BACKGROUND & AIMS: OBJECTIVE:To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS:Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS:The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR. CONCLUSION:FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.

背景与目标：

BACKGROUND & AIMS: :The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp(90) of the catfish GnRH receptor can be substituted by a neutral Asn(90) without abolishing receptor signalling if another negatively charged Glu(93) is introduced in a proximal region of the receptor interior, thereby mimicking the Glu(90)-Lys(121) salt bridge of mammalian GnRH receptors.

背景与目标： : 跨膜结构域2中Asp残基的带负电荷的侧链可能在受体信号传导中起重要作用，因为它在整个g蛋白偶联受体家族中高度保守的，除了哺乳动物促性腺激素释放激素 (GnRH) 受体。在本文中，我们表明，如果在受体的近端区域引入另一个带负电的Glu(93)，则cat鱼GnRH受体的保守Asp(90) 可以被中性Asn(90) 取代，而不会消除受体信号传导内部，从而模仿哺乳动物GnRH受体的Glu(90)-Lys(121) 盐桥。

BACKGROUND & AIMS: :G protein-coupled receptors (GPCRs) coupled to activation of Gs, such as the PTH1 receptor (PTH1R), have long been known to regulate skeletal function and homeostasis. However, the role of GPCRs coupled to other G proteins such as Gi is not well established. We used the tet-off system to regulate the expression of an activated Gi-coupled GPCR (Ro1) in osteoblasts in vivo. Skeletal phenotypes were assessed in mice expressing Ro1 from conception, from late stages of embryogenesis, and after weaning. Long bones were assessed histologically and by microcomputed tomography. Expression of Ro1 from conception resulted in neonatal lethality that was associated with reduced bone mineralization. Expression of Ro1 starting at late embryogenesis resulted in a severe trabecular bone deficit at 12 wk of age (>51% reduction in trabecular bone volume fraction in the proximal tibia compared with sex-matched control littermates; n = 11; P < 0.01). Ro1 expression for 8 wk beginning at 4 wk of age resulted in a more than 20% reduction in trabecular bone volume fraction compared with sex-matched control littermates (n = 16; P < 0.01). Bone histomorphometry revealed that Ro1 expression is associated with reduced rates of bone formation and mineral apposition without a significant change in osteoblast or osteoclast surface. Our results indicate that signaling by a Gi-coupled GPCR in osteoblasts leads to osteopenia resulting from a reduction in trabecular bone formation. The severity of the phenotype is related to the timing and duration of Ro1 expression during growth and development. The skeletal phenotype in Ro1 mice bears some similarity to that produced by knockout of Gs-alpha expression in osteoblasts and thus may be due at least in part to Gi-mediated inhibition of adenylyl cyclase.

背景与目标： : g蛋白偶联受体 (GPCRs) 与Gs的激活 (例如PTH1受体 (PTH1R)) 偶联，长期以来一直已知可以调节骨骼功能和体内平衡。但是，与其他g蛋白 (例如Gi) 偶联的GPCRs的作用尚不明确。我们使用tet-off系统调节体内成骨细胞中活化的Gi偶联GPCR (Ro1) 的表达。在受孕，胚胎发生后期和断奶后表达Ro1的小鼠中评估了骨骼表型。通过组织学和显微计算机断层扫描对长骨进行评估。受孕后Ro1的表达导致新生儿致死，这与骨矿化减少有关。在胚胎发生晚期开始的Ro1的表达导致在12周龄时严重的小梁骨缺损 (与性别匹配的同窝对照相比，胫骨近端的小梁骨体积分数> 51% 减少; n = 11; P <0.01)。与性别匹配的同窝对照组相比，从4周龄开始的8周Ro1表达导致小梁骨体积分数减少20% 以上 (n = 16; P <0.01)。骨组织形态计量学显示，Ro1表达与骨形成和矿物质并置率降低有关，而成骨细胞或破骨细胞表面没有明显变化。我们的结果表明，成骨细胞中Gi偶联GPCR的信号传导导致骨小梁骨形成减少导致骨质减少。表型的严重程度与生长发育过程中Ro1表达的时间和持续时间有关。Ro1小鼠的骨骼表型与成骨细胞中Gs-α 表达的敲除产生的表型相似，因此可能至少部分归因于Gi介导的腺苷酸环化酶的抑制。

