bluuebunny/crossref_metadata_2025_split huggingface dataset:
Advances in direct DNA sequencing for mutation scanning
Authors: Lundeberg Joakim , Williams Cecilia , Ahmadian, Asfhin , Uhlén Mathias 
Published: November 2023

Abstract
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

Abstract
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

Abstract
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 &lt; 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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).
Genomic DNA preparation
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
The protein-tyrosine phosphatase (PTP) family of enzymes comprises a large number (&gt;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

Abstract
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.