• The gastrointestinal syndrome and mucosal clonogenic cells: relationships between target cell sensitivities, LD50 and cell survival, and their modification by antibiotics.

      Hendry, Jolyon H; Potten, Christopher S; Roberts, N P; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1983-10)
      The sensitivity of the target cells responsible for the gastrointestinal syndrome in mice was deduced from the steepness of the dose-survival curve for mice assessed on Day 7 after irradiation. The D0 value was 1.25 +/- 0.22 Gy, virtually identical to the value of 1.23 +/- 0.08 measured for microcolony-forming cells (clonogens) over about the same range of dose in concurrent experiments. The survival of clonogens was similar when assayed in mice surviving to Days 3, 4, or 5, but clonogenic sensitivity was lower when assessed on Day 7. This was shown at one dose to be due largely to a selection of mice with high colony counts with only a small contribution from crypt budding. The LD50 for mice corresponded to a surviving fraction of crypts of about 0.35. An injection of 5 mg streptomycin sulphate ip daily for 5 days after irradiation increased the latent period by about 1 day, increased the LD50 by about 1.4 Gy, but did not significantly change the survival of clonogens. These studies are the first to test and satisfy the interpretation of a dose-response curve for animal survival in terms of "target cell" survival, where measurements of both are made over a similar range of dose in concurrent experiments.
    • A GATA-2/estrogen receptor chimera functions as a ligand-dependent negative regulator of self-renewal.

      Heyworth, Clare M; Gale, Karin; Dexter, T Michael; May, Gillian; Enver, Tariq; Cancer Research Campaign (CRC) Section of Hematopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester M20 4BX, UK. (1999-07-15)
      The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is functionally implicated in their survival and proliferation. We have used estrogen and tamoxifen-inducible forms of GATA-2 to modulate the levels of GATA-2 in the IL-3-dependent multipotential hematopoietic progenitor cell model FDCP mix. Ligand-dependent induction of exogenous GATA-2 activity did not rescue cells deprived of IL-3 from apoptosis. However, induction of GATA-2 activity in cells cultured in IL-3 blocked factor-dependent self-renewal but not factor-dependent survival: Cells undergo cell cycle arrest and cease proliferating but do not apoptose. This was accompanied by differentiation down the monocytic and granulocytic pathways. Differentiation occurred in the presence of IL-3 and did not require addition of exogenous differentiation growth factors such as G-CSF or GM-CSF normally required to induce granulomonocytic differentiation of FDCP-mix cells. Conversely, EPO-dependent erythroid differentiation was inhibited by GATA-2 activation. These biological effects were obtained with levels of exogenous GATA-2 representing less than twofold increases over endogenous GATA-2 levels and were not observed in cells overexpressing GATA-1/ER. Similar effects on proliferation and differentiation were also observed in primary progenitor cells, freshly isolated from murine bone marrow and transduced with a GATA-2/ER-containing retrovirus. Taken together, these data suggest that threshold activities of GATA-2 in hematopoietic progenitor cells are a critical determinant in influencing self-renewal versus differentiation outcomes.
    • Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development

      Pimanda, John E; Ottersbach, Katrin; Knezevic, Kathy; Kinston, Sarah; Chan, Wan Y I; Wilson, Nicola K; Landry, Josette-Renee; Wood, Andrew D; Kolb-Kokocinski, Anja; Green, Anthony R; et al. (2007-11-06)
      Conservation of the vertebrate body plan has been attributed to the evolutionary stability of gene-regulatory networks (GRNs). We describe a regulatory circuit made up of Gata2, Fli1, and Scl/Tal1 and their enhancers, Gata2-3, Fli1+12, and Scl+19, that operates during specification of hematopoiesis in the mouse embryo. We show that the Fli1+12 enhancer, like the Gata2-3 and Scl+19 enhancers, targets hematopoietic stem cells (HSCs) and relies on a combination of Ets, Gata, and E-Box motifs. We show that the Gata2-3 enhancer also uses a similar cluster of motifs and that Gata2, Fli1, and Scl are expressed in embryonic day-11.5 dorsal aorta where HSCs originate and in fetal liver where they multiply. The three HSC enhancers in these tissues and in ES cell-derived hemangioblast equivalents are bound by each of these transcription factors (TFs) and form a fully connected triad that constitutes a previously undescribed example of both this network motif in mammalian development and a GRN kernel operating during the specification of a mammalian stem cell.
    • GEFs: Dual regulation of Rac1 signaling.