BACKGROUND & AIMS: :Hippocampal CA1 pyramidal neurons are normally quiescent but can fire spontaneously when stimulated by muscarinic agonists. In brain slice recordings from mouse CA1 pyramidal neurons, we examined the ionic basis of this activity using interleaved current-clamp and voltage-clamp experiments. Both in control and after muscarinic stimulation, the steady-state current-voltage curve was dominated by inward TTX-sensitive persistent sodium current (I(NaP)) that activated near -75 mV and increased steeply with depolarization. In control, total membrane current was net outward (hyperpolarizing) near -70 mV so that cells had a stable resting potential. Muscarinic stimulation activated a small nonselective cation current so that total membrane current near -70 mV shifted to become barely net inward (depolarizing). The small depolarization triggers regenerative activation of I(NaP), which then depolarizes the cell from -70 mV to spike threshold. We quantified the relative contributions of I(NaP), hyperpolarization-activated cation current (I(h)), and calcium current to pacemaking by using the cell's own firing as a voltage command along with specific blockers. TTX-sensitive sodium current was substantial throughout the entire interspike interval, increasing as the membrane potential approached threshold, while both Ih and calcium current were minimal. Thus, spontaneous activity is driven primarily by activation of I(NaP) in a positive feedback loop starting near -70 mV and providing increasing inward current to threshold. These results show that the pacemaking "engine" from I(NaP) is an inherent property of CA1 pyramidal neurons that can be engaged or disengaged by small shifts in net membrane current near -70 mV, as by muscarinic stimulation.

背景与目标： : 海马CA1锥体神经元通常处于静止状态，但在毒蕈碱激动剂刺激下会自发发射。在来自小鼠CA1锥体神经元的脑切片记录中，我们使用交错的电流钳和电压钳实验检查了这种活动的离子基础。在对照和毒蕈碱刺激后，稳态电流-电压曲线均由内向TTX敏感的持续钠电流 (I(NaP)) 主导，该电流在75 mV附近激活并随着去极化而急剧增加。在对照中，总膜电流净向外 (超极化) 接近70 mV，使得细胞具有稳定的静息电位。毒蕈碱刺激激活小的非选择性阳离子电流，使得接近70 mV的总膜电流移动，几乎变为净向内 (去极化)。小的去极化触发I(NaP) 的再生激活，然后将细胞从-70 mV去极化到尖峰阈值。我们通过使用细胞自身的放电作为电压命令以及特定的阻滞剂，量化了I(NaP)，超极化激活的阳离子电流 (I(h)) 和钙电流对起搏的相对贡献。TTX敏感的钠电流在整个峰间间隔内都很大，随着膜电位接近阈值而增加，而Ih和钙电流都很小。因此，自发活动主要由正反馈回路中的I(NaP) 的激活驱动，该正反馈回路开始接近70 mV并提供增加的内向电流至阈值。这些结果表明，来自I(NaP) 的起搏 “引擎” 是CA1锥体神经元的固有特性，可以通过接近70 mV的净膜电流的小偏移 (如通过毒蕈碱刺激) 来接合或脱离。

BACKGROUND & AIMS: :Genetic epidemiologists routinely assess disease susceptibility in relation to haplotypes, that is, combinations of alleles on a single chromosome. We study statistical methods for inferring haplotype-related disease risk using single nucleotide polymorphism (SNP) genotype data from matched case-control studies, where controls are individually matched to cases on some selected factors. Assuming a logistic regression model for haplotype-disease association, we propose two conditional likelihood approaches that address the issue that haplotypes cannot be inferred with certainty from SNP genotype data (phase ambiguity). One approach is based on the likelihood of disease status conditioned on the total number of cases, genotypes, and other covariates within each matching stratum, and the other is based on the joint likelihood of disease status and genotypes conditioned only on the total number of cases and other covariates. The joint-likelihood approach is generally more efficient, particularly for assessing haplotype-environment interactions. Simulation studies demonstrated that the first approach was more robust to model assumptions on the diplotype distribution conditioned on environmental risk variables and matching factors in the control population. We applied the two methods to analyze a matched case-control study of prostate cancer.

