Background splicing as a predictor of aberrant splicing in genetic disease.

Mutations of splice sites, auxiliary splicing elements and the splicing machinery cause a wide range of genetic disease. Here we report that many of the complex effects of splicing mutations can be predicted from background splicing information, with emphasis on BRCA1, BRCA2 and DMD. Background splicing arises from very low level splicing between rarely used background splice sites and from low-level exon skipping between intron splice sites.

Glycosylated Nanoparticles Derived from RAFT Polymerization for Effective Drug Delivery to Macrophages.

The functional group tolerance and simplicity of reversible addition fragmentation chain transfer (RAFT) polymerization enable its use in the preparation of a wide range of functional polymer architectures for a variety of applications, including drug delivery. Given the role of tumor-associated macrophages (TAMs) in cancer and their dependence on the tyrosine kinase receptor FMS (CSF-1R), the key aim of this work was to achieve effective delivery of an FMS inhibitor to cells using a polymer delivery system. Such a system has the potential to exploit biological features specific to macrophages and therefore provide enhanced selectivity.

Construction of a doxycycline inducible lentivirus that expresses stem cell-specific miR-302 cluster.

Introduction: The polycistronic miR-302 cluster encodes five miRNA genes that have an important role in the regulation of embryonic stem cell function. Studies showed that the miR-302 cluster can reprogram both mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) with high efficiency. The aim of this study was to generate an inducible lentivirus that expresses miR-302 cluster in order to further investigate somatic cell reprogramming by these miRNAs.

Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer.

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.

IsomiRs have functional importance.

Since the inception of deep sequencing, isomiRs are consistently observed to be produced by most miRNA genes in a variety of cell types. IsomiRs appear as a variation in length from the canonical sequence annotated in miRBase, due to an addition or deletion of one or more nucleotides at the 5(') or 3(') ends or both. As the seed sequence is located at the 5(') end of the microRNA, the target mRNA will be theoretically different.

5' isomiR variation is of functional and evolutionary importance.

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3' and/or 5' end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types.

Lentiviral vector transduction of spermatozoa as a tool for the study of early development.

Spermatozoa and lentiviruses are two of nature's most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies.

Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa.

Transgenic technologies conventionally rely on the oocyte as a substrate for genetic modification. Owing to their accessibility, however, male germ cells, including mature sperm, have material advantages for use in transgenesis. Here we have exploited lentiviruses to generate transgenic animals via the male germline.

Cryptic splice sites and split genes.

We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species.

Polyomavirus middle T-antigen is a transmembrane protein that binds signaling proteins in discrete subcellular membrane sites.

Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside.

Novel imatinib derivatives with altered specificity between Bcr-Abl and FMS, KIT, and PDGF receptors.

Imatinib is a clinically important ATP analogue inhibitor that targets the tyrosine kinase domain of the intracellular Abl kinase and the PDGF receptor family. Imatinib has revolutionised the treatment of chronic myeloid leukaemia, which is caused by the oncogene Bcr-Abl and certain solid tumours that harbor oncogenic mutations of the PDGF receptor family. As a leading kinase inhibitor, imatinib also provides an excellent model system to investigate how changes in drug design impact biological activity, which is an important consideration for rational drug design.

Harnessing the tumour-derived cytokine, CSF-1, to co-stimulate T-cell growth and activation.

Aberrant growth factor production is a prevalent mechanism in tumourigenesis. If T-cells responded positively to a cancer-derived cytokine, this might result in selective enhancement of function within the tumour microenvironment. Here, we have chosen colony-stimulating factor-1 (CSF-1) as a candidate to test this concept.

FMS receptor for M-CSF (CSF-1) is sensitive to the kinase inhibitor imatinib and mutation of Asp-802 to Val confers resistance.

The kinase inhibitor imatinib is used in the treatment of chronic myeloid leukaemia, where it targets the intracellular Bcr-Abl tyrosine kinase, and gastrointestinal stromal tumours, where it targets either the KIT or PDGF tyrosine kinase receptors. Here, we report that imatinib is also an effective inhibitor of the closely related FMS receptor for macrophage colony stimulating factor and that mutation of Asp 802 of FMS to Val confers imatinib resistance. Imatinib readily reverted the transformed phenotype of haemopoietic and fibroblast cell lines that express the oncogene v-fms and also inhibited the growth of the Bacl.

Switching on kinases: oncogenic activation of BRAF and the PDGFR family.

The cytoplasmic serine/threonine kinase BRAF and receptor tyrosine kinases of the platelet-derived growth factor receptor (PDGFR) family are frequently activated in cancer by mutations of an equivalent amino acid. Structural studies have provided important insights into why these very different kinases share similar oncogenic hot spots and why the PDGFR juxtamembrane region is also a frequent oncogenic target. This research has implications for other kinases that are mutated in human tumours and for the treatment of cancer using kinase inhibitors.

Exon junction sequences as cryptic splice sites: implications for intron origin.

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns.

Mae mediates MAP kinase phosphorylation of Ets transcription factors in Drosophila.

The evolutionarily conserved Ras/mitogen-activated protein kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors. Here we report the cloning and characterization of a new component of this signal transduction pathway called Mae (for modulator of the activity of Ets).

Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase.

We have developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT-PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation development and to correlate this with embryo sex.

Evidence that downregulation of the M-CSF receptor is not dependent upon receptor kinase activity.

The downregulation of tyrosine kinase receptors attenuates signalling and is thought to be dependent upon intrinsic receptor kinase activity, largely because down-regulation is inhibited by a kinase-inactivating mutation of an invariant lysine residue of the receptors for EGF, insulin, M-CSF and PDGF. We confirmed that this mutation inhibited the degradation of the M-CSF receptor. However, two different kinase inactivating mutations of the invariant amino acids Gly 591 and Glu 633 did not prevent M-CSF-induced receptor degradation, so demonstrating that receptor kinase activity is not essential for this process.

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation.

Expression of a receptor for human macrophage-colony stimulating factor (M-CSF or CSF-1), containing a point mutation which changes an aspartate to a valine at position 802 of the activating loop of the kinase domain, potently transforms the haemopoietic cell line FDC-P1 yet prevents Rat-2 fibroblast transformation. In order to understand this apparent paradox, aspartate 802 was changed by cassette mutagenesis to each of the other 19 amino acids. All hydrophobic amino acid substitutions were transforming when tested in FDC-P1 cells yet inactivating when tested in Rat-2 fibroblasts.

Biological activity of the receptor for macrophage colony-stimulating factor in the human endometrial cancer cell line, Ishikawa.

Previously we found that the Ishikawa endometrial cancer cell line expresses macrophage colony-stimulating factor (M-CSF) and c-fms transcripts and that its proliferation is enhanced by the addition of recombinant M-CSF. This suggested that Ishikawa cells are constitutively stimulated by M-CSF. In support of this we now show that Ishikawa cells secrete M-CSF and that known stimulators of M-CSF production increase the amount detected in Ishikawa cell conditioned medium.