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1.
Oh SC Nam SY Kwon HC Kim CM Seo JS Seong RH Jang YJ Chung YH Chung HY 《Molecules and cells》2001,11(2):192-197
We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required. 相似文献
2.
Yang H Williams BO Hinds PW Shih TS Jacks T Bronson RT Livingston DM 《Molecular and cellular biology》2002,22(9):3103-3110
Rb(+/+):Rb(-/-) chimeric mice are healthy until early in adulthood when they develop lethal pituitary tumors composed solely of Rb(-/-) cells. In an effort to delineate the minimal structures of the retinoblastoma protein necessary for RB tumor suppression function, chimeric animals derived from stably transfected RB(-/-) embryonic stem (ES) cells were generated. One such ES cell transfectant expressed a human RB allele encoding a stable, truncated nuclear derivative lacking residues 1 to 378 (Delta 1-378). Others encoded either wild-type human RB or an internally deleted derivative of the Delta 1-378 mutant. All gave rise to viable chimeric animals with comparable degrees of chimerism. However, unlike control mice derived, in part, from naive Rb(-/-) ES cells or from ES cells transformed by the double RB mutant, Delta 1-378/Delta exon22, animals derived from either wild-type RB- or Delta 1-378 RB-producing ES cells failed to develop pituitary tumors. Thus, in this setting, a substantial fraction of the RB sequence is unnecessary for RB-mediated tumor suppression. 相似文献
3.
In order to gain insight into the catalytic mechanism of Fe-type nitrile hydratases (NHase), the pH and temperature dependence of the kinetic parameters k cat, K m, and k cat/ K m along with the solvent isotope effect were examined for the Fe-type NHase from Comamonas testosteroni Ni1 ( CtNHase). CtNHase was found to exhibit a bell-shaped curve for plots of relative activity vs pH over pH values 4-10 for the hydration of acrylonitrile and was found to display maximal activity at pH approximately 7.2. Fits of these data provided a p K ES1 value of 6.1 +/- 0.1, a p K ES2 value of 9.1 +/- 0.2 ( k' cat = 10.1 +/- 0.3 s (-1)), a p K E1 value of 6.2 +/- 0.1, and a p K E2 value of 9.2 +/- 0.1 ( k' cat/ K' m of 2.0 +/- 0.2 s (-1) mM (-1)). Proton inventory studies indicate that two protons are transferred in the rate-limiting step of the reaction at pH 7.2. Since CtNHase is stable to 25 degrees C, an Arrhenius plot was constructed by plotting ln( k cat) vs 1/ T, providing an E a of 33.3 +/- 1.5 kJ/mol. Delta H degrees of ionization values were also determined, thus helping to identify the ionizing groups exhibiting the p K ES1 and p K ES2 values. Based on Delta H degrees ion data, p K ES1 is assigned to betaTyr68 while p K ES2 is assigned to betaArg52, betaArg157, or alphaSer116 (NHases are alpha 2beta 2 heterotetramers). Given the strong similarities in the kinetic data obtained for both Co- and Fe-type NHase enzymes, both types of NHase enzymes likely hydrate nitriles in a similar fashion. 相似文献
4.
Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis. 相似文献
5.
Combined effect of CCR5-Delta32 heterozygosity and the CCR5 promoter polymorphism -2459 A/G on CCR5 expression and resistance to human immunodeficiency virus type 1 transmission 总被引:1,自引:0,他引:1
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Hladik F Liu H Speelmon E Livingston-Rosanoff D Wilson S Sakchalathorn P Hwangbo Y Greene B Zhu T McElrath MJ 《Journal of virology》2005,79(18):11677-11684
Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) Delta32, CCR5 promoter -2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4+ T cells and CD14+ monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 -2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P = 0.02). This increase was mostly attributable to a higher frequency of the -2459 A/G versus the -2459 A/A genotype in individuals heterozygous for the delta32 allele (P = 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF delta32/CCR5 -2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 -2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r = 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF delta32/wt-CCR5 -2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1. 相似文献
6.
