bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of in situ PCR techniques to human tissues
Authors: Bagasra Omar , Bobroski Lisa E , Amjad Muhammed , Pomerantz, Roger J , Hansen John
Published: November 2023

Since we first reported our findings regarding in situ amplification of the HIV-1 gag gene in an HIV-1-infected cell line in March 1990 (1), there has been an explosion of research in the area of in situ polymerase chain reaction (in situ PCR). There are over 300 publications describing various forms of in situ gene amplifications (reviewed in 2,3,13) , identifying various infectious agents, tumour marker genes, cytokines, growth factors and their receptors, and other genetic elements of interest, in peer-reviewed journals (1–25). The solution-based PCR method for amplifying defined gene sequences has proved a valuable tool not only for basic researchers but also for clinical scientists. Using even a minute amount of DNA or RNA and choosing a thermostable enzyme from a large variety of sources, the gene of interest can be amplified and then analysed and/or sequenced. Thus genes or segments of gene sequences present only in a small sample of cells or a small fraction of mixed cellular populations can be examined. However, one of the major drawbacks of solution-based PCR is that it does not allow the association of amplified signals of a specific gene segment with the histological cell type(s) (13,14). For example, it would be advantageous to determine what types of cells in the peripheral blood carry aberrant genes in a leukaemia patient and what percentage of leukaemia cells is present after various forms of anti tumour therapy.
Applications and modifications of PCR in situ hybridization
Authors: Patterson Bruce K
Published: November 2023

The localization of genes in specific cells and tissues has been instrumental in many disciplines for defining disease pathogenesis. This pursuit has united scientists studying such diverse topics as oncogene expression, viral infections, and plant pathology. In its earliest incarnation, molecular biologists met with pathologists to use large subgenomic probes to detect genes in situ. This process relied on the intrinsic amplification of genes during transcription into mRNA to accumulate enough target to be detected.
Mitochondrial DNA isolation, separation, and detection of fragments
Authors: White P Scott , Tatum Owatha L , Tegelstrom Hakan , Densmore III Llewellyn D
Published: November 2023

The 'revolution' that began in the 1980s with the application of mitochondrial DNA (mtDNA) analyses to address questions of molecular evolutionary and population biology has now largely become the paradigm for such work (1). Studies ranging in scope from purely systematic problems to identification and recognition of species to documentation of genetic changes in populations that have occurred over a very short period of time, are based on both indirect or direct estimates of nucleic acid variation. While progress continues to be made in analysing coding regions of nuclear DNA, the sequences retained in organellar genomes are still the most widely studied by evolutionary biologists. Restriction site or fragment analyses of mtDNA and direct sequencing of specific regions of the mitochondrial genome following amplification by the polymerase chain reaction (PCR) are currently the methods of choice for the majority of population level studies. The reasons for the increasingly wide spread application of mtDNA markers in studies of animal populations are many; however, there are also a few noteworthy drawbacks (reviewed in ref. 1). Figure 1 shows the typical vertebrate gene order of mtDNA, with birds having a single rearrangement involving ND 6 (a subunit of NAD dehydrogenase) and tRNAGlu not found in other vertebrates examined to date. Other minor rearrangements have been reported, and gene order can provide important synapomorphies (shared derived characters) for deciphering some relationships.
Silane-modified surfaces for biomaterial immobilization
Authors: Shriver-lake Lisa C
Published: November 2023

Biosensors, affinity chromatography, and many bio analytical methods require a high density of functional molecules, low non-specific protein adsorption, long-term stability, and durability (1). Proteins, including antibodies and enzymes, and cells have been immobilized onto solid supports for these and other applications over the last 30 years. In the biosensor arena, optical transduction methods are increasingly employed. With this increase, solid supports such as fused silica and quartz are appropriate substrates for immobilization. Non-specific protein adsorption to these substrates has been a major problem.
Antibodies as immobilization reagents
Authors: Egodage Kamal L , Wilson George S
Published: November 2023

Immobilized antibodies have been used extensively as reagents for the isolation of chemical and biological molecules where there are no naturally occurring ligands. Due to high specificity of the antibodies, the immobilization of specific antigens on different matrices in the required orientation and in high concentration, for use in analytical and biological applications, is possible. The ability to elicit an immune response to a hapten by conjugation to a carrier molecule extends the applicability of antibodies as immobilizing ligands to small molecules. Although polyclonal antibodies have been used as capture matrices, the advent of monoclonal antibody technology has extended and enhanced the use of this approach. Antibodies covalently immobilized to different solid matrices have been used for extraction of biomolecules in plasma (1–3), and for purification of enzymes (4—8), and receptors (9–11). They have also been used as reagents in purification, concentration, and analysis of environmental samples (12, 13). Immobilized antibodies have also been used to prepare immunoreactors for flow injection imrnunoassays (14-16) and to prepare enzyme reactors (17–19).
Bioanalytical applications of self-assembled monolayers
Authors: Liedberg Bo , Cooper Jonathan M
Published: November 2023

