13 results found
Chandrashekran A, Casimir C, Dibb N, et al., 2016, Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer., Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools., Publisher: Humana Press, Pages: 95-106, ISBN: 978-1-4939-3751-6
Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice.
Tan GC, Chan E, Molnar A, et al., 2014, 5 ' isomiR variation is of functional and evolutionary importance, Nucleic Acids Research, Vol: 42, Pages: 9424-9435, ISSN: 1362-4962
We have sequenced miRNA libraries from human embryonic,neural and foetal mesenchymal stem cells.We report that the majority of miRNA genes encodemature isomers that vary in size by one ormore bases at the 3 and/or 5 end of the miRNA.Northern blotting for individual miRNAs showed thatthe proportions of isomiRs expressed by a singlemiRNA gene often differ between cell and tissuetypes. IsomiRs were readily co-immunoprecipitatedwith Argonaute proteins in vivo and were active inluciferase assays, indicating that they are functional.Bioinformatics analysis predicts substantial differencesin targeting between miRNAs with minor 5differences and in support of this we report that a 5isomiR-9–1 gained the ability to inhibit the expressionof DNMT3B and NCAM2 but lost the ability toinhibit CDH1 in vitro. This result was confirmed bythe use of isomiR-specific sponges. Our analysis ofthe miRGator database indicates that a small percentageof human miRNA genes express isomiRs asthe dominant transcript in certain cell types and analysisof miRBase shows that 5 isomiRs have replacedcanonical miRNAs many times during evolution. Thisstrongly indicates that isomiRs are of functional importanceand have contributed to the evolution ofmiRNA genes.INT
Witney TH, Carroll L, Alam IS, et al., 2014, A novel radiotracer to image glycogen metabolism in tumors by positron emission tomography, Cancer Research, Vol: 74, Pages: 1319-1328, ISSN: 0008-5472
The high rate of glucose uptake to fuel the bioenergetic and anabolic demands of proliferating cancer cells is well recognized and is exploited with 18F-2-fluoro-2-deoxy-d-glucose positron emission tomography (18F-FDG–PET) to image tumors clinically. In contrast, enhanced glucose storage as glycogen (glycogenesis) in cancer is less well understood and the availability of a noninvasive method to image glycogen in vivo could provide important biologic insights. Here, we demonstrate that 18F-N-(methyl-(2-fluoroethyl)-1H-[1,2,3]triazole-4-yl)glucosamine (18F-NFTG) annotates glycogenesis in cancer cells and tumors in vivo, measured by PET. Specificity of glycogen labeling was demonstrated by isolating 18F-NFTG–associated glycogen and with stable knockdown of glycogen synthase 1, which inhibited 18F-NFTG uptake, whereas oncogene (Rab25) activation–associated glycogen synthesis led to increased uptake. We further show that the rate of glycogenesis is cell-cycle regulated, enhanced during the nonproliferative state of cancer cells. We demonstrate that glycogen levels, 18F-NFTG, but not 18F-FDG uptake, increase proportionally with cell density and G1–G0 arrest, with potential application in the assessment of activation of oncogenic pathways related to glycogenesis and the detection of posttreatment tumor quiescence.
Chandrashekran A, Sarkar R, Thrasher A, et al., 2014, Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa, FASEB JOURNAL, Vol: 28, Pages: 569-576, ISSN: 0892-6638
Chandrashekran A, Isa I, Dudhia J, et al., 2014, Lentiviral vector transduction of spermatozoa as a tool for the study of early development, FEBS OPEN BIO, Vol: 4, Pages: 266-275, ISSN: 2211-5463
Pisaneschi F, Witney T, Iddon L, et al., 2013, Synthesis and evaluation of ICL-CCIC-37, a new probe for the PET imaging of the fatty acid synthesis pathway in tumors., JOURNAL OF LABELLED COMPOUNDS & RADIOPHARMACEUTICALS, Vol: 56, Pages: S408-S408, ISSN: 0362-4803
Witney TH, Chandrashekran A, Fortt R, et al., 2013, Noninvasive imaging of tumor apoptosis in NSCLC with F-18-ICMT-11 positron emission tomography, AACR/SNMMI Joint Conference on State-of-the-Art Molecular Imaging in Cancer Biology and Therapy, Publisher: SOC NUCLEAR MEDICINE INC, Pages: 27-27, ISSN: 0161-5505
Lavial F, Bessonnard S, Ohnishi Y, et al., 2012, Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development, GENES & DEVELOPMENT, Vol: 26, Pages: 1445-1458, ISSN: 0890-9369
Alder O, Lavial F, Helness A, et al., 2010, Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment, Development, Vol: 137, Pages: 2483-2492, ISSN: 0950-1991
Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. We show that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, our results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development.
Taribagil SS, Chandrashekran A, Stamp G, et al., 2004, P13-kinase signalling pathway is involved in adipogenic differentiation of fetal liver derived mesenchymal stem cells, Annual Meeting of the American-Society-for-Cell-Biology, Publisher: AMER SOC CELL BIOLOGY, Pages: 338A-338A, ISSN: 1059-1524
Chandrashekran A, Gordon MY, Darling D, et al., 2004, Growth factor displayed on the surface of retroviral particles without manipulation of envelope proteins is biologically active and can enhance transduction, JOURNAL OF GENE MEDICINE, Vol: 6, Pages: 1189-1196, ISSN: 1099-498X
Chandrashekran A, Gordon MY, Casimir C, 2004, Targeted retroviral transduction of c-kit(+) hematopoietic cells using novel ligand display technology, BLOOD, Vol: 104, Pages: 2697-2703, ISSN: 0006-4971
Alenzi FQB, Marley SB, Lewis JL, et al., 2002, A role for the Fas/Fas ligand apoptotic pathway in regulating myeloid progenitor cell kinetics, EXPERIMENTAL HEMATOLOGY, Vol: 30, Pages: 1428-1435, ISSN: 0301-472X
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