can be obtained by crossing of mice harboring the floxed allele to one of the “deleter” strains (96, 97, see Chapter 11), which express are ubiquitously in the early embryo.
LoxP-containing transgenes
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
As discussed in the previous chapters the Cre/loxP system offers a variety of possibilities in the manipulation of targeted endogenous genes - both in the ES cell genome and mice derived from them. Moreover, Cre recombinase can be also used to activate (or inactivate) in vivo loxP interrupted, randomly integrated transgenes introduced into the mouse gerrnline either by pronucleus injection or the ES cell route. Similar to the conditional gene targeting strategy (Figure 13), transgene expression can be controlled by the pattern of Cre expression in vivo, i.e., in a tissue¬ specific or temporal manner via Cre-mediated recombination (59, 60). Especially attractive with this type of transgene manipulation is the Cremediated activation of (trans)gene expression. In this strategy, two transgenic strains, one expressing Cre and the other containing the recombination target must be generated and crossed together thereby allowing the synthesis of the protein encoded by the transgene only in double transgenic animals.
ES cell culture and transfection
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
When maintaining ES cells in culture the primary consideration is to prevent their spontaneous differentiation. Any homologous recombinants you obtain will be of no use if the cells are mistreated and lose their ability to colonize the germ cells of the developing chimeric embryo (the exception to this is the possible use of chimeric analysis or blastocyst complementation - see Chapter 21). Therefore great care and consideration should be given to the propagation of these cells in tissue culture. The previous chapter gives a few guidelines used in the routine handling and maintenance of the ES cell lines used in our laboratory. ES cells used for electroporation should be growing exponentially since the transfection efficiency and perhaps also the frequency at which homologous recombination occurs appears to be increased in optimally growing cells compared to dense ES cultures. For the same reason we prefer to feed ES cultures with fresh medium a few hours prior to transfection. For gene targeting purposes the fraction of cells being in S phase at the point of transfection might be critical as homologous recombination, but not random integration was found to depend on the cell cycle, peaking when DNA is introduced into the nucleus during S phase, as tested in rat-20 cells (161). Thus, ES cells should be plated out in defined numbers 1−2 days before transfection and frequently inspected to meet the point when the cultures contain sufficient cells for transfection but are still in the optimal growth phase.
Macrophage secretory products
Authors: Allavena Paola , Bianchi Giancarlo , Luini Walter , Doni Andrea , Transidico Pietro , Sozzani Silvano 
Published: November 2023

Abstract
Mononuclear phagocytes play a fundamental role in tissue remodelling as a first line of resistance against pathogens and in the activation of specific immunity. These functions of cells of the monocyte/macrophage lineages are mediated to a large extent by the production of secretory molecules. The secretory capacity of mononuclear phagocytes is enormous and the repertoire of secreted molecules is vast and diverse, ranging from lipid mediators, to enzymes, cytokines, and their receptors (1). A simplified classification and view of macrophage secreted molecules is provided in Table 1.
Three-dimensional (3D) reconstruction from serial sections
Authors: Verbeek Fons J 
Published: November 2023

Abstract
This chapter describes the theory and application of three-dimensional (3D) reconstruction from serial sections. The technique has a long history, starting by sculpturing from sections via photomicrographs to a wax model and evolving to a virtual 3D model exclusively existing in computer memory. The latter method is, at least to some extent, automated and therefore referred to as computer-assisted reconstruction (CAR). CAR is commonly applied in the laboratory. However, computerizing the reconstruction process has implications for laboratory practice, and clear and unambiguous terminology is important and necessary. The biological problems to be solved with 3D reconstruction tend to be complex. Ideally, finding solutions is an interaction between the life sciences and computer sciences. The computer1 is the means to solve the problems raised, serving as the objective assistant to the process, and solutions are formulated in terms of applied physics since the whole procedure depends on the formation and manipulation of the microscope image. Computer science provides the computational resources because 3D reconstruction requires effective algorithms for processing, as well as for data management. Interpretation of the data of course remains the exclusive domain of the biologist.