The ‘self-assembly’ of thin molecular films of biological or chemical moieties onto surfaces is a process which is of enormous interest in diverse areas of technology, due to the apparent simplicity by which a solid support can be modified or functionalized. Consequently, there has been a tremendous growth in the interest shown in the use of these systems during recent years, with a recognition of their importance in the development of novel surface coatings. It is now dear that, when the deposition procedure is carefully controlled, it is possible to ‘spontaneously’ deposit a single layer of particular species (or, indeed, a mixed monolayer of different species) on a surface, giving rise to the use of the term ‘self-assembled monolayer’ or SAM. Although it is the purpose of this chapter to describe the application of SAMs to bioanalytical methodologies, it should also be acknowledged that the uses of these techniques are numerous and now impinge on many areas of modern materials science. For example, ultrathin SAMs, with predefined compositions have now been used in order to obtain a deeper understanding of interfacial phenomena like adhesion, lubrication, wetting, nucleation, and crystal growth (1, 2).
Protein adsorption: friend or foe?
Authors: Johnston Erika , Ratner Buddy D
Published: November 2023

The phenomenon of protein adsorption is widely observed in nature and in technology. For example, consider marine fouling, contact lenses, food processing equipment, cell growth surfaces, and biosensors. Also, the biocompatibility of medical implants may be driven by the surface adsorbed layer of proteins. When a biological solution comes in contact with a clean surface, proteins and other surface active molecules diffuse rapidly from the bulk liquid and become reversibly or irreversibly adsorbed to the surface (1). Although the compositional mixture, conformation, and binding strength of the adsorbed proteins may vary with time, the conditioning layer is persistent and will mediate all future interactions between the surface and components in the surrounding liquid. The effects attributable to conditioning layers can be separated into two types: those directly associated with the conditioning layer itself (changes in permeability, lubricity, texture, etc.) and those due to cells (e.g. macrophages, platelets, bacteria) that are activated or attracted by the adsorbed material.
Micropatterning cell adhesiveness
Authors: Clark Peter
Published: November 2023

The ability to micro pattern adhesiveness for cells has been exploited by a number of research groups to provide model surfaces for investigating cell behaviour, and for devising technologies in cell and tissue engineering (1–11). These techniques have been developed to make surfaces which provide differentially adhesive cues for cultured cells, i.e. areas which are permissive for cell adhesion and locomotion, with adjacent non-permissive areas. The techniques were developed using a variety of approaches, but have in common the use of, at least at some stages, the micro fabrication technologies of the electronics industries. Though a brief description of the facilities required will be given, it is not within the scope of this chapter to deal with the details of the methods for standard micro fabrication, or for micro-mask formation, which today usually involves computerized ‘writing’, e.g. electron-beam writing of electron-sensitive resists. It will be assumed that anyone embarking on adhesion micro patterning will have access to masks and to micro fabrication facilities, and the protocols will reflect this. The electronics and physics departments of many universities have such facilities. Similarly, it will be assumed that experience of general cell culture and access to the appropriate facilities is available; only general background and non-standard cell culture methods will be detailed.
Sol-gel matrices for protein entrapment
Authors: Dave Bakul C , Dunn Bruce , Valentine Joan S , Zink Jeffrey I
Published: November 2023

Traditional inorganic glasses are not generally considered to be a suitable host matrix for immobilization of biological molecules. Consequently, stabilization of proteins and enzymes within inorganic silica glasses is very unusual (1). This breakthrough in immobilization methods has been made possible by the sol-gel method of synthesizing inorganic solids (2). The sol-gel approach is a solution-based synthesis technique for making inorganic materials at low temperature. The main advantage of the sol-gel method is that inorganic oxide glasses are synthesized by hydrolysis of simple alkoxo precursors (3). In the case of silica-based systems, the hydrolysed precursors then condense together to form a predominantly SiO2 glass framework. In this way, SiO2-based silica glass can be conveniently fabricated using Si(OR)4 precursors at room temperature without the extreme temperatures required for conventional glass melting methods. Sol-gel-derived silica glasses are highly porous structures containing an interstitial solvent phase. A high molecular weight dopant molecule added to the initial reaction mixture becomes trapped in the interconnected porous network as the final SiO2 glass structure forms (4). In addition, since the pore diameters are much less than the wavelength of light, the sol-gel glasses are optically transparent. This enables one to monitor spectroscopically the nature of events associated with encapsulation and reactivity of the dopant molecules (5).
Immobilization of ‘smart’ polymer-protein conjugates
Authors: Stayton Patrick S , Hoffman Allan S
Published: November 2023