背景与目标： 遗传流行病学家通常评估与单倍型 (即单个染色体上等位基因的组合) 相关的疾病易感性。我们研究了使用来自匹配病例对照研究的单核苷酸多态性 (SNP) 基因型数据来推断单倍型相关疾病风险的统计方法，其中对照在某些选定因素上与病例单独匹配。假设单倍型-疾病关联的逻辑回归模型，我们提出了两种条件似然方法，以解决无法从SNP基因型数据 (阶段模糊性) 确定性推断单倍型的问题。一种方法是基于疾病状态的可能性，以每个匹配层中的病例总数，基因型和其他协变量为条件，另一种方法是基于疾病状态和基因型的联合可能性，仅以病例总数和其他协变量为条件。联合似然方法通常更有效，特别是对于评估单倍型-环境相互作用。模拟研究表明，第一种方法对以环境风险变量和对照人群中的匹配因子为条件的二倍体型分布的模型假设更稳健。我们应用这两种方法分析了一项匹配的前列腺癌病例对照研究。

BACKGROUND & AIMS: :If acquired associations are to accurately represent real relevance relations, there is motivation for the hypothesis that learning will, in some circumstances, be more appropriately modelled, not as direct dependence, but as conditional independence. In a serial compound conditioning experiment, two groups of rats were presented with a conditioned stimulus (CS1) that imperfectly (50%) predicted food, and was itself imperfectly predicted by a CS2. Groups differed in the proportion of CS2 presentations that were ultimately followed by food (25% versus 75%). Thus, the information presented regarding the relevance of CS2 to food was ambiguous between direct dependence and conditional independence (given CS1). If rats learnt that food was conditionally independent of CS2, given CS1, subjects of both groups should thereafter respond similarly to CS2 alone. Contrary to the conditionality hypothesis, subjects attended to the direct food predictability of CS2, suggesting that rats treat even distal stimuli in a CS sequence as immediately relevant to food, not conditional on an intermediate stimulus. These results urge caution in representing indirect associations as conditional associations, accentuate the theoretical weight of the Markov condition in graphical models, and challenge theories to articulate the conditions under which animals are expected to learn conditional associations, if ever.

背景与目标： : 如果获得的关联要准确地表示真实的关联关系，则存在这样的假设的动机，即在某些情况下，学习将被更适当地建模，而不是作为直接依赖，而是作为条件独立性。在连续复合条件调节实验中，向两组大鼠提供了条件刺激 (CS1)，该条件刺激不完全 (50%) 预测食物，而cs2本身则不完全预测食物。组在CS2呈现的比例上有所不同，最终跟随食物 (25% 与75%)。因此，关于CS2与食物相关性的信息在直接依赖和条件独立性之间是模棱两可的 (给定CS1)。如果大鼠得知食物有条件地独立于CS1，则两组的受试者此后应与单独的CS2类似。与条件性假设相反，受试者关注了CS2的直接食物可预测性，这表明大鼠甚至将CS序列中的远端刺激视为与食物立即相关，而不是以中间刺激为条件。这些结果敦促在将间接关联表示为条件关联时要谨慎，在图形模型中强调马尔可夫条件的理论权重，并挑战理论以阐明期望动物学习条件关联的条件 (如果有的话)。

BACKGROUND & AIMS: :A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.