      Marei, Hadir; Malliri, Angeliki; Cell Signaling Group, Cancer Research UK Manchester Institute, The University of Manchester , Manchester M20 4BX (2016)
      GEFs play a critical role in regulating Rac1 signaling. They serve as signaling nodes converting upstream signals into downstream Rac1-driven cellular responses. Through associating with membrane-bound Rac1, GEFs facilitate the exchange of GDP for GTP, thereby activating Rac1. As a result, Rac1 undergoes conformational changes that mediate its interaction with downstream effectors, linking Rac1 to a multitude of physiological and pathological processes. Interestingly, at least 20 GEFs have been shown to activate Rac1, suggesting a more complex role of GEFs in regulating Rac1 signaling apart from promoting the exchange of GDP for GTP. Indeed, accumulating evidence implicates GEFs in directing the specificity of Rac1-driven signaling cascades, although the underlying mechanisms were poorly defined. Recently, through conducting a comparative study, we highlighted the role of two Rac-specific GEFs, Tiam1 and P-Rex1, in dictating the biological outcome downstream of Rac1. Importantly, further proteomic analysis uncovered a GEF activity-independent role of both GEFs in modulating the Rac1 interactome, which results in the stimulation of GEF-specific signaling cascades. Here, we provide an overview of our recent findings and discuss the role of GEFs as master regulators of Rac1 signaling with a particular focus on GEF-mediated modulation of cell migration following Rac1 activation.
    • A gel-free quantitative proteomics analysis of factors released from hypoxic-conditioned placentae.

      Blankley, R T; Robinson, N J; Aplin, J D; Crocker, I P; Gaskell, S J; Whetton, Anthony D; Baker, P N; Myers, J E; Maternal and Fetal Health Research Group, St Mary's Hospital, Manchester, UK. richard.blankley-2@manchester.ac.uk (2010-03)
      Characterizing the protein factors released from placentae during pathogenesis remains a key objective toward understanding preeclampsia and related pregnancy disorders. Gel-free proteomics technologies applied to placental explant-conditioned media offers the potential of identifying these factors. Relative quantification mass spectrometry using isobaric tagging for relative and absolute quantification (iTRAQ) labeling was employed to compare the ''secretome'' between healthy term placental tissue cultured under both normoxic and hypoxic oxygen tensions. Of the 499 proteins identified, 45 were differentially expressed (P < .01 level), including interleukin 8 (IL-8) which was significantly upregulated under hypoxia. Global protein level changes are suggestive of decreased extracellular matrix remodeling under the same conditions. A significant enrichment of soluble liberated placental factors is achieved using this model system. Identifying these changes resulting from hypoxic conditioning is hypothesis generating and may provide new mechanistic insights into preeclampsia.
    • Gemcitabine plus best supportive care (BSC) vs BSC in inoperable non-small cell lung cancer--a randomized trial with quality of life as the primary outcome. UK NSCLC Gemcitabine Group. Non-Small Cell Lung Cancer.

