bluuebunny/crossref_metadata_2025 huggingface dataset:
The tumour suppressor gene p53
Authors: Gangopadhyay Suman B , Abraham Jacinth , Lin Yun Ping , Benchimol Sam
Published: November 2023

The p53 tumour suppressor gene is frequently a target for recessive mutations in a wide range of human and rodent malignancies. The loss of wild-type p53 expression in tumour cells appears to provide these cells with a selective growth advantage in vivo. The existence of mice that are devoid of p53 expression indicates that p53 is not required for normal cell growth or development. However, the increased incidence of tumours in p53 null mice as well as in mutant p53 transgenic mice compared with their normal littermates demonstrates the critical role of p53 in suppressing tumour growth. Two models have been proposed to explain the role of p53 as a tumour suppressor. In one model, p53 participates in the cellular response to DNA damage by delaying cell cycle progression at a Gl checkpoint (see Chapter 7).
Clinical relevance of oncogenes
Authors: Sikora Karol , Pandha Hardey
Published: November 2023

In most developed countries, one person in three will develop cancer during their lifetime. This will increase to one in two by the year 2010, reflecting shifts in the age characteristics of the population. The disease represents a considerable public health problem. The term ‘cancer ‘ comprises over 200 disorders which differ in their genetic basis, aetiology, clinical characteristics, patterns of progression, and final outcome to sufficient degrees to be classified as separate entities. Genetic alterations are at the very centre of tumorigenesis so that, at a cellular level, cancer can be designated a genetic disorder.
Kinetoplast DNA: structure and replication
Authors: Englund Paul T , Guilbride D Lys , hwa kuo-yuan , Johnson catharine e. , Li Congjun , Rocco laura J , Torri AL F
Published: November 2023

Trypanosomatid protozoa are parasites which are responsible for important human diseases in tropical areas of the world. Examples are Trypanosoma brucei, an African trypanosome, T. cruzi, a South American trypanosome, and Leishmania. These parasites are among the most primitive of the eukaryotes and, because of their ancient lineage, it is not surprising that they have unusual biological properties. They are among the first eukaryotes to have mitochondria and one of their most remarkable features is their mitochondrial DNA, known as kinetoplast DNA (kDNA). Unlike any other DNA in nature, kDNA is organized into a network containing several thousand topologically interlocked DNA circles. The circles are of two types. There are several thousand small minicircles and a few dozen larger maxicircles. Figure 1 shows an electron micrograph of part of a kDNA network from the trypanosomatid Crithidia fasciculata. Each cell contains a single network within the matrix of its single mitochondrion (for reviews, see Refs. 1-5).
Developtnental regulation of gene expression in African trypanosotnes
Authors: Pays Etienne , Vanhamme Luc
Published: November 2023

The parasitic way of life of many trypanosomatids requires frequent, drastic, and rapid adaptations to the different environments encountered during their development. In particular, Trypanosoma and Leishmania are cyclically transmitted from different insects to mammals, where some species proliferate intracellularly and others extracellularly. In their mammalian hosts, the parasites live at 37°C and have to protect themselves against lytic factors and cells. They are able not only to recognize and use specific components of the host, such as growth factors, cytokines and surface receptors, but also to produce molecules that influence the activity of mammalian cells, for instance to modify the phenotype of macrophages and lymphocytes. When taken up by insect vectors, they undergo a cold shock as well as a complete change of the extracellular milieu, and must interact with totally different cells. The adaptations of the parasite are achieved through extensive re-programming of gene expression, comparable to that occurring during cell differentiation in metazoans. In this chapter, we review the information available on the genetic mechanisms involved in these differentiation processes.
Trans-splicing in trypanosotnatid protozoa
Authors: Ullu Elisabetta , Tschudi Christian , Gunzl Arthur
Published: November 2023

Trans-splicing is an intermolecular RNA processing reaction in which exons from two separate RNA molecules are joined together to form a mature species (Fig. 1). In one form of trans-splicing, the partners of this reaction are the pre-mRNA and a small RNA, the spliced leader (SL) RNA. The SL sequence, which represents the 5’ end of the SL RNA, is joined to the 3’ splice site located just at the boundary of the mature mRNA within the polycistronic pre-mRNA. The first evidence for this type of trans-splicing (or SL addition) was obtained in trypanosomatid protozoa in 1982 (1), when it was noted that an identical 39-nucleotide sequence is present at the very 5’ end of transcripts coding for different variant surface glycoproteins in Trypanosoma brucei. This sequence, the SL or mini-exon, was subsequently found at the 5’ terminus of every mRNA of trypanosomatids. Critical evidence in support of the trans-splicing model was the detection of various predicted splicing intermediates in T. brucei cells (2--4). It is now clear that trans-splicing and cis-splicing (removal of intervening sequences from pre-mRNA) are fundamentally similar processes. More recently, the list of organisms endowed with trans-splicing has grown to include nematodes (5) as well as trematodes (6, 7) and euglenoids (8).
Mechanisins of drug resistance in protozoan parasites
Authors: Wirth Dyann F , Cowman Alan
Published: November 2023