The remarkable molecular recognition capabilities of proteins provide the basis for many important clinical, industrial, and laboratory applications. Key existing technologies include separations, hiosensors, bio transformations, diagnostics, and targeted drug delivery. The central event in protein-mediated recognition processes is typically a protein:ligand binding step, which is characterized by a combination of high specificity and affinity, and in the case of enzymes by a high affinity for the reaction coordinate transition state. While these molecular recognition capabilities have proven extremely useful, many practical problems limit the utility of proteins because nature has not optimized them for the device environment.
Fluorophore-labelled saccharide electrophoresis for the analysis of glycoproteins
Authors: Jackson Peter
Published: November 2023

It has become apparent that the carbohydrate m01etles of glycoproteins (glycones) have important and varied functions (1-3) and this knowledge has stimulated considerable advances in methods for their purification and analysis. However these procedures are not routine. Current analytical strategies usually involve the release of the free saccharide (glycan) from the glycoprotein or glycopeptide followed by chromatographic isolation and structural determination using a variety of techniques including compositional analysis, chemical and enzymatic degradation, lectin binding, mass spectrometry, and nuclear magnetic resonance (4-10).
Designing new agonists/antagonists of growth factor receptors-the rational design of a superantagonist of the IL-6 receptor
Authors: Ciliberto G , Lahm A , Paonessa G , Savino R , Toniatti C
Published: November 2023

Growth factors and cytokines assemble multisubunit receptor complexes stabilized by a variety of protein:protein interactions (1), each of which plays a distinctive role in signalling and activating biological responses. Identifying functional epitopes on the cytokine surface thus opens up possibilities for engineering variants with predefined properties for potential use as therapeutic agents. We have applied this concept to the rational design of receptor antagonists of human interleukin-6 (IL-6).
A chimeric approach for studying receptor binding domains in EGF-like molecules
Authors: Van Zoelen E J J , Lenferink A E G , Vugt M J H Van , Van de Poll M L M
Published: November 2023

The use of chimeric proteins has gained increasing popularity in studies on the structure-function relationship of biologically active peptides. Using recombinant DNA technology, proteins can readily be made in which specific domains have been exchanged for corresponding domains from other proteins. The use of chimeras obtained by such a domain-exchange approach has been shown to be particularly fruitful when comparing the biological properties of two structurally related proteins. It is essential that the two proteins of inter est have different biological activities, and that a direct assay is available to distinguish these activities. That is the reason why this approach has been used particularly for proteins with specific binding or signalling activity, including polypeptide growth factors, growth factor receptors, and nuclear transcription factors.
Defining growth factor function through tissue-specific expression of dominant-negative receptor mutants
Authors: Werner Sabine
Published: November 2023

Elucidating the function of growth factors and their receptors during development and in the adult organism is one of the most challenging tasks in growth factor biology. A few years ago, only descriptive expression data were available which suggested multiple roles of growth factors in development. However, the development of transgenic mouse technologies has provided new insights into the function of growth factors and their receptors in vivo. These technologies allow gain-of-function experiments (over-expression of ligands or receptors) as well as loss-of-function experiments (gene knock-outs by homologous recombination in embryonic stem cells). Knock-out studies have shed light on the function of many growth factors in embryonic development. While these studies can reveal the earliest essential function of a particular growth factor or its receptor, the specific role of these genes at later stages of development may be obscured due to the early embryonic lethality. In addition, the loss of one member of a gene family may be compensated for by overlapping expression of another, related gene (1). Such a compensation could be particularly important for growth factors which are members of a large family of related ligands, e.g. the fibroblast growth factors (FGFs) and the epidermal growth factor (EGF) receptor ligands. This hypothesis is supported by the very ‘mild’ phenotypes of FGF-5, FGF-7, and transforming growth factor-a (TGFa) knock-out mice (2-5). These animals reveal only a defect in the hair, although FGF-5, FGF-7, and TGFa are widely expressed in the embryo and-in the case ofFGF-7 and TGFa-also in the adult animal (6-8).
Practice and benefits of microcolumn HPLC
Authors: Yang Frank J
Published: November 2023

The application of microcolumn HPLC have grown significantly in recent years, especially since the current trends in HPLC are to employ continuously smaller diameter columns in order to reduce solvent consumption, to enhance sample detection, to allow a direct interface with mass spectrometers, and to facilitate sample limited applications and hyphenated techniques. Microcolumn HPLC is the HPLC of choice for the most modern HPLC bench-top MS applications, being used for several ionization techniques including particle beam (1-3), continuous flow FAB (4-9), EI/CI (10-13), and electrospray and nanospray interfaces (14-20). The major driving force behind the rapid growth of microcolumn HPLC in the past few years has been the commercialization of bench-top low cost mass spectrometers with electrospray and nanospray interfaces. At reduced flow rates, a few µ1/min and down to sub-µl/min, maximum sensitivity and baseline stability can be achieved. Thus, it can be expected that microcolumn HPLC-MS will become even more popular than capillary GCMS in the near future as the ability to connect microcolumn HPLC directly to the mass spectrometer will be propitiated by the development of low cost, easy to use, high sensitivity, and high reliability mass spectrometer, and supported by the low flow rates used in microcolumn HPLC.
Detection devices
Authors: Scott Raymond P W
Published: November 2023