      Anderson, Heather; Hopwood, Penelope; Stephens, R J; Thatcher, Nick; Cottier, B; Nicholson, M; Milroy, Robert; Maughan, T S; Falk, S J; Bond, M G; et al. (2000-08)
      Three hundred patients with symptomatic, locally advanced or metastatic NSCLC not requiring immediate radiotherapy were enrolled into this randomized multicentre trial comparing gemcitabine + BSC vs BSC alone. Patients allocated gemcitabine received 1000 mg/m2 on days 1, 8 and 15 of a 28-day cycle, for a maximum of six cycles. The main aim of this trial was to compare patient assessment of a predefined subset of commonly reported symptoms (SS14) from the EORTC QLQ-C30 and LC13 scales. The primary end-points were defined as (1) the percentage change in mean SS14 score between baseline and 2 months and (2) the proportion of patients with a marked (> or = 25%) improvement in SS14 score between baseline and 2 months sustained for > or =4 weeks. The secondary objectives were to compare treatments with respect to overall survival, and multidimensional QL parameters. The treatment groups were balanced with regard to age, gender, Karnofsky performance status (KPS) and disease stage (40% had metastatic disease). The percentage change in mean SS14 score from baseline to 2 months was a 10% decrease (i.e. improvement) for gemcitabine plus BSC and a 1% increase (i.e. deterioration) for BSC alone (P = 0.113, two-sample t-test). A sustained (> or = 4 weeks) improvement (> or =25%) on SS14 was recorded in a significantly higher proportion of gemcitabine + BSC patients (22%) than in BSC alone patients (9%) (P = 0.0014, Pearson's chi-squared test). The QLQ-C30 and L13 subscales showed greater improvement in the gemcitabine plus BSC arm (in 11 domains) than in the BSC arm (one symptom item). There was greater deterioration in the BSC alone arm (six domains/items) than in the gemcitabine + BSC arm (three QL domains). Tumour response occurred in 19% (95% CI 13-27) of gemcitabine patients. There was no difference in overall survival: median 5.7 months (95% CI 4.6-7.6) for gemcitabine + BSC patients and 5.9 months (95% CI 5.0-7.9) (log-rank, P = 0.84) for BSC patients, and 1 -year survival was 25% for gemcitabine + BSC and 22% for BSC. Overall, 74 (49%) gemcitabine + BSC patients and 119 (79%) BSC patients received palliative radiotherapy. The median time to radiotherapy was 29 weeks for gemcitabine + BSC patients and 3.8 weeks for BSC. Patients treated with gemcitabine + BSC reported better QL and reduced disease-related symptoms compared with those receiving BSC alone. These improvements in patient-assessed QL were significant in magnitude and were sustained.
    • The gene for the naevoid basal cell carcinoma syndrome acts as a tumour-suppressor gene in medulloblastoma.

      Cowan, Richard A; Hoban, Paul R; Kelsey, Anna M; Birch, Jillian M; Gattamaneni, Rao; Evans, D Gareth R; CRC Department of Cancer Genetics, Christie Hospital, Manchester, UK. (1997)
      Individuals with naevoid basal cell carcinoma (Gorlin) syndrome are at increased risk of developing medulloblastoma in childhood. We have shown that approximately 5% of patients with Gorlin syndrome will develop this complication in the first few years of life, and in addition 10% of patients with medulloblastoma diagnosed at age 2 years or under have Gorlin syndrome. One out of three medulloblastomas occurring in patients with Gorlin syndrome was shown to have lost the wild-type allele on 9q, indicating that the Gorlin locus probably acts as a tumour suppressor in the development of this tumour. We have also confirmed this role in a basal cell carcinoma (BCC) from the same individual. Information from these families would suggest that Gorlin syndrome is more common than previously recognized and may not always be diagnosed on clinical grounds alone even in middle life.
    • Gene function prediction using semantic similarity clustering and enrichment analysis in the malaria parasite Plasmodium falciparum.

      Tedder, P M R; Bradford, James R; Needham, C J; McConkey, G A; Bulpitt, A J; Westhead, D R; Institute of Molecular and Cellular Biology, University of Leeds, Leeds, UK. (2010-10-01)
      MOTIVATION: Functional genomics data provides a rich source of information that can be used in the annotation of the thousands of genes of unknown function found in most sequenced genomes. However, previous gene function prediction programs are mostly produced for relatively well-annotated organisms that often have a large amount of functional genomics data. Here, we present a novel method for predicting gene function that uses clustering of genes by semantic similarity, a naïve Bayes classifier and 'enrichment analysis' to predict gene function for a genome that is less well annotated but does has a severe effect on human health, that of the malaria parasite Plasmodium falciparum. RESULTS: Predictions for the molecular function, biological process and cellular component of P.falciparum genes were created from eight different datasets with a combined prediction also being produced. The high-confidence predictions produced by the combined prediction were compared to those produced by a simple K-nearest neighbour classifier approach and were shown to improve accuracy and coverage. Finally, two case studies are described, which investigate two biological processes in more detail, that of translation initiation and invasion of the host cell. AVAILABILITY: Predictions produced are available at http://www.bioinformatics.leeds.ac.uk/∼bio5pmrt/PAGODA.
    • Gene therapy of patient-derived T lymphocytes to target and eradicate colorectal hepatic metastases.

