Differentiation of AFS cells derived from the EGFP gene transgenic porcine fetuses

We have obtained the EGFP (enhanced green fluorescence protein) gene transgenic porcine fetuses before. The aims of this study were (i) to determine whether stem cells could be isolated from amniotic fluid of the transgenic porcine fetuses, and (ii) to determine if these stem cells could express EGFP and differentiate in vitro. The results demonstrated that stem cells could be isolated from amniotic fluid of the EGFP gene transgenic porcine fetuses and could express EGFP and differentiate in vitro. Undifferentiated AFSs (amniotic fluid-derived stem cells) expressed POU5F1, THY1 and SOX2, while the following differentiation cells expressed markers for chondrogenic (COL2A1), osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte (GFAP), oligodendrocyte (GALC) and neuron (NF, ENO2 and MAP).     

The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation

SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53–Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53–Mdm2 pathway.     

人工锌指核酸酶突变EGFP基因的功能分析

锌指核酸酶技术在基因定点修饰中具有效率高和特异性好等特点,并成功应用于数十种生物。目前,该技术是否能应用羊上尚未报道。为了敲除转基因山羊标记基因 (EGFP),构建了一对针对EGFP外显子上的锌指核酸酶表达载体,将其电转染至转EGFP基因胎儿成纤维细胞中,研究了锌指核酸酶突变EGFP基因的效率和方式,利用基因显微注射单细胞获得获得的转基因 (EGFP) 细胞系作为锌指核酸酶的靶细胞。结果显示,通过锌指核酸酶的突变作用,转染后的细胞发绿色荧光比例下降,测序结果显示在EGFP外显子中插入1个碱基G,导致编码EGFP基因的阅读框改变,从而起到基因突变的作用。结果表明,文中构建的锌指核酸酶对EGFP基因有突变作用,可以为以后获得无标记基因供核细胞进行体细胞核移植生产克隆羊奠定基础。     

猪胰岛素启动子调控EGFP表达载体的优化及体外验证

目的获得猪胰岛素启动子调控外源基因的高效表达载体,为制备转基因猪胰岛特异性表达目的基因奠定基础。方法利用猪胰岛素启动子（PIP,包含5＇调控区、第一外显子、第一内含子及第二外显子ATG前的序列共1.5 kb）构建表达载体,连接PIP和EGFP的酶切位点HindⅢ设计在起始密码子前,命名为PIP-Hind IIIEGFP。鉴于酶切位点的插入位置可能会影响外源基因的表达效率,对载体进行了优化：将Hind III酶切位点删除,实现PIP和EGFP无缝连接,载体命名PIP-EGFP;将第一内含子3＇端的内含子剪接受体位点（splicing acceptor site,SA）突变为Hind III内切酶识别位点,命名为PIP-SA（M）-EGFP。三种载体分别电转染小鼠胰岛素瘤β细胞株MIN-6细胞、猪耳成纤维细胞以及猪肾细胞,48 h后通过荧光强度、流式分析、RT-PCR及Western blot检测,验证载体的表达效率。结果转染细胞后,三种载体都仅在MIN-6胰岛β细胞表达绿色荧光。RT-PCR及其产物测序结果显示三种载体的胰岛素启动子第一内含子的剪切存在差异：载体PIP-Hind III-EGFP和PIP-EGFP的内含子存在剪切和不剪切两种情况,剪切不稳定;载体PIP-SA（M）-EGFP的SA位点突变后内含子不剪切,流式细胞及Western blot检测显示,与另外两种载体相比,该载体目的蛋白的表达量最高。结论通过突变胰岛素启动子第一内含子的3＇端的剪接受体位点,成功获得了胰岛β细胞的高效表达载体,可用于制备胰岛特异表达外源基因的转基因猪。     

Eukaryotic expression and secretion of EGFP-labeled annexin A5

The Ca²⁺-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 μg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.     