Drug resistance in microorganisms is an increasing problem in world health today. Commonly used antimicrobial agents are no longer effective and the rate at which resistance develops, often to multiple drugs, has increased the urgency for a solution to this problem. Drug-resistant strains have been found among almost all infectious organisms from viruses to multicellular parasitic nematodes. The impact of drug-resistant infectious diseases on world health is just beginning to be assessed. Certain antimicrobial agents are no longer usable as resistance is so widespread. An example of this trend is the antimalarial drug chloroquine; in many malariaendemic areas, chloroquine resistance is so prevalent that the drug is no longer recommended. Yet, antimalarials are the most commonly purchased drugs in the world. Resistance to other antimalarials is emerging faster than new drugs are being developed. Similar trends are apparent in other infectious diseases and the problem of drug resistance promises to be a major challenge facing the world in the later part of this decade and into the 21st century.
Prokaryotic DNA topology and gene expression
Authors: Drlica Karl , Wu Erden-Dalai , Chen Chang-Rung , Wang Jian-Ying , Zhao Xilin , Xu Chen , Qiu Lin , Malik Muhammad , Kayman, Samuel , Friedman S Marvin
Published: November 2023

Systems that regulate initiation of transcription are built on the binding of specific proteins to their cognate DNA sites. Complexity is generated by interactions of the primary DNA recognition proteins with themselves and with other proteins, sometimes through formation of distinct structures that either block or facilitate transcription. All of these interactions occur on a chromosome that undergoes structural changes mediated by other proteins (topoisomerases and DNA bending proteins) that alter the interactions between specific regulatory proteins and DNA. The aim of this chapter is to provide a framework for understanding how the dynamics of chromosome topology influences gene expression.
Two-component systems
Authors: Atkinson Mariette R , Ninfa Alexander J
Published: November 2023

In this chapter, we will discuss the signal transduction systems known as the two-component systems. This review covers the literature in this area through July 1995. Signal transduction by these systems requires at least one each of two different types of conserved protein domains, known as the histidine protein kinase domain (HPK) and response regulator domain (RR). Signal transduction by the HPK and RR domains involves the reversible phosphorylation of each of these domains and the transfer of phosphoryl groups between HPK and RR domains. Phosphorylation of the response regulator domain results in an altered conformation that permits a regulatory target to be engaged, either by the RR domain itself or, more frequently, by additional protein domains linked to the RR domain that are controlled by the conformational state of the RR domain. Various regulatory mechanisms, some of which act through the HPK domain and others acting directly on the RR domain, regulate the intracellular concentration of the phosphorylated form of the RR domain in response to stimuli. When the intracellular concentration of the phosphorlated form of the RR domain reaches a critical level, the regulatory target is engaged, triggering a physiological adaptation. Thus, the overall logic employed by the two-component systems is that a targeted physiological adaptation is regulated via the control of the concentration of the phosphorylated form of the RR domain. Different mechanisms of regulation of these phosphorylation and phosphotransfer reactions, in as much as these are currently known, will be discussed in this review.
Purification of sequence-specific DNA-binding proteins by affinity chromatography
Authors: Kaplan Ken B , Sorger Peter K
Published: November 2023

This chapter describes methods for the purification of sequence-specific DNA-binding proteins by DNA affinity chromatography. Although it deals exclusively with yeast proteins. the majority of techniques presented should also be useful in the analysis of a broad range of sequence-specific DNA binding proteins from a variety of sources. including animal cells.
Chemical modification
Authors: Imoto Taiji , Yamada Hidenori
Published: November 2023

Chemical modification is one of the most useful methods of identifying the functional groups of a protein. Whether or not the various types of amino acid side chains are involved in a protein’s function can be determined readily by whether or not chemical modification of that type of amino acid affects the function. This requires only chemical reactions that are reasonably specific for each type of amino acid side chain. Suitably specific reactions are available for aspartic acid, glutamic acid, histidine, lysine, arginine, methionine, tryptophan, tyrosine. and cysteine residues. A few, less satisfactory reactions are available for serine, threonine, asparagine. and glutamine residues. hut none arc available for glycine, alanine, valine, leucine, isoleucine, phenylalanine, and proline, which have no reactive groups on their side chains. Fortunately, reactive groups are usually required for important functional roles, so chemical modification is able to test all the likely functional amino acids.
Monitoring protein expression
Authors: Colyer John
Published: November 2023