Wang H Gilner JB Bautch VL Wang DZ Wainwright BJ Kirby SL Patterson C 《The Journal of biological chemistry》2007,282(1):782-791
Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation. 相似文献
7.
Expression of kFGF, which belongs to the family of fibroblast growth factor genes, is restricted to undifferentiated embryonal carcinoma and embryonic stem cells. Stem cell specific expression of kFGF is controlled by a distally localized enhancer, conferring both positive and negative regulation to the kFGF and tk promoters. This enhancer contains a consensus octamer binding sequence that controls positive regulation in EC and ES cells. The octamer sequence binds Oct1 and Oct4 in nuclear extracts from undifferentiated EC cells, while only Oct1 is bound in nuclear extracts from RA differentiated cells. These results suggest that the kFGF gene is a target for positive regulation by Oct4 and implicate Oct4 as target for regulation by the retinoic acid receptors. 相似文献
8.
Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells. 总被引:7,自引:1,他引:6
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We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a substrate for detecting the presence of immunoglobulin heavy chain constant region (CH) gene switch (S) recombination activity in murine pre-B cells. ZN(Smu/S gamma 2b)tk1 contains a neomycin (neo) resistance gene in addition to the herpes simplex virus thymidine kinase (Htk) gene which is positioned between murine Smu and S gamma 2b sequences. Stable acquisition of the ZN(Smu/S gamma 2b)tk1 vector was selected in G-418 and switch region recombination within these proviruses was selected by resistance to the drug bromodeoxyuridine (BUdR). Fluctuation analyses of ZN(Smu/S gamma 2b)tk1 infected 18-8tk- and 38B9tk- pre-B lines revealed Htk gene inactivations with apparent frequencies of 5 X 10(-5) and 1 X 10(-5) events/cell/generation, respectively, while G-418 resistant Ltk- fibroblasts lost the HTK phenotype at an apparent rate of 4 X 10(-8). Southern blot analysis demonstrated that switch recombination caused the deletion of the Htk gene in all pre-B clones examined while the loss of Htk in Ltk- clones was not mediated by S region recombination. In 21 out of 24 pre-B clones, the recombinations involved the tandemly repetitive portions of the Smu and S gamma 2b sequences. These results demonstrate that the CH gene S region segments inserted into ZN(Smu/S gamma 2b)tk1 are sufficient for B-cell-specific recombination/deletion within the S region tandem repeats. 相似文献
9.
Frank Y. T. Sin 《Molecular biology reports》1987,12(2):79-83
The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed. 相似文献
10.
C. Bandeira-Duarte C. A. M. Carvalho E. J. Cragoe Jr A. P. Carvalho 《Neurochemical research》1990,15(3):313-320
Preparations of synaptosomes isolated in sucrose or in Na+-rich media were compared with respect to internal pH (pH1), internal Ca2+ concentration ([Ca2+]i), membrane potential and45Ca2+ uptake due to K+ depolarization and Na+/Ca2+ exchange. We found that synaptosomes isolated in sucrose media have a pHi of 6.77±0.04 and a [Ca2+]i of about 260 nM, whereas synaptosomes isolated in Na+-rich ionic media have a pHi of 6.96±0.07 and a [Ca2+]i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca2+ only by voltage sensitive calcium channels (VSCC'S) when K+-depolarized, while the Na+-rich synaptosomes take up45Ca2+ both by VSCC'S and by Na+/Ca2+ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca2+ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca2+ uptake by VSCC'S, but not by Na+/Ca2+ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca2+ flux through channels and through Na+/Ca2+ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca2+ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF
2,7-Biscarboxyethyl-5(6)-carboxyfluorescein
- BCECF/AM
acetoxymethyl ester of BCECF
- [Ca2+]i
Internal free calcium ion concentration
- CBZ-DMB
5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil
- DMB
2, 4-dimethylbenzamil
- DMSO
dimethyl sulfoxide
- Indo-1/AM
acetoxymethyl ester of Indo-1
- MES
2-|N-Morpholino|ethanesulfonic acid
- NMG
N-methyl-D-glucamine
- pHi
internal pH
- TPP+
tetraphenylphosphonium
- p
plasma membrane potential 相似文献
11.