背景与目标： : 描述了一种新颖的染色体工程技术，该技术可使酿酒酵母单倍体细胞中的自然染色体有条件分裂。该技术包括将归巢核酸内切酶PI-SceI的识别序列引入酿酒酵母基因组，并在MET3启动子的控制下条件表达编码PI-SceI酶的基因。为了测试该技术，我们使用了我们最近开发的自主复制序列 (ARS) 添加的聚合酶链反应介导的染色体分裂 (ARS-PCS) 方法，将GLC7上游的V染色体分裂。随后在GLC7基因座的下游引入了PI-SceI的识别序列。在诱导PI-SceI编码基因后分析了分裂。大约50% 的测试克隆具有预期的微型染色体，仅包含GLC7基因，这表明任何期望的染色体区域都可以通过使用该方法转化为新染色体。由于该技术允许在特定的选择条件下随后诱导随机染色体丢失事件之前初始构建包含多个构建体的菌株，因此我们建议该技术可能适用于通过组合丢失的微型染色体来重建酿酒酵母基因组。

BACKGROUND & AIMS: A dynamic approach to the stochastic modelling of reliability systems is further explored. This modelling approach is particularly appropriate for load-sharing, software reliability, and multivariate failure-time models, where component failure characteristics are affected by their degree of use, amount of load, or extent of stresses experienced. This approach incorporates the intuitive notion that when a set of components in a coherent system fail at a certain time, there is a 'jump' from one structure function to another which governs the residual lifetimes of the remaining functioning components, and since the component lifetimes are intrinsically affected by the structure function which they constitute, then at such a failure time there should also be a jump in the stochastic structure of the lifetimes of the remaining components. For such dynamically-modelled systems, the stochastic characteristics of their jump times are studied. These properties of the jump times allow us to obtain the properties of the lifetime of the system. In particular, for a Markov dynamic model, specific expressions for the exact distribution function of the jump times are obtained for a general coherent system, a parallel system, and a series-parallel system. We derive a new family of distribution functions which describes the distributions of the jump times for a dynamically-modelled system.

背景与目标： 进一步探索了可靠性系统随机建模的动态方法。这种建模方法特别适用于负载分担，软件可靠性和多变量故障时间模型，其中组件故障特征受其使用程度，负载量或所经历的应力程度的影响。这种方法结合了一个直观的概念，即当相干系统中的一组组件在某个时间发生故障时，从一个结构函数到另一个结构函数的 “跳跃”，这控制了其余功能组件的剩余寿命，并且由于组件寿命受到它们构成的结构函数的内在影响，然后，在这样的故障时间，其余组件的寿命的随机结构也应该发生跳跃。对于此类动态建模系统，研究了其跳跃时间的随机特征。跳跃时间的这些属性使我们能够获得系统寿命的属性。特别是，对于马尔可夫动态模型，对于一般相干系统，并行系统和串并联系统，可以获得跳跃时间的精确分布函数的特定表达式。我们推导了一个新的分布函数族，该函数描述了动态建模系统的跳跃时间分布。

BACKGROUND & AIMS: Caenorhabditis elegans vulval development culminates during exit from the L4-to-adult molt with the formation of an opening through the adult hypodermis and cuticle that is used for egg laying and mating. Vulva formation requires the heterochronic gene lin-29, which triggers hypodermal cell terminal differentiation during the final molt. lin-29 mutants are unable to lay eggs or mate because no vulval opening forms; instead, a protrusion forms at the site of the vulva. We demonstrate through analysis of genetic mosaics that lin-29 is absolutely required in a small subset of lateral hypodermal seam cells, adjacent to the vulva, for wild-type vulva formation and egg laying. However, lin-29 function is not strictly limited to the lateral hypodermis. First, LIN-29 accumulates in many non-hypodermal cells with known roles in vulva formation or egg laying. Second, animals homozygous for one lin-29 allele, ga94, have the vulval defect and cannot lay eggs, despite having a terminally differentiated adult lateral hypodermis. Finally, vulval morphogenesis and egg laying requires lin-29 activity within the EMS lineage, a lineage that does not generate hypodermal cells.