The methods available to monitor protein expression are numerous and can be employed in combination to explore a particular aspect of protein expression. A cursory examination of the issue of protein expression might leave one with the impression that there are only a few, somewhat straightforward objectives.
Consequences of genomic imprinting for fetal development
Authors: Fundele Reinald H , Suran M Azim , Allen Nicholas D
Published: November 2023

Mouse embryos with only paternal (androgenetic; ag) or maternal (gynogenetic; gg and parthenogenetic; pg) genomes both suffer embryonic lethality but with opposite phenotypes (1-5). This is because of the presence of imprinted genes in the mammalian genome whose expression or repression state is dependent on their parental origin. In addition, the analysis of uniparental isodisomies where specific chromosomes are derived from one parent only has led to the identification of specific chromosome regions that harbour imprinted genes (6-9). In these genetic experiments, there is again the appearance of embryonic and postnatal mice with opposite phenotypes (6, 8).
The cloning of tumour suppressor genes
Authors: Housman David E
Published: November 2023

The identification of a class of genes whose action is required to regulate cell growth and whose absence leads to unregulated cell proliferation can be attributed to two lines of experimental investigation. The term tumour suppressor gene is most clearly associated with the pioneering somatic cell genetic work of Dr Harris and his collaborators and contemporaries in the late 1970s. Terms such as anti-oncogene were first associated with the work of Knudson (1971) and his collaborators who carried out a statistical analysis of the age of onset of childhood cancers and suggested that a specific small number of rate-limiting events was involved in the aetiology of childhood cancers and that hereditary predisposition could reduce the number of such steps to a single rate-limiting event. These two intellectual pathways in fact crossed and merged in the late 1980s and early 1990s.
The road to monoclonal antibodies
Authors: Cotton R G H
Published: November 2023

This article aims to highlight the contribution of cell fusion to the monoclonal antibody technique, and to describe the scientific background to this technique and the early days of its application. The scientific background will be approached both from the broader context of immunogenetics as well as from the local aspect in Cambridge from 1970 to 1974. This summary illustrates the natural progression of scientific findings and techniques towards the development of the monoclonal antibody technique.
The genetic analysis of human cancer
Authors: Stanbridge Eric J
Published: November 2023

Another possibility is that in every normal cell there is a specific arrangement for inhibiting, which allows the process of division to begin only when the inhibition has been overcome by a special stimulus ... i.e. definite chromosomes which inhibit division.Cells of tumours with unlimited growth would arise if those inhibiting chromosomes were eliminated.
Genes that suppress the action of mutated ras genes
Authors: Makoto Noda
Published: November 2023

Three major types of approach have been taken to address the question of how activated ras oncogenes induce malignant cell transformation: enzymology, genetics, and cell biology. Ras proteins have intrinsic guanine nucleotide-binding activity as well as GTP-hydrolysing (GTPase) activity (for review see Barbacid (1987)). Therefore, cellular factors which affect these activities in the test tube can be sought. The GTPase activating protein (GAP) and the guanine nucleotide-exchange factors (GNEFs) have been identified in this fashion (McCormick 1989; Downward et al. 1990; Huang et al. 1990; Wolfman and Macara 1990). Oncogenically mutated (‘activated’) Ras proteins show either decreased intrinsic GTPase activity or poor susceptibility to GAP (reviewed in Barbacid (1987) and Krengel et al. (1990)) and, therefore, GAP probably serves as a negative regulator and GNEFs as positive regulators of Ras. Although enzymology can yield precise results, it has its limitations. For instance, interaction between purified proteins does not necessarily reflect the physiological functions of the proteins in vivo. Also, the applicability of this technique is confined to the molecules directly interacting with Ras proteins.
Unfinished business
Authors: Harris Henry
Published: November 2023

The original experiments that revealed the existence of normal cellular genes that had the ability to suppress malignancy also revealed that this suppression was not achieved by mechanisms that inhibited the multiplication of cells in vitro (Harris et al. 1969). For the fusion of tumour cells with normal fibroblasts generated hybrids that multiplied vigorously in culture but that were unable to produce tumours; and when chromosome segregation permitted the re-emergence of the malignant phenotype, there was often little or no difference in growth rate between the non-malignant hybrids and the malignant segregants derived from them (Harris 1971). It was therefore clear that something happened when the cells were injected into the animal that did not happen when the cells were grown in vitro.
Construction and use of somatic cell hybrids
Authors: Naylor Susan l.
Published: November 2023

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

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

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

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

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

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

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

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

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

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 (<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

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

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

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

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

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

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

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

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

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

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.
Mouse breeding for the generation of ES cell chimeras
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

For ES cell injection blastocysts are needed at day 3.5 after fertilization therefore breeding must be started one day earlier, or 4.5 days prior to injection date. For example, to start injections on a Monday mice breeding must be set up on Thursday of the preceding week.
Macrophage secretory products
Authors: Allavena Paola , Bianchi Giancarlo , Luini Walter , Doni Andrea , Transidico Pietro , Sozzani Silvano
Published: November 2023