E-box元件修饰端粒酶核心启动子序列及体外转录活性分析

人类端粒酶启动子(hTERT启动子)在肿瘤基因治疗中的有效性已经得到了证实. 然而,hTERT启动子有限的肿瘤靶向转录活性困扰着它的临床应用.早期研究已经揭示,核心hTERT启动子上的-34位E-box元件与该启动子的肿瘤靶向转录活性有关.为进一步探索核心hTERT启动子序列3′端富余E-box元件是否能提高启动子的肿瘤靶向转录能力,用化学合成方法在野生型hTERT(WT-hTERT)核心启动子片段(编码蛋白起始子ATG上游-268 bp～-10 bp)的3′端接入3个E-box序列, 构建成修饰型hTERT(Mod-hTERT)启动子. 然后,分别用WT-hTERT和Mod-hTERT启动子去调控增强型绿色荧光蛋白(EGFP)及荧光素酶报告基因在293FT、HepGⅡ、SGC7901、U2OS、以及原代培养人成纤维细胞(PHF)中表达. 结果表明, 在Mod-hTERT启动子的各实验组细胞中,能够在端粒酶阳性的293FT、HepGⅡ及 SGC7901细胞组中观测到EGFP的表达,而在端粒酶阴性的U2OS及PHF细胞组中没有观测到EGFP的表达;在端粒酶阳性的293FT、HepGⅡ和SGC7901细胞株中,Mod-hTERT启动子调控下的荧光素酶活性要高于WT-hTERT启动子组（P＜0.01）; 而在端粒酶阴性的U2OS细胞组中,Mod-hTERT启动子调控下的荧光素酶活性则低于WT-hTERT启动子组（P＜0.01); 在PHF细胞组中,Mod-hTERT启动子组与WT-hTERT启动子组的荧光素酶活性差异不显著(P＞0.05).研究提示,在3′端增加E-box元件可以提高核心hTERT启动子序列的肿瘤靶向转录活性.     

表达HIV-1 gag重组鸡痘病毒的构建与筛选

构建并筛选表达HIV-1 gag蛋白的重组鸡痘病毒,并对其进行鉴定。首先设计引物通过PCR技术扩增HIV-1 gag基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的鸡痘病毒穿梭载体pTKET中,获得重组质粒pTKET-HIV gag。然后将其与鸡痘病毒FPV282E4株共转染原代鸡胚成纤维细胞(CEF)进行同源重组,以增强型绿色荧光蛋白(EGFP)为筛选标记,通过噬斑筛选获得重组病毒,应用PCR,RT-PCR,Western blot方法对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组鸡痘病毒基因组中,RT-PCR,Western blot结果表明HIV-1 gag在感染细胞内成功表达且具有抗原性。连续传代20次,PCR,RT-PCR,Western blot均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,而且病毒已经纯化。结论:成功获得表达HIV-1gag的重组鸡痘病毒,为进一步免疫试验研究奠定基础。     

Twice Daily Melatonin Peaks in Siberian but not Syrian Hamsters under 24 h Light:Dark:Light:Dark Cycles

The daily pattern of blood-borne melatonin varies seasonally under the control of a multi-oscillator circadian pacemaker. Here we examine patterns of melatonin secretion and locomotor activity in Siberian and Syrian hamsters entrained to bimodal LDLD8:4:8:4 and LD20:4 lighting schedules that facilitate novel temporal arrangements of component circadian oscillators. Under LDLD, both species robustly bifurcated wheel-running activity in distinct day scotophase (DS) and night scotophase (NS) bouts. Siberian hamsters displayed significant melatonin increases during each scotophase in LDLD, and in the single daily scotophase of LD20:4. The bimodal melatonin secretion pattern persisted in acutely extended 16 h scotophases. Syrian hamsters, in contrast, showed no significant increases in plasma melatonin during either scotophase of LDLD8:4:8:4 or in LD20:4. In this species, detectable levels were observed only when the DS of LDLD was acutely extended to yield 16 h of darkness. Established species differences in the phase lag of nocturnal melatonin secretion relative to activity onset may underlie the above contrast: In non-bifurcated entrainment to 24 h LD cycles, Siberian hamsters show increased melatonin secretion within ～2 h after activity onset, whereas in Syrian hamsters, detectable melatonin secretion phase lags activity onset and the L/D transition by at least 4?h. The present results provide new evidence indicating multi-oscillator regulation of the waveform of melatonin secretion, specifically, the circadian control of the onset, offset and duration of nocturnal secretion.     

甘菊DFL::EGFP高效融合蛋白表达载体的构建

增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)是一种优化的突变型GFP,DFL是从甘菊中分离出的LFY基因的同源序列。为了研究DFL基因的功能和表达模式,研究利用小片段克隆法将linker序列插入到EGFP基因5′端启始密码子前面,在pBI121载体的CaMV35S启动子的3′端后面插入一段多克隆位点,成功地构建了pBI-DFL-EGFP表达载体。通过设计特异引物,利用PCR技术扩增得了到拟南芥LFY基因的启动子序列,用粘性末端PCR技术将pBI-DFL-EGFP表达载体中CaMV35S启动子替换成LFY基因启动子,构建成了pLFY-DFL-EGFP表达载体。用含有pBI-DFL-EGFP和pLFY-DFL-EGFP质粒的农杆菌侵染洋葱表皮细胞,在荧光显微镜下分别用蓝光激发,均观测到了荧光。这一结果表明,融合蛋白DFL∷EGFP表达载体构建成功,同时还证明了通过PCR技术克隆到的LFY启动子序列具有启动子功能。