12.
Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells. 总被引:6,自引:3,他引:3
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Mouse Ltk- cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk+ segregants at an average rate of 10(-8) events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10(-9). One such cell line produced homeologous recombinants at a rate of 10(-8). Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation. 相似文献
13.
Carter JR Durocher JJ Kern RP 《American journal of physiology. Regulatory, integrative and comparative physiology》2008,295(6):R1898-R1903
Sympathetic neural responses to mental stress are well documented but controversial, whereas sympathetic neural responses to emotional stress are unknown. The purpose of this study was to investigate neural and cardiovascular responses to emotional stress evoked by negative pictures and reexamine the relationship between muscle sympathetic nerve activity (MSNA) and perceived stress. Mean arterial pressure (MAP), heart rate (HR), MSNA, and perceived stress levels were recorded in 18 men during three randomized trials: 1) neutral pictures, 2) negative pictures, and 3) mental stress. MAP and HR increased during mental stress (Delta14 +/- 2 mmHg and Delta15 +/- 2 beats/min, P < 0.001) but did not change during viewing of negative or neutral pictures. MSNA did not change during viewing of neutral (Delta1 +/- 1 burst/min, n = 16) or negative (Delta0 +/- 1 burst/min, n = 16) pictures or during mental stress (Delta1 +/- 2 burst/min, n = 13). Perceived stress levels were higher during mental stress (3 +/- 0 arbitrary units) than during viewing negative pictures (2 +/- 0 arbitrary units, P < 0.001). Perceived stress levels were not correlated to changes in MSNA during negative pictures (r = 0.10, P = 0.84) or mental stress (r = 0.36, P = 0.23). In conclusion, our results demonstrate robust increases in MAP and HR during mental stress, but not during emotional stress evoked by negative pictures. Although the influence of mental stress on MSNA remains unresolved, our findings challenge the concept that perceived stress levels modulate MSNA during mental stress. 相似文献
14.
Generation of a floxed Presenilin-1 (PS1) allele involved two recombination events in the embryonic stem (ES) cells. First, a targeting vector containing a loxP site in intron 1 and a floxed CMV-HYG/TK double selection cassette in intron 3 was integrated into the PS1 locus by homologous recombination. The use of a negative selection cassette, PGK-DTA, dramatically increased the recombination efficiency within the targeted locus (75-fold). Second, an expression vector encoding Cre recombinase was introduced to excise the floxed CMV-HYG/TK cassette via site-specific recombination. However, all five ES cell clones testing positive for the proper removal of the CMV-HYG/TK cassette also contained a proportion of ES cells in which recombination had occurred between the distal loxP sites in introns 1 and 3, resulting in excision of the entire floxed region. It is therefore critical to screen for possible recombination events involving all 3 loxP sites, in order to identify ES cells clones bearing high proportions of the desired ES cells. genesis 26:5-8, 2000. 相似文献
15.
Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: a transient gene-integration marker for ES cells. 总被引:12,自引:1,他引:11
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Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice carrying specifically designed mutations in the germline. Puromycin can completely kill ES cells within 24 to 48 h whereas G418 and hygromycin cannot. We have, therefore, proposed that the puromycin N-acetyltransferase ( pac ) gene, may be utilized as a transient gene-integration marker. Using a circular expression vector of cre and pac genes, Cre-mediated mutant cells were effectively enriched by pulse treatment of puromycin without stable integration of their genes. We have thus demonstrated the first application of pac as a transient gene-integration marker for ES cells. 相似文献
16.