背景与目标： 秀丽隐杆线虫的外阴发育在从L4-to-adult蜕皮中退出时达到高潮，形成穿过成年皮下组织和角质层的开口，用于产卵和交配。外阴的形成需要异慢性基因lin-29，这在最终蜕皮过程中触发皮下细胞末端分化。lin-29突变体无法产卵或交配，因为没有外阴开口形成; 相反，在外阴部位形成突起。我们通过对遗传马赛克的分析证明，对于野生型外阴形成和产卵，在与外阴相邻的一小部分外侧皮下接缝细胞中绝对需要lin-29。然而，lin-29功能并不严格限于外侧皮下组织。首先，LIN-29在许多非皮下细胞中积累，在外阴形成或产卵中具有已知作用。其次，一个lin-29等位基因ga94纯合的动物具有外阴缺陷，尽管具有终末分化的成年外侧皮下组织，但无法产卵。最后，外阴形态发生和产卵需要EMS谱系内的lin-29活动，该谱系不会产生皮下细胞。

BACKGROUND & AIMS: :The structure of non-Gaussian autoregressive schemes is described. Asymptotically efficient methods for the estimation of the coefficients of the models are described under appropriate conditions, some of which relate to smoothness and positivity of the density function f of the independent random variables generating the process. The principal interest is in nonminimum phase models.

背景与目标： : 描述了非高斯自回归方案的结构。在适当的条件下描述了估计模型系数的渐近有效方法，其中一些与生成过程的独立随机变量的密度函数f的平滑度和正性有关。主要兴趣在于非最小相位模型。

BACKGROUND & AIMS: BACKGROUND:Traditionally, the placement of the tibial component in total knee arthroplasty (TKA) has focused on maximizing coverage of the tibial surface. However, the degree to which maximal coverage affects correct rotational placement of symmetric and asymmetric tibial components has not been well defined and might represent an implant design issue worthy of further inquiry. QUESTIONS/PURPOSES:Using four commercially available tibial components (two symmetric, two asymmetric), we sought to determine (1) the overall amount of malrotation that would occur if components were placed for maximal tibial coverage; and (2) whether the asymmetric designs would result in less malrotation than the symmetric designs when placed for maximal coverage in a computer model using CT reconstructions. METHODS:CT reconstructions of 30 tibial specimens were used to generate three-dimensional tibia reconstructions with attention to the tibial anatomic axis, the tibial tubercle, and the resected tibial surface. Using strict criteria, four commercially available tibial designs (two symmetric, two asymmetric) were placed on the resected tibial surface. The resulting component rotation was examined. RESULTS:Among all four designs, 70% of all tibial components placed in orientation maximizing fit to resection surface were internally malrotated (average 9°). The asymmetric designs had fewer cases of malrotation (28% and 52% for the two asymmetric designs, 100% and 96% for the two symmetric designs; p < 0.001) and less malrotation on average (2° and 5° for the asymmetric designs, 14° for both symmetric designs; p < 0.001). CONCLUSIONS:Maximizing tibial coverage resulted in implant malrotation in a large percentage of cases. Given similar amounts of tibial coverage, correct rotational positioning was more likely to occur with the asymmetric designs. CLINICAL RELEVANCE:Malrotation of components is an important cause of failure in TKA. Priority should be given to correct tibial rotational positioning. This study suggested that it is easier to balance rotation and coverage with asymmetric tibial baseplates; clinical research will need to determine whether the observed difference affects patellar tracking, loosening rates, or the likelihood of revisions after TKA.

背景与目标：

BACKGROUND & AIMS: :Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.