In chromatography, the detection device is as essential to a successful analysis as the distribution system that produces the separation, and over the years there has been a continuous synergistic development between these two essential components of the chromatograph. As the columns and thin-layer plates have become more efficient, the solute bands have become reduced in size, and the solute concentrations smaller. These changes have educed the development of detectors, with higher sensitivities, and smaller sensor volumes. The improved detectors, in turn, have evoked improvements in column technology which have produced higher separating efficiencies. It is clear that the design of the detecting device is critical in high resolution chromatography.
Lectins as affinity probes
Authors: Sheldon Philip S
Published: November 2023

In 1888, Hermann Stillmark described the agglutination of erythrocytes by an extract from castor beans. Soon after this initial observation, the proteins which we now know as lectins, began to be used as tools in biomolecular research (1). Paul Ehrlich carried out pioneering studies in immunology using plant agglutinins and toxins as model antigens. Concanavalin A (Con A) from jack beans was the first lectin to be purified and crystallized. The observations that Con A precipitated glycogen and that sucrose inhibited haemagglutination led to the proposal that haemagglutination was a consequence of interactions between Con A and carbohydrate on the cell surface. As it emerged that haemagglutination was species- and blood type-specific, the term lectin became used to refer to plant agglutinins which were blood group-specific.
Strategies and methods for purification of Ca2+ -binding proteins
Authors: Fitzpatrick Sandra L , Waisman Da Yid M
Published: November 2023

Caz+ has been demonstrated to be the key second messenger in a variety of stimulus-response systems. These Caz+ regulated processes include muscle contraction, secretory events such as exocytosis, cell cycle regulation, cellular differentiation, and gene transcription. The Ca2+-binding proteins play a fundamental role in the Ca2+ signalling pathway: they transduce changes in intracellular Ca2+ into cellular activation. For example, Ca2+-dependent modulatory proteins such as calmodulin or annexin II tetramer undergo conformational changes in response to increases in intracellular Ca2+. The interaction of the Ca2+ -binding protein with Caz+ results in the generation of a conformational change in the protein and when in this active conformation these proteins can bind to and affect a variety of intracellular proteins or enzymes. Alternatively, Ca2+ -binding proteins directly participate in cellular signal transduction by containing in the structure of the Ca2+ -binding protein both Ca2+-binding domains and catalytic domains. For example, certain isozymes of protein kinase C contain Ca2+/phospholipid-binding domains and a catalytic domain.
Cationic Liposomes in Gene Delivery
Authors: Sullivan Sean , Gong Yan , Hughes Jeffrey
Published: November 2023

Gene therapy provides a new paradigm for the treatment of human diseases. Although gene therapy trials have been initiated worldwide for greater than two decades, little has been achieved clinically in curing diseases. A major hurdle is the lack of efficient delivery systems for in vitro and in vivo studies. An ideal system should have the following characteristics: be targetable, biodegradable, nontoxic, and stable for storage. Gene delivery systems are generally categorized into viral and plasmid-based vectors. Plasmid-based systems are also extremely useful for in vitro studies and will be the focus of the chapter.
lsothermic Titration Calorimetry (ITC)
Authors: Lasch J
Published: November 2023

This chapter covers a technique which measures the heats associated with the binding of surfactants to liposomes. It starts with the technique and sample handling and proceeds to the estimation of critical surfactant parameters. The estimation of these parameters is outlined in three main sections comprising the CMC, surfactant partitioning into lipid membranes, and liposome solubilization. Surfactants-induced transitions of lipid bilayers take place in a four-stage process (1). Surfactants added to liposomes are incorporated into the lipid membranes up to a critical (saturating) surfactant/lipid ratio, Rsat· Increasing the ratio above Rsat, the lipid bilayers are progressively destroyed and mixed vesicles start to coexist with mixed micelles. When the total content of surfactant in the aggregates reaches R501, all membranes are dissolved leaving only mixed micelles and the solubilization is complete. The micellar intermediate structures are cylindrical mixed micelles (CMM), lipid-rich spherical or ellipsoidal mixed micelles (SMM or EMM), and finally lipid-poor mixed micelles (1) (Figure 1).
Detection of sequence variation using primer extension
Authors: Syvänen A -C
Published: November 2023

Most of the currently used methods for detecting single nucleotide polymorphisms or point mutations are based on amplification of the target DNA by the PCR technique or another amplification method, which allows sensitive and specific analysis of the target DNA sequence by a variety of methods. Previously known sequence variants are distinguished in the amplified fragments by hybridization with sequence-specific oligonucleotide (SSO) probes (see chapter 7) or with the aid of nucleic acid modifying enzymes, such as restriction enzymes, DNA ligases, or DNA polymerases (see chapters 8 and 9).
Advances in direct DNA sequencing for mutation scanning
Authors: Lundeberg Joakim , Williams Cecilia , Ahmadian, Asfhin , Uhlén Mathias
Published: November 2023