      Sheen, Aali J; Irlam, Joely J; Kirillova, Natalia; Guest, Ryan D; Sherlock, David J; Hawkins, Robert E; Gilham, David E; Cancer Research United Kingdom, Department of Medical Oncology, Paterson Institute for Cancer Research, University of Manchester, United Kingdom. (2003-06)
      PURPOSE: The overall aim of this study was to develop a novel treatment for colorectal cancer based on the use of gene therapy. Genetic modification of T lymphocytes has been used to specifically target and kill tumor cell lines directly. To test the efficacy of this method with clinically relevant materials, this study investigated the potential of T lymphocytes derived from patients with advanced colorectal disease to target autologous primary tumor material. METHODS T lymphocytes isolated preoperatively were modified genetically with recombinant retroviruses encoding CD3zeta-based chimeric immune receptors and were tested for functional activity against freshly isolated autologous tumor cells harvested from hepatic colorectal metastases. RESULTS: Patient-derived T cells were successfully transduced, and chimeric immune receptor expression was confirmed. Carcinoembryonic antigen expression on freshly isolated colorectal tumor cells was also demonstrated by molecular and immunohistochemical techniques. T cells expressing the anticarcinoembryonic antigen receptor were specifically activated by coculture with disaggregated or intact, diced tumor, whereas control non-carcinoembryonic antigen-targeted T-cell populations failed to activate. CONCLUSIONS: These results indicate that gene-targeted primary T lymphocytes depict specific functional activity against autologous colorectal tumor cells. This evidence indicates that chimeric immune receptor-expressing T cells may be able to circumvent the mechanisms used by tumor cells to avoid immune cell activity in vivo. This study emphasizes the potential of this approach as a therapy for carcinoembryonic antigen-expressing primary colorectal tumor and its metastases.
    • Gene transfer to augment the therapeutic index of anticancer chemotherapy.

      Baum, C; Margison, Geoffrey P; Eckert, H G; Fairbairn, Leslie J; Ostertag, W; Rafferty, Joseph A (1996-01)
    • Gene-specific linear trends constrain transcriptional variability of the toll-like receptor signaling

      Bagnall, J.; Rowe, W.; Alachkar, N.; Roberts, J.; England, H.; Clark, Christopher; Platt, M.; Jackson, D. A.; Muldoon, M.; Paszek, P.; et al. (2020)
      Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1β involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.
    • General criteria for radioassays by colloid scintillation counting under optimal conditions.

      Fox, Brian W; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1974-05)
    • A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

      Lyon, Malcolm; Gallagher, John T; Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom. (1990-02-15)
      A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.
    • General report on the European Union concerted action workshop on 11q23, London, UK, May 1997.

      Secker-Walker, L M; Harrison, Christine J; Department of Haematology, The Royal Free Hospital School of Medicine, London, UK. (1998-05)
      Seventeen cytogenetic laboratories in eight European countries contributed karyotypic, hematological, clinical and follow-up data from 550 patients with an acquired abnormality of 11q23. The patients had acute lymphoblastic leukemia (254), acute myeloid leukemia (250), unspecified, undifferentiated, biphenotypic acute leukemia or myeloproliferative disorder (18 cases together), or myelodysplastic syndrome (MDS) (28). The patients were classified by cytogenetic subgroup as t(4;11) (183 cases), t(6;11) (30) cases), t(9;11) (125 cases), t(10;11) (20 cases), t(11;19) (53 cases), 'other' abnormalities of 11q23 (82 cases) and del(11)(q23) (57 cases). Manuscripts were prepared on each cytogenetic subgroup, on MDS, on secondary hematological malignancies (40 cases) and on 11q23-translocation derivatives. For each subgroup the following aspects were investigated: associated clinical features, additional karyotypic change, distribution between hematological subtypes and between different age groups, prognosis at different age groups, and the impact of bone marrow transplantation on survival. The Workshop confirmed some previous findings from smaller studies, challenged others, identified new chromosomal partners and threw new light on less well documented aspects of 11q23 malignancies. The large number of cases investigated in a coordinated manner gives authoritative support to the findings. The Workshop thus demonstrates the value of collaborative European studies in the cytogenetics of malignancy.
    • Generalizability of potential biomarkers of response to CTLA-4 and PD-1 blockade therapy in cancer

