Targeted insertion of an IRES Cre into the Hnf4alpha locus: Cre-mediated recombination in the liver, kidney, and gut epithelium

The Hnf4alpha gene belongs to a family of trancriptional regulators required for liver development and function. Hnf4alpha is also expressed in other tissues, including the newly formed visceral endoderm of the early postimplantation embryo, and later in embryogenesis in the gut epithelium and the kidney. The regulatory sequences involved in controlling expression of Hnf4alpha at these diverse sites are not clearly understood. Here we used homologous recombination to introduce Cre recombinase coding sequences into the endogenous Hnf4alpha locus. Crossing Hnf4alpha(Creex2/+) mice with R26R partners allowed us to follow the pattern of Cre-mediated recombination. Our results show that recombination of the reporter allele closely follows endogenous Hnf4alpha expression, but with a slight temporal delay. Thus, the Hnf4alpha(Creex2) strain should prove useful for conditionally deleting gene activity in the liver, gut epithelium, or kidney.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

Excision of the Frt-flanked neo (R) cassette from the CD19cre knock-in transgene reduces Cre-mediated recombination

Intercross of mice transgenic for Flp-recombinase with the CD19cre mouse strain leads to excision of the Frt-flanked neo ^R cassette from the CD19cre knock-in transgene. This significantly reduces the expression level of Cre by the CD19cre transgene and consequently decreases the extent of Cre-mediated recombination of loxP-flanked alleles, most likely due to the fact that this neo ^R cassette contains polyoma enhancer sequences. We wish to draw attention to this finding, since the Flp-deleter mouse strain is commonly used to remove Frt-flanked selection cassettes in vivo from conditional alleles. Therefore conditional alleles have to be separated from the Flp-deleter transgene by breeding before crosses with CD19cre mice are initiated. In addition our findings suggest that gene expression from the CD19 locus can be increased by the insertion of exogenous enhancer sequences, without compromising B cell specificity.     

Peptide vector for gene delivery with high affinity for phosphatidylserine.

Since phosphatidylserine (PS) is known to translocate to the external face of the plasma membrane when the cell membrane becomes disordered, we decided to focus our attention on PS as a target molecule for gene delivery. In this paper, the novel peptide Td3701 was designed, synthesized, and characterized for its physico-chemico-biological properties. Td3701 simultaneously exhibited both characters as a DNA carrier and a sensor probe for active targeting, which seemed to be triggered by structural changes in the presence of PS. This is a very unique character among nonviral vectors, and it is believed that Td3701 could be used for selective gene delivery.     

Improvement of peptide vectors for gene delivery with active targeting profiles for phosphatidylserine

A cationic peptide, Td3701, which was derived from factor VIII that has affinity with phosphatidylserine (PS), showed efficient transfection ability for cells that express PS on the cell surface. PS is exposed on tumor cell surfaces therefore we have focused on PS as the target molecule for tumor specific gene delivery. In this article, to improve transfection efficiency and specificity in targeting tumor cells, some amino acid residues of Td3701 were replaced. The resulting peptide, Td3717, shows higher transfection efficiency (more than 30 times that of Td3701). The transfection efficiency was dependent on the amount of PS on the cell surface, suggesting that Td3717 bound with plasmid DNA could recognize PS on the cell surface. Td3717 is expected to be useful as an efficient gene carrier molecule specific to PS‐presenting tumor cells. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOSalpha beta-finger is largely exposed to antibodies

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.     

Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a , one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T₀) transgenic knock-in plants obtained were found to carry only one copy of GUS , with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter -fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.     

Steroidal constituents of rice (Rryza sativa) hulls with algicidal and herbicidal activity against blue-green algae and duckweed

Two new compounds, 14-methyl stigmast-9(11)-en-3alpha-ol-3beta-D-glucopyranoside (1) and cholest-11-en-3beta, 6beta, 7alpha, 22beta-tetraol-24-one-3beta-palmitoleate (2), along with the known compound beta-sitosteryl-3beta-D-glucopyranosyl-6'-linoleiate (3), were isolated from the methanolic extract of rice (Oryza sativa) hulls. The structures of the two new compounds were elucidated using one- and two-dimensional NMR in combination with IR, EI/MS, FAB/MS, HR-EI/MS and HR-FAB/MS. In bioassays with blue-green algae, Microcystis aeruginosa UTEX 2388 and duckweed, Lemna paucicostata Hegelm 381, the efficacy of bioactivity of the two new compounds linearly increased as the concentration increased from 0.3 to 300 IgM. Compared with momilactone A, compounds 1 and 2 showed similar and higher inhibitory activities against the growth of M. aeruginosa at a concentration of 300 microM. However, compound 2 was similar to momilactone A in inhibiting L. paucicostata growth at a concentration of 300 microM. As a result, compound 2 appears to have a strong potential for the environmentally friendly control of weed and algae that are harmful to water-logged rice.     

Allelic variants interaction of CLOCK gene and G-protein beta3 subunit gene with diurnal preference

The 3111 C/T single nucleotide polymorphism (SNP) in the CLOCK gene and the 825C/T SNP in the G-protein β3 subunit gene (GNB3) have been reported to influence diurnal preference. This study has attempted to characterize the association between the CLOCK gene and GNB3 polymorphisms and diurnal preference in healthy Korean college students. All subjects completed the 13-item Composite Scale for Morningness (CSM). The interaction between the 3111 C/T SNP in the CLOCK gene and the 825 C/T SNP in the GNB3 gene significantly influenced diurnal preference, according to the CSM Performance subscore (F=10.94, p=0.001). However, when the different polymorphisms of the two genes were analyzed independently, no direct correlations with diurnal preference were detected. The CLOCK gene 3111 C/T SNP and GNB3 gene 825 C/T SNP were found to manifest a gene-gene interaction that affects diurnal preference.     

Effects of different intensities of extremely low frequency pulsed electromagnetic fields on formation of osteoclast-like cells

Over the past 30 years, the beneficial therapeutic effects of selected low energy, time varying electromagnetic fields (EMF) have been documented with increasing frequency to treat therapeutically resistant problems of the musculoskeletal system. However, the underlying mechanisms at a cellular level are still not completely understood. In this study, the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)(2)D(3), were examined. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8, 8.7, and 12.2 micro V/cm rms) and stimulation times (0.5, 2, and 8 h/day). Recruitment and authentication of osteoclast-like cells were evaluated, respectively, by determining multinuclear, tartrate resistant acid phosphatase (TRAP) positive cells on day 8 of culture and by the pit formation assay. During the experiments, cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and prostaglandin-E(2) (PGE(2)) were assayed using the enzyme linked immunosorbent assay (ELISA). These findings suggest that ELF-PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast-like cells in bone marrow culture, depending on the induced electric field intensity. In addition, consistent correlations were observed between TNF-alpha, IL-1beta, and osteoclast-like cell number after exposure to different induced electric field intensities of ELF-PEMF. This in vitro study could be considered as groundwork for in vivo ELF-PEMF clinical applications on some osteoclast-associated bone diseases.