The rate of discovery for disease-causing genes and underlying mutations is accelerating, and a number of genes, such as the tumour suppressor gene p53, cumulatively affect large numbers of individuals in the general population. Screening for single nucleotide changes and less frequent frame-shift losses/ gains of one to a few nucleotides is most readily accomplished by direct DNA sequencing. The transformation of DNA sequencing from research to clinical laboratories will probably lead to routine procedures for prognostic and therapeutic genes in specialized laboratories within ten years (1, 2). The primary issues to be addressed for applying direct DNA sequencing as a clinical tool include automation, optimal methods to obtain high quality data, efficient multiplex amplification stategies, quality control, and procedures to deal with very small samples.
Two colour in situ hybridization
Authors: Jowett T
Published: November 2023

Once the expression pattern of a gene has been established it is often necessary to relate it to that of other genes expressed at the same period of development. Transcripts from different genes may be expressed in com¬ plementary or overlapping domains. The temporal and spatial pattern of expression of different genes can be related by performing in situ hybridization on consecutive sections of tissue but it is also possible to perform hybridiza¬ tions with two probes and to visualize the signals separately in the same tissue.
Detection of genomic sequences by fluorescence in situ hybridization to chromosomes
Authors: Kearney Lyndal
Published: November 2023

Since the first report over ten years ago (1), fluorescence in situ hybridization (FISH) has fulfilled its’ promise as a powerful tool in genome analysis. The type of genomic sequence that can be used as a probe now ranges from small sequence tagged sites (STSs) to whole genomes (reviewed in ref. 2). With the advent of new labelling and imaging systems (3, 4), the sensitivity now approaches that of earlier isotopic methods, with the ability to detect hybridized sequences < 1 kb in size. Recently, the possibility of 24 colour FISH has been achieved, enabling the simultaneous analysis of the entire chromosome complement in a single multicolour hybridization experiment (5, 6). The chromosomal targets for FISH are equally varied, from metaphase chromosomes to extended strands of DNA (reviewed in ref. 7). High-resolution metaphase chromosomes can be used to resolve two sequences approximately one megabase (Mb) apart, while interphase mapping allows the resolution of sequences 100 kb apart (8). Techniques using extended DNA fibres have revolutionized fine mapping, allowing the ordering and orientation of sequences 1 kb apart (9-12).
Immunohistochemistry
Authors: Ormerod Michael G , Imrie Susanne F
Published: November 2023

Immunohistochemistry utilizes antibodies to localize specific products in tissue sections. Briefly, a tissue section is incubated with a labelled antibody, the section is washed, and the site of reaction of the antibody is identified by visualizing the label. A variety of histochemical techniques have been used for many years to identify certain constituents of cells. These methods lack the specificity obtained from an antibody and it is this property which enables a worker to map precisely the distribution of a particular product in a tissue. Aside from its power as a tool in research, immunohistochemistry has found an increasing role in diagnostic histopathology with particular application to the diagnosis of tumours. The range of applications of the method has been advanced immeasurably by the development of technology for the production of mono clonal antibodies. This has produced a wide range of antibodies, many of which have specificities which would have been difficult, if not impossible, to obtain from conventional antisera.
Gene targeting strategies
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

Gene targeting, defined as the introduction of site-specific modifications into the mouse genome by homologous recombination, is generally used for the production of mutant animals to study gene function in vivo. Since homologous recombination of foreign DNA with endogenous genomic sequences is a relatively infrequent event in mammalian cells, compared to its random integration, the only efficient gene targeting method presently established utilizes pluripotent murine embryonic stem cell lines. Using these cells, the selection of rare, homologous recombinant ES cell clones in vitro can be accomplished. When such genetically modified ES cells are introduced into a preimplantation embryo they can contribute, even after extensive in vitro manipulation, to all cell lineages (including germ cells) of the resulting chimeric animal. The breeding of germline chimeras, which transmit an ES cell derived mutant chromosome(s) to their progeny, allows the establishment of an animal heterozygous for your genetic alteration and, importantly, by further breeding a homozygous mutant mouse strain. The necessity of chimera production may be bypassed in the future when spermatogonial stem cell lines become available (17, 18).
The Cre/loxP recombination system
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

The Pl bacteriophage Cre recombinase recognizes and mediates site-specific recombination between 34 bp sequences referred to as loxP (locus of crossover (x) in Pl) (35). The loxP sequence consists of two 13 bp inverted repeats interrupted by an 8 bp nonpalindromic sequence which dictates the orientation of the overall sequence (Figure 3a) (36, 37). When two loxP sites are placed in the same orientation on a linear DNA molecule, a Cre-mediated intramolecular recombination event results in the excision of the loxP-flanked, or ‘floxed’, sequence as a circular molecule with one loxP site left on each reaction product (Figure 3b). The reverse reaction, an intermolecular recombination event, will result in the integration of the circular DNA molecule into the linear molecule, each possessing one loxP site. Alternatively, an intermolecular recombination event between two linear molecules as substrates (again, each possessing one loxP site) results in the reciprocal exchange of the regions flanking the loxP sites (Figure 3c). If the two loxP sites are placed in opposing orientation the floxed sequence will be inverted (Figure 3d) (38-40).
Large deletions
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