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

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.
Model-based methods in analysis of biomedical images
Authors: Cootes Tim
Published: November 2023

Biomedical images usually contain complex objects, which will vary in appearance significantly from one image to another. Attempting to measure or detect the presence of particular structures in such images can be a daunting task. The inherent variability will thwart naive schemes. However, by using models which can cope with the variability it is possible to successfully analyse complex images. Here we will consider a number of methods where the model represents the expected shape and local greylevel structure of a target object in an image. Model-based methods make use of a prior model of what is expected in the image, and typically attempt to find the best match of the model to the data in a new image. Having matched the model, one can then make measurements or test whether the target is actually present.
Assay of membrane transport in cells and membrane vesicles
Authors: Jarvis Simon M
Published: November 2023

Membrane transport is a vectorial process involving the translocation of ions or solutes from one compartment to another. With the exception of simple diffusion, specific proteins catalyse transport. The last decade has seen an explosion in studies applying molecular biology techniques to the identification, classification, localization, and regulation of the transport proteins (see other chapters in this book). An ultimate goal of many studies is to relate structure to function. Transport function is characterized by studying properties of substrate specificity, species of co-substrates and counter-substrates, inhibitors, activators, pH dependence, permeant concentration dependence, and temperature dependence. These functional characteristics rely upon measurements of the initial rates of transport.
Expression of foreign transport proteins in yeast
Authors: Sauer N , Stolz J
Published: November 2023

Over the last few years we have learned from sequencing projects for various organisms that up to 5% of all nuclear genes encode putative transport proteins, emphasizing the importance of these proteins for compartmentation and transmembrane fluxes. The number of cloned transporter genes is increasing exponentially, and the functional analysis of these transporters is an inalienable requirement for understanding their specific functions and their influence on the underlying physiological processes. For that reason, expression of transporter genes or cDNAs in heterologous expression systems has become increasingly important, and expression in yeast has, in particular, turned out to be extremely useful. Besides functional analysis, which includes determination of substrate specificities, ion and pH-dependences, KM values, electrochemical properties, and energy-dependences, expression in yeast has also been the basis for the biochemical characterization of recombinant transport proteins. Numerous well-characterized yeast promoters and yeast mutants represent excellent tools for the large-scale preparation of transport proteins and also for the identification of transporter genes by complementation analyses.
Two-dimensional crystallization of membrane proteins
Authors: Ringler Philippe , Heymann Bernard , Engel Andreas
Published: November 2023

While structure determination of soluble proteins by X-ray crystallography and nuclear magnetic resonance (NMR) imaging has progressed at a remarkable rate, it has been far less successful for membrane proteins, as manifested by the small number solved to atomic resolution. The fact that membrane proteins must be solubilized using detergents leads to large complexes unsuitable for study by NMR. Also, solubilized membrane proteins are often destabilized by the detergent and are thus unsuitable for 3-D (three-dimensional) crystallization. 3-D crystals that have allowed atomic resolution by X-ray crystallography have so far all been obtained from proteins known for their high structural stability. These include four bacterial porins (for a review, see ref. 1), the photosynthetic reaction centre (2), two cytochrome c oxidases (3, 4), two forms of the bovine mitochondrial cytochrome bc1 complex (5, 6), the purple bacterium light-harvesting complex II (7), bacteriorhodopsin (8), and a microbial K+ channel (9). In addition, a membrane inserting toxin (a-haemolysin) has recently been crystallized in 3-D (10).
Mapping genomes
Authors: Denny Paul , Brown stephen D M
Published: November 2023

It is an exciting time in mammalian genetics, as the power of molecular genetic and genomic tools allows a genetic approach to many different biological problems in both mouse and man. The rapid pace of development means, however, that this chapter will quickly become out of date, so we advise the reader to consult the literature for novel and improved methods. The methods described here outline our approach to the generation of genetic and physical maps. There are alternative approaches but there is insufficient space to pro vide a completely comprehensive set of protocols. We describe the use of genetic and genomic resources to construct maps de nova as well as the use of published genetic maps for the construction of higher resolution physical maps such as for positional cloning of an unknown gene.
Mutagenesis of the mouse germline
Authors: Justice Monica J
Published: November 2023

Concern about genetic risk from radiation and chemicals has, in the past, directed the research of germline mutagens in the mouse. Now, however, the Human Genome Project compels functional studies of the mammalian genome (1). This requires new mutations, changing the focus for mouse mutagenesis from studies of genetic risk to isolating new variants at a high frequency (2,3). The value of mutagenesis in the mouse is compounded by the highly developed comparative genetic linkage map between mouse and human, which allows mammalian gene functions to be defined in conserved regions and provides mouse models of human diseases.