Thrombopoietin (Tpo) and its receptor, c-mpl, are expressed in murine embryonic stem (ES) cells. ES cells are maintained in a pluripotent state by leukemia inhibitory factor (LIF) via activation of the Janus kinase (Jak)-STAT3 signaling pathway. Tpo, like LIF, activates STAT3. We report that Tpo increases the number of undifferentiated colonies derived from wild type or Shp-2 mutant (Shp-2(Delta46-110)) ES cells. Tpo plus LIF acted synergistically on the Shp-2(Delta46-110) ES cells to maintain undifferentiated colonies but no evidence of synergism via Jak-STAT3 activation was detected. Collectively, these data suggest that Tpo can play a role in preventing ES cell differentiation via Jak-STAT3 activation and perhaps via novel pathways that are enhanced in the absence of functional Shp-2. 相似文献
17.
18.
Animal studies indicate that opioids inhibit the firing rate of vestibular neurons, which are important in mediating the vestibulosympathetic reflex. Furthermore, this inhibition appears to be greater in more mature rats. In the present study, we tested the hypotheses that opioids inhibit the vestibulosympathetic reflex in humans and that endogenous opioids contribute to the age-related impairment of the vestibulosympathetic reflex. These hypotheses were tested by measuring muscle sympathetic nerve activity (MSNA), arterial blood pressure, and heart rate responses to otolith organ engagement during head-down rotation (HDR) in young (24 +/- 2 yr old) and older (63 +/- 2 yr) subjects before and after administration of either an opioid-receptor antagonist (16 mg naloxone in 9 young and 8 older subjects) or an opioid-receptor agonist (60 mg codeine in 7 young and 7 older subjects). Naloxone did not augment the reflex increase in MSNA during HDR in young (Delta7 +/- 2 vs. Delta4 +/- 2 bursts/min and Delta81 +/- 23 vs. Delta60 +/- 24% change in burst frequency and total MSNA before and after naloxone, respectively) or older subjects (Delta2 +/- 2 vs. Delta1 +/- 2 burst/min and Delta8 +/- 7 vs. Delta8 +/- 9% before and after naloxone). Similarly, codeine did not attenuate the increase in MSNA during HDR in young (Delta8 +/- 1 vs. Delta7 +/- 2 bursts/min and Delta53 +/- 4 vs. Delta64 +/- 16% before and after codeine) or older subjects (Delta6 +/- 4 vs. Delta3 +/- 3 bursts/min and Delta38 +/- 21 vs. Delta33 +/- 20%). Mean arterial blood pressure and heart rate responses to HDR were not altered by either naloxone or codeine. These data do not provide experimental support for the concept that opioids modulate the vestibulosympathetic reflex in humans. Moreover, endogenous opioids do not appear to contribute the age-associated impairment of the vestibulosympathetic reflex. 相似文献
19.
Genetic ablation experiments are used to resolve problems regarding cell lineages and the in vivo function of certain groups of cells. We describe a two-component conditional ablation technology using a mouse carrying an X-linked puDeltatk transgene, which is only activated in cells expressing Cre. Ablation of the Cre-expressing cells can be temporally regulated by the time of ganciclovir (GCV) administration. This strategy was demonstrated using a Col2Cre transgenic line. Differentiating chondrocytes in bigenic animals could be ablated at different developmental stages resulting in disorganized growth plates and dwarfism. Macrocephaly, macroglossia and umbilical hernia were also observed in ablated 18.5 dpc embryos. Crosses between the puDeltatk selector transgenic line and existing cre lines will facilitate numerous temporally regulated tissue-specific ablation experiments. 相似文献
20.
Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis 总被引:2,自引:0,他引:2
Fico A Paglialunga F Cigliano L Abrescia P Verde P Martini G Iaccarino I Filosa S 《Cell death and differentiation》2004,11(8):823-831
Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pd Delta) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pd Delta and wild-type (wt) ES cells. We found that G6pd Delta ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-X(L). Bcl-X(L) does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Interestingly, ERK activation has opposite outcomes in G6pd Delta ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level. 相似文献