      Bortone, D.; Vensko, S.; Entwistle, S.; Cogdill, A.; Monette, A.; Najjar, Y.; Sweis, R.; Tschernia, N.; Wennerberg, E.; Bommareddy, P.; et al. (2020)
      Background Multiple genomics-based biomarkers of response to immune checkpoint inhibition have been reported or proposed, including tumor mutation/neoantigen frequency, PD-L1 expression, T cell receptor repertoire clonality, interferon gene signature expression, HLA expression, and others.1 Although genomics associations of response have been reported, the primary studies have used a variety of data generation and processing techniques. There is a need for data harmonization and assessment of generalizability of potential biomarkers across multiple datasets. Methods We acquired patient-level RNA sequencing FASTQ data files from 10 data sets reported in seven pan-cancer PD-1 and CTLA-4 immune checkpoint inhibition trials with matched clinical annotations.2–7 We applied a common bioinformatics workflow for quality control, mapping to reference (STAR), generating gene expression matrices (SALMON), T cell receptor repertoire inference (MiXCR), extraction of immune gene signatures and immune subtypes,8 and differential gene expression analysis (DESeq2). We analyzed i) immunogenomics features proposed as biomarkers, and ii) gene expression signatures built from each trial for association with overall survival across the set of trials using univariable Cox proportional hazards regression. In all, we assessed 9 total immunogenomics features/signatures. P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Results Of the 9 immunogenomics features assessed, cytolytic activity score and expression of the Follicular Dendritic Cell Secreted Protein gene (FDCSP) were associated with survival in two of seven studies, respectively (adjusted p < 0.05) (figure 1). No proposed biomarkers were significantly associated with survival in more than two studies. The sets of genes significantly associated with clinical benefit across the studies were highly disjoint, with only three genes significant in three studies and thirteen genes significant in two studies (figure 2). No genes were significantly associated with clinical benefit in more than three of seven studies
    • Generation of a contig comprising YACs and BACs within chromosome region 1p13.1.

      Brintnell, Bill; Hey, Yvonne; Jones, David; Hoggard, Nigel; James, Louise A; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, UK. (1997-03)
      Chromosome region 1p13 is known to show loss of heterozygosity (LOH) in a number of human tumor types, including breast. We have generated a contig comprising YACs and BACs spanning part of 1p13.1 which includes the smallest region of overlapping loss identified in our earlier studies. The contig is anchored to the genetic map by a number of microsatellite markers, and by the use of CEPH YACs. We have excluded a number of candidate genes from this region, and we have oriented the contig with respect to the centromere and a number of other genes and markers on 1p13. This resource will be valuable in mapping the target for LOH in breast and other tumors, and may also be useful for the genetic analysis of other genes or diseases known to map to this region.
    • Generation of a mouse SWATH-MS spectral library to quantify 10148 proteins involved in cell reprogramming

      Ulanga, U.; Russell, M.; Patassini, Stefano; Brazzatti, J.; Graham, C.; Whetton, Anthony D; Graham, R. L. J.; Clinical Proteomics Research Group, Division of Molecular and Clinical Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road Manchester, Manchester, U (2021)
      Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods. One such technique is SWATH-MS, which in the main requires prior acquisition of mass spectra to generate an assay reference library. In stem cell research, it has been shown pluripotency can be induced starting with a fibroblast population. In so doing major changes in expressed proteins is inevitable. Here we have created a reference library to underpin such studies. This is inclusive of an extensively documented script to enable replication of library generation from the raw data. The documented script facilitates reuse of data and adaptation of the library to novel applications. The resulting library provides deep coverage of the mouse proteome. The library covers 29519 proteins (53% of the proteome) of which 7435 (13%) are supported by a proteotypic peptide.
    • Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.

      Thomson, Graeme J; Watson, Amanda J; Caldecott, K; Denneny, Olive; Depledge, Paul; Hamilton, Nicola S; Hopkins, Gemma V; Jordan, Allan M; Morrow, Christopher J; Raoof, Ali; et al. (2013-02-12)
      Topoisomerases regulate DNA topology by the transient cleavage and re-ligation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase which repairs this topoisomerase-mediated DNA damage, thus playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in an increased susceptibility and sensitivity to Topo II-induced DNA double strand breaks, thereby revealing Tdp2 as a potentially attractive anti-cancer target. No drug-like inhibitors of Tdp2 have been identified to date and assays suitable for high throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogenous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. Additionally, a new and specific Tdp2 antibody is described. Together these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.
    • Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM).

      Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Sanderson, Helen S; Gardiner, Fiona; Kiseleva, Elena; Goldberg, Martin W; Drummond, Sheona P; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk (2007)
      This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy (SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes (NEs) and nuclear pore complexes (NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at -80 degrees C. Demembranated sperm chromatin should take only approximately 2-3 h to prepare and can be stored at -80 degrees C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.