Small genomic deletions (1-2 kb) are often created by replacing genomic sequence with the selection marker gene included in a replacement type gene targeting vector. This strategy has been used to generate deletions up to 19 kb in length in the hprt locus with no decrease in the frequency of homologous recombination compared to smaller replacements in the same locus (93). The majority of recombination products, however, obtained in these latter experiments were found to have an aberrant structure with a rate of correctly targeted ES cell clones of 10 - 30%. In contrast to this single step method for generating genomic deletions, the Cre/loxP system requires that two loxP sites first be introduced by homologous recombination into a locus and the actual deletion is created in a second step by transient expression of Cre recombinase in targeted ES cell clones. Thus, the strategy for generating deletions of up to approximately 10 kb, using a single targeting vector, is the same as described in the section on the flax-and-delete approach (see Chapter 9). Compared to the conventional single step method, the Cre/loxP system has the advantage that virtually no unexpected recombination products are obtained in the Cre-mediated deletion step and that the selection marker is removed from the targeted locus. Moreover, unlike other methods, the use of the Cre/loxP system offers the unique possibility to generate very large deletions by the introduction of loxP sites into different chromosomal positions using two gene targeting vectors sequentially, each transferring one loxP site. This general strategy has proven successful in deleting DNA segments in ES cells ranging in size from 200 kb (94) to 3-4 cM (95). The potential applications of large deletions include the targeted inactivation of large genes, the removal of whole gene clusters from the genome, and genetic screening for recessive mutations.
Chromosomal translocations
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

To generate chromosomal translocations in ES cells using the Cre/loxP system two consecutive homologous recombination events are necessary. In the first step, a positive selection marker gene with one loxP site (e.g. loxP-neo) must be introduced by homologous recombination on one chromosome and a second selection marker with another loxP site (e.g. loxP-hygro) on the second of the two chromosomes. Alternatively to using two different selection markers, the same loxP-flanked marker gene could be used in both targeting vectors if the floxed marker is deleted after the first targeting step leaving one loxP site behind. Depending on the aim of the individual experiment the loxP sites must be placed into introns if a fusion protein should be generated or outside of transcription units to exchange only regulatory regions, e.g. mimicking naturally occurring translocations. In any case, both loxP sites should have the same orientation, with respect to the centromeres, to retain chromosomal integrity. After identification of homologous recombinants the Cre¬ mediated chromosomal translocation should be obtained by transient expression of Cre recombinase in ES cells (see Chapter 21).
Conditional gene targeting
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

We understand the term ‘conditional gene targeting’ as a restricted gene modification in the mouse, limited to either certain cell types (tissue specific), to only a portion of its lifetime (temporally-specific), or both (11, 12, 61). This contrasts with the conventional gene targeting strategy which creates a permanently modified allele in all cells of an animal from the onset of development (Figure 2). While the strategies discussed in this section are based on the Cre/loxP bacteriophage recombinase system, their applicability clearly extends to the other members of the A integrase family which have the common feature of an independence on host cofactors (e.g. FLP/FRT). It must be emphasized, however, that the Cre/loxP system has already proven its usefulness and efficiency in conditional gene targeting whereas the other recombinase systems have thus far appeared less than optimal.
ES cells - handling and use
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

Pluripotent embryonic cells (ES cells) were first established directly from murine blastocysts in 1981 (136, 137) after unsuccessful early attempts to obtain germline chimerism with teratocarcinoma (EC) cells (138, 139 for review), and later shown to efficiently produce germline chimeras (140) even after transfection (141, 142). Since then, a fairly large number of ES cell lines have been generated and the lines currently often used for gene targeting experiments include D3 (143), E14 (144, 145), ABl (146), Rl (147), Jl (148) and CCE (140) which are all derived from 129 substrains or 129 hybrids. A germline competent hprt-negative line which can be used with HPRT as selection marker has also been described (149). There is a smaller number of less frequently used ES lines derived from other strains like ES623 (143) and B6-III (150) which were established from C57BL/6 mice. For the derivation and establishment of new ES cell lines see Robertson (14) and Abbondanzo, et al. (151).
Thawing ES clones from 48 well plates
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

After identification of homologous recombinants by Southern analysis, plates must be thawed and appropriate clones recovered. Again there are different ways to thaw the frozen plates which differ slightly in the time required for the thaw.
Genomic DNA preparation
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