背景与目标： : 细菌中条件性基因沉默的可靠方法一直难以捉摸。为了改善表达的反义rna (asRNAs) 的沉默，我们系统地改变了几个设计参数，并针对大肠杆菌中的多个报告基因和必需基因。事实证明，成对的末端 (PT) 设计是有效的，其中侧翼反向重复创建成对的dsRNA末端。针对乙酸盐激酶-磷酸转乙酰酶操纵子 (ackA-pta) 内的ackA基因的ptasrna触发了靶mRNA衰减，并且具有高遗传外显率的AckA活性78% 降低。Ptasrna丰富且稳定，并通过独立于RNase III的机制起作用，该机制需要大量化学计量过量的asRNA。有条件的ackA沉默减少了碳向乙酸盐的通量，并增加了异源基因的表达。PT设计还改善了必需fabI基因的沉默。完全的抗fabI PTasRNA诱导可防止生长并部分诱导对FabI抑制剂敏感的细胞。Ptasrna具有功能基因组学，抗菌发现和代谢通量控制的潜力。

BACKGROUND & AIMS: :We previously developed the technique of conditional reprogramming (CR), which allows primary epithelial cells from fresh or cryopreserved specimens to be propagated long-term in vitro, while maintaining their genetic stability and differentiation potential. This method requires a combination of irradiated fibroblast feeder cells and a Rho-associated kinase (ROCK) inhibitor. In the present study, we demonstrate increased levels of full-length p53 and its natural isoform, Δ133p53α, in conditionally reprogrammed epithelial cells from primary prostate, foreskin, ectocervical, and mammary tissues. Increased Δ133p53α expression is critical for CR since cell proliferation is rapidly inhibited following siRNA knockdown of endogenous Δ133p53α. Importantly, overexpression of Δ133p53α consistently delays the onset of cellular senescence of primary cells when cultured under non-CR conditions in normal keratinocyte growth medium (KGM). More significantly, the combination of Δ133p53α overexpression and ROCK inhibitor, without feeder cells, enables primary epithelial cells to be propagated long-term in vitro. We also show that Δ133p53α overexpression induces hTERT expression and telomerase activity and that siRNA knockdown of hTERT causes rapid inhibition of cell proliferation, indicating a critical role of hTERT for mediating the effects of Δ133p53α. Altogether, these data demonstrate a functional and regulatory link between p53 pathways and hTERT expression during the conditional reprogramming of primary epithelial cells.

背景与目标： : 我们先前开发了条件重编程 (CR) 技术，该技术允许新鲜或冷冻保存的标本中的原代上皮细胞在体外长期繁殖，同时保持其遗传稳定性和分化潜力。此方法需要辐照的成纤维细胞饲养细胞和Rho相关激酶 (ROCK) 抑制剂的组合。在本研究中，我们证明了来自原发性前列腺，包皮，宫颈外和乳腺组织的有条件重编程的上皮细胞中全长p53及其天然同工型 Δ133p53α 的水平增加。增加的 Δ133p53α 表达对于CR至关重要，因为在siRNA敲低内源性 Δ133p53α 后，细胞增殖被迅速抑制。重要的是，在正常角质形成细胞生长培养基 (KGM) 中，在非CR条件下培养时，Δ133p53α 的过表达始终会延迟原代细胞的细胞衰老。更重要的是，Δ133p53α 过表达和ROCK抑制剂的组合 (没有饲养细胞) 使原代上皮细胞能够在体外长期繁殖。我们还表明，Δ133p53α 过表达诱导hTERT表达和端粒酶活性，hTERT的siRNA敲低导致细胞增殖的快速抑制，表明hTERT在介导 Δ133p53α 作用中的关键作用。总之，这些数据证明了在原代上皮细胞的条件重编程过程中，p53途径与hTERT表达之间的功能和调节联系。

