Targeted gene modification in mouse ES cells using integrase‐defective lentiviral vectors

Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase‐defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector‐mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild‐type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild‐type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 ± 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene‐manipulation in therapeutic applications. genesis 47:217–223, 2009. © 2009 Wiley‐Liss, Inc.     

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1 fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation.

Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.     

Solvent polarity effects on supramolecular chirality of a polyfluorene‐thiophene copolymer

This study demonstrates the supramolecular chirality control of a conjugated polymer via solvent polarity. We designed and synthesized a chiral polyfluorene‐thiophene copolymer having two different chiral side chains at the 9‐position of the fluorene unit. Chiral cyclic and alkyl ethers with different polarities were selected as the chiral side chains. The sign of the circular dichroism spectra in the visible wavelength region was affected by the solvent system, resulting from the change of supramolecular structure. The estimation of the solubility parameter revealed that the solubility difference of the side chains contributed to the change of the circular dichroism sign, which was also observed in spin‐coated films prepared from good solvents having different polarities.     

Higher susceptibility to osmolality of the medaka (Oryzias latipes) mutants in orthologue genes of mammalian skin transglutaminases

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.     

FTY720 (Gilenya) phosphate selectivity of sphingosine 1-phosphate receptor subtype 1 (S1P1) G protein-coupled receptor requires motifs in intracellular loop 1 and transmembrane domain 2

FTY720 phosphate (FTY720P) is a high potency agonist for all the endothelial differentiation gene family sphingosine 1-phosphate (S1P) receptors except S1P receptor subtype 2 (S1P(2)). To map the distinguishing features of S1P(2) ligand recognition, we applied a computational modeling-guided mutagenesis strategy that was based on the high degree of sequence homology between S1P(1) and S1P(2). S1P(2) point mutants of the ligand-binding pocket were characterized. The head group-interacting residues Arg3.28, Glu3.29, and Lys7.34 were essential for activation. Mutation of residues Ala3.32, Leu3.36, Val5.41, Phe6.44, Trp6.48, Ser7.42, and Ser7.46, predicted to interact with the S1P hydrophobic tail, impaired activation by S1P. Replacing individual or multiple residues in the ligand-binding pocket of S1P(2) with S1P(1) sequence did not impart activation by FTY720P. Chimeric S1P(1)/S1P(2) receptors were generated and characterized for activation by S1P or FTY720P. The S1P(2) chimera with S1P(1) sequence from the N terminus to transmembrane domain 2 (TM2) was activated by FTY720P, and the S1P(2)(IC1-TM2)(S1P1) domain insertion chimera showed S1P(1)-like activation. Twelve residues in this domain, distributed in four motifs a-d, differ between S1P(1) and S1P(2). Insertion of (78)RPMYY in motif b alone or simultaneous swapping of five other residues in motifs c and d from S1P(1) into S1P(2) introduced FTY720P responsiveness. Molecular dynamics calculations indicate that FTY720P binding selectivity is a function of the entropic contribution to the binding free energy rather than enthalpic contributions and that preferred agonists retain substantial flexibility when bound. After exposure to FTY720P, the S1P(2)(IC1-TM2)(S1P1) receptor recycled to the plasma membrane, indicating that additional structural elements are required for the selective degradative trafficking of S1P(1).     

Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle–eye–brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of α-DG, and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^?/? muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1^?/? satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^?/? myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of α-DG is important for maintenance of the proliferative activity of satellite cells in vivo.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).