There are several methods for extracting genomic DNA from tissue culture cells. Presented here are two methods: a relatively new method which is’very simple, quick and convenient (no phenol) and a reliable, although perhaps outdated (i.e., phenol/chloroform), method. Genomic DNA can be extracted from either tissue culture cells (or mouse tails) using either method. The simplified, non-phenol method presented here was developed with gene targeting in mind since large numbers of genomic extractions are required from both tissue culture cells and mouse tails in the course of an experiment (163). Genomic DNA isolated from ES cells and tails in this manner has been used successfully in Southern analysis and PCR amplification.
ere-mediated (neomycin) deletion in ES cells
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

As discussed in Section 1, a major advantage to using the Cre/loxP recombinase system in conjunction with gene targeting is the ability to remove the floxed selection marker (as well as any additional loxP-flanked sequences) after homologous recombinants have been identified. This is clearly important when studying gene regulation to avoid the influence of the selection marker, and its own regulatory elements, on the regulation of the gene of interest (20). Removing the selection marker (e.g neo gene) from homologous recombinant clones also allows for the selection of ES cells which are homozygous or hemizygous (if Cre-mediated deletion deletes the allele) for the desired genetic modification after retransfecting these clones with the initial targeting vector.
Injection, holding, and transfer needles
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

If you will inject ES cells into blastocysts, then before injections begin you will need to prepare your own stock of needles for embryo manipulation. You should devote as much time as necessary beforehand to generate a comfortable stock of all needles. As equipment for the production of microneedles you need a (simple) needle puller (e.g. Kopf 720), a stereomicroscope (dissecting scope; e.g. Wild MSA, lSX oculars), a microforge (e.g. de Fonbrune, Bachofer), a microscope with 400x magnification and a bunsen burner or ethanol burner.
Blastocyst injedion
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

Generally, a microscope with up to 300−400x magnification, two micromanipulators, two micrometer syringes, tubings, needle holders and injection chambers are needed for blastocyst injections. For the collection and handling of blastocysts a stereomicroscope (-50x magnification) is needed and a 37°C incubator (preferably with CO2 supply) for blastocyst culture. Since there are many suppliers of micromanipulation instruments many different configurations are possible. The set-up we use, and regard as comfortable, consists of a Leitz Labovert inverted microscope equipped with lOx oculars and lOx, 16x, 32x and 40x phase contrast objectives. This scope is mounted on a Leitz ground plate together with two Leitz micromanipulators with Leitz or Eppendorf needle holders. As syringes we use AGLA micrometer syringe holders (Vetter) equipped with Hamilton syringes (100μl to control injection needle, #710 or #1710, Hamilton; 250 μl to control holder needle, #725 or 1725) which are connected via thick-walled tubing to the needle holders (micrometer syringes can be also purchased from Narishige).
Analysis of macrophage activity in vivo
Authors: Rooijen Nico van , Kesteren-Hendrikx Esther van
Published: November 2023

In mammals, macrophages have developed into multifunctional cells. Apart from their scavenger role in the clearance of non-self materials such as micro-organisms and altered-self materials such as apoptotic cells, senescent erythrocytes, immune complexes, and inflammatory products, they play a crucial role in the regulation of both innate and acquired immunity. Whereas the former activity is based on phagocytosis and intracellular degradation, the latter activity largely depends on the production and secretion of a panel of regulatory molecules such as cytokines, chemokines, and nitrogen oxide (NO).
Microscope image acquisition
Authors: Ellenberger Stephanie L , Young Ian T
Published: November 2023

Modern microscopes allow the attachment of a camera. Technology is avail-able to send acquired images directly to a computer for further processing. In these images object features such as length, area, or intensity can be measured with appropriate software. The measurement results can be used to classify objects or to determine the quality of an object. The automatic determination of object properties is called automated quantitative microscopy. Progress in medicine and industry has stimulated the development of fast and accurate automated microscope systems which process a great number of objects in a short time, for example to monitor the treatment of cancer patients or to inspect a large number of printed circuit boards. Another advantage of automated systems besides speed and accuracy is that results are reproducible if the set-up is unchanged. It is also easy to store results electronically for further processing at a later time or for comparison with other results.
Image analysis: quantitative interpretation of chromosome images
Authors: Castleman Kenneth R
Published: November 2023

In this chapter we illustrate how a set of image analysis techniques can be integrated to form a solution for a problem. One problem that illustrates this particularly well is chromosome analysis. Since the early 1960s, the field of cytogenetics has provided an important application area for digital image analysis techniques. Indeed, many of the methods now widely used in other fields were first applied to chromosomes. The object of automatic karyotyping is to recognize, in a microscope image, each of the 46 chromosomes from the nucleus of a human cell, and place them into a standardized arrangement called a karyotype. From this rearranged image the geneticist can make a diagnosis.
3D analysis: registration of biomedical images
Authors: Rueckert Daniel , Hawkes David J
Published: November 2023