BACKGROUND & AIMS: :The conditional systems Tet-on and Geneswitch were compared and optimized for the tissue-specific expression of transgenes and manipulation of life span in adult Drosophila. Two versions of Tet-on system reverse-tetracycline-Trans-Activator (rtTA) were compared: the original rtTA, and rtTAM2-alt containing mutations designed to optimize regulation and expression. The rtTAM2-alt version gave less leaky expression of target constructs in the absence of doxycyline, however the absolute level of expression that could be achieved was less than that produced by rtTA, in contrast to a previous report. Existing UAS-rtTAM2-alt insertions were re-balanced, and combined with several tissue-general and tissue-specific GAL4 driver lines to yield tissue-specific, doxycyline-inducible transgene expression over three orders of magnitude. The Geneswitch (GS) system also had low background, but the absolute level of expression was low relative to Tet-on. Consequently, actin5C-GS multi-insert chromosomes were generated and higher-level expression was achieved without increased background. Moderate level over-expression of MnSOD has beneficial effects on life span. Here high-level over-expression of MnSOD was found to have toxic effects. In contrast, motor-neuron-specific over-expression of MnSOD had no detectable effect on life span. The results suggest that motor-neuron tissue is not the essential tissue for either MnSOD induced longevity or toxicity in adult males.

背景与目标： : 比较了条件系统Tet-on和Geneswitch，并优化了成年果蝇转基因的组织特异性表达和寿命控制。比较了Tet-on系统反向四环素反式激活因子 (rtTA) 的两个版本: 原始的rtTA和包含旨在优化调节和表达的突变的rtTAM2-alt。在没有强力环的情况下，rtTAM2-alt版本的目标构建体的泄漏表达较少，但是与先前的报告相反，可以达到的绝对表达水平低于rtTA产生的表达水平。重新平衡现有的UAS-rtTAM2-alt插入，并与几个组织一般和组织特异性GAL4驱动系结合，以产生组织特异性，强力霉素诱导的转基因表达，超过三个数量级。Geneswitch (GS) 系统的背景也较低，但相对于Tet-on，绝对表达水平较低。因此，产生了actin5C-GS多插入染色体，并且在没有增加背景的情况下实现了更高水平的表达。MnSOD的中度过表达对寿命有有益影响。在这里，发现MnSOD的高水平过表达具有毒性作用。相反，运动神经元特异性MnSOD的过度表达对寿命没有可检测的影响。结果表明，运动神经元组织不是MnSOD诱导的成年男性寿命或毒性的必需组织。

BACKGROUND & AIMS: :During somatic reprogramming, Yamanaka's pioneer factors regulate a complex sequence of molecular events leading to the activation of a network of pluripotency factors, ultimately resulting in the acquisition and maintenance of a pluripotent state. Here, we show that, contrary to the pluripotency factors studied so far, overexpression of Mybl2 inhibits somatic reprogramming. Our results demonstrate that Mybl2 levels are crucial to the dynamics of the reprogramming process. Mybl2 overexpression changes chromatin conformation, affecting the accessibility of pioneer factors to the chromatin and promoting accessibility for early immediate response genes known to be reprogramming blockers. These changes in the chromatin landscape ultimately lead to a deregulation of key genes that are important for the mesenchymal-to-epithelial transition. This work defines Mybl2 level as a gatekeeper for the initiation of reprogramming, providing further insights into the tight regulation and required coordination of molecular events that are necessary for changes in cell fate identity during the reprogramming process.

背景与目标： : 在体细胞重编程过程中，Yamanaka的先驱因子调节复杂的分子事件序列，导致多能性因子网络的激活，最终导致多能性状态的获得和维持。在这里，我们表明，与迄今为止研究的多能性因素相反，Mybl2的过表达抑制了体细胞重编程。我们的结果表明，Mybl2水平对于重编程过程的动态至关重要。Mybl2过表达改变染色质构象，影响先驱因子对染色质的可及性，并促进已知为重编程阻滞剂的早期即时反应基因的可及性。染色质景观中的这些变化最终导致关键基因的失调，这对于间充质到上皮的转变很重要。这项工作将Mybl2水平定义为启动重编程的看门人，从而提供了对分子事件的严格调节和所需协调的进一步见解，这些分子事件是重编程过程中细胞命运身份变化所必需的。