The analysis of medical images plays an increasingly important role in many clinical applications. Different imaging modalities provide anatomical information about the underlying tissues such as the X-ray attenuation coefficient from X-ray computed tomography (CT) and proton density or proton relaxation times from magnetic resonance (MR) imaging. The images allow clinicians to gather information about the size, shape, and spatial relationship between anatomical structures and any pathology, if present. Other imaging modalities provide functional information such as the blood flow or glucose metabolism from positron emission tomography (PET) or single-photon emission tomography (SPECT), and permit clinicians to study the relation-ship between anatomy and physiology. Finally, histological images provide another important source of information which depicts structures at a microscopic level of resolution.
Data processing
Authors: Nix* Barry , Wild† David
Published: November 2023

Immunoassay developers need to be aware of the concepts involved in calibration curve-fitting and data interpolation, if only because of the ever-present risk of inaccurate results caused by the computational procedure itself. Although the computerized curve-fitting algorithms used are often highly sophisticated and have improved in recent years, they are still not universally applicable or totally reliable and can introduce bias or poor precision to assay data. Researchers may need to become involved in curve-fitting in a number of situations, such as:
Identification of substrates for protein—tyrosine phosphatases
Authors: Garton Andrew J , Flint Andrew J , Tonks Nicholas K
Published: November 2023

The protein-tyrosine phosphatase (PTP) family of enzymes comprises a large number (>75) of structurally diverse enzymes involved in regulating intracellular signalling processes through the dephosphorylation of phosphotyrosine-containing substrate proteins (1, 2). Genetic studies have now clearly demonstrated that individual PTPs fulfil specific, non-redundant roles in regulating mammalian signal transduction processes (3-5). It therefore appears likely that individual PTPs recognize only a limited range of tyrosine-phosphorylated substrate proteins in vivo. However, genetic studies generally provide little information regarding the biochemical functions of the gene products under investigation. Thus, in order to understand fully the physiological functions of individual PTPs, it will be necessary to employ additional approaches to identify the biologically relevant substrates for a particular enzyme. This has proven to be an extremely elusive goal, which has been significantly hampered by the rellatively low substrate specificity displayed by many PTPs in vitro. Indeed, early investigations of PTP substrate specificity using a small number of artificial substrates led to the proposal that intracellular targetting of PTPs via their non-catalytic segments may be the primary determinant of substrate specificity in vivo, whereas isolated PTP catalytic domains may be rather promiscuous in their substrate preferences (6). However, although subcellular location clearly plays an important role in determining the range of substrates available to many PTPs (7), evidence is now mounting that PTP catalytic domains are themselves capable of demonstrating discernible, and sometimes exquisite, substrate specificity (8).
Yeast as a system for the study of nuclear receptor function
Authors: Garabedian M J , Iniguez-Lluhí J A , Kralli A
Published: November 2023

Nuclear receptor function can be reconstituted in yeast. enabling a wide variety of genetic approaches to be taken toward defining the mechanisms of signal transduction and transcriptional regulation. This chapter describes vectors for the expression of nuclear receptors in yeast, reporter genes. yeast host strains, and simple assays that monitor receptor transcriptional activity. Methods that couple random mutagenesis with phenotypic screens to generate receptors defective in particular receptor functions (e.g. DNA and hormone binding or transcriptional activation), are also described. In addition, we give an overview or strategies for the identification of non-receptor components that may act on receptors. The experimental advantages of yeast invite a detailed genetic analysis of vertebrate nuclear receptor functions and should further illuminate the mechanisms of signal transduction and transcriptional regulation by this important class of regulatory molecules.
G proteins I: Gs and Gi
Authors: Mumby Susanne M
Published: November 2023

Cells respond with remarkable speed and specificity to the many environmental cues that they encounter. Signal transduction mediated by heterotrimeric G proteins is a common strategy among eukaryotes to relay extracellular information to the interior of the cell. This strategy is employed in physiological functions ranging from mating in yeast to vision in humans. External primary messengers, such as light, odorants, hormones, chemoattractants, and neurotransmitters, stimulate antennae-like receptors that activate G proteins. The activity of second-messenger-producing effectors are, in turn, modulated by the activated G proteins. A relay diagram of G-protein signalling is shown in Fig. 1. The specific examples shown, hormone-sensitive adenylyl cyclase and visual transduction (Fig. 1, lines a-c), are the most extensively studied systems, on which much of our current knowledge of G-protein-mediated signal transduction is based.

Signal transduction is initiated at the plasma membrane by G-protein-coupled receptors, characterized by amino-acid sequences predicted to form seven membrane spans (Fig. 2). Extracellular-facing aspects of the receptor bind agonist and intracellular portions thus are enabled to interact fruitfully with a cognate G protein positioned at the inner face of the plasma membrane. G proteins are composed of a, [3, and ‘Y subunits whose deduced amino-acid sequences lack clear hydrophobic stretches that would be anticipated to span the bilayer. Instead, G proteins are modified by lipids that facilitate their interaction with proteins and/ or lipids comprising the plasma membrane. Effectors, including adenylyl cyclase and ion channels, span the membrane (Fig. 2) but others do not. Light-activated cGMP phosphodiesterase (PDE), for example, is found in cytosolic and membranous fractions of retinal cells.