Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.     

脑源性神经营养因子受体trkB在NIH 3T3细胞上的表达

构建了克隆有大鼠脑源性神经营养因子（BDNF）受体trkB全长基因的真核表达载体pcDNA3.1( )-rat trkB.用脂质体介导法将重组载体转入小鼠NIH3T3细胞,在mRNA和蛋白质水平检测到了trkB基因在用G418筛选到的抗性NIH 3T3细胞中的表达,表达的trkB蛋白定位于细胞膜上。BDNF能够剂量依赖性地促进NIH 3T3－trkB细胞的增殖,说明表达的trkB是有功能的。该表达trkB和NIH3T3细胞为研究BDNF的生理功能、活性测定和从噬菌体展示肽库中筛选BDNF肽提供了一个简便的细胞模型。     

应用二维电泳和质谱技术初步研究NAG7基因的功能

为了进一步研究鼻咽癌相关基因NAG7的功能,NAG7基因编码框的cDNA片段被亚克隆至pcDNA3.1( )的表达载体,通过脂质体转染入鼻咽癌细胞系HNE1。抽提HNE1细胞和已转染了NAG7基因的HNE1细胞总蛋白质,用二维凝胶电泳分离蛋白质,获得二维凝胶图谱,PDQuest软件包分析后,确立表达上调的蛋白质斑点进行质谱分析,得到的肽质指纹经蛋白质数据库分析鉴定蛋白质。共分析了12个蛋白质点,其中3个未有质谱结果,已鉴定的9个蛋白质包括生长捕获特异蛋白、DNA结合蛋白、c-myc启动子结合蛋白及胱冬肽酶（caspase)6等蛋白质。通过对这些蛋白质性质和功能的讨论,发现它们参与了细胞周期调控、转录调节及细胞凋亡等重要事件。因此,VAG7很可能是通过介导上述蛋白质的表达上调而发挥作用。     

Dendritic cell vaccination and immune monitoring

We exploited dendritic cells (DC) to vaccinate melanoma patients. We recently demonstrated a statistical significant correlation between favorable clinical outcome and the presence of vaccine-related tumor antigen-specific T cells in delayed type hypersensitivity (DTH) skin biopsies. However, favorable clinical outcome is only observed in a minority of the treated patients. Therefore, it is obvious that current DC-based protocols need to be improved. For this reason, we study in small proof of principle trials the fate, interactions and effectiveness of the injected DC.     

Akt基因转染对骨髓间充质干细胞缺氧时凋亡和增殖的影响

目的采用Akt基因转染鼠骨髓MSCs探讨Akt基因是否减轻MSCs缺氧时的凋亡和提高缺氧时的增殖能力,即耐缺氧能力。方法将转染和未转染Akt基因的MSCs置于94%N2、1%O2和5%CO2缺氧箱中37℃孵育不同时间（常氧、缺氧0.5h、1h、2h、4h和8h）后,Annexin V/PI双染法行流式细胞仪分析凋亡率（apoptoticrate,AR）和死亡率（deadrate,DR）、MTT法分析细胞增殖状态、Rt-PCR和Western blot等检测Akt和p-Akt表达以及放射同位素法检测MSCs对氚标-葡萄糖（^3H-G）的摄取等。结果1.Akt基因显著降低MSCs缺氧时AR和DR（P〈0.01）,而各缺氧时间点没有统计学意义（P〉0.05）;2.Akt基因显著增高MSCs常氧和缺氧（与未转染Akt基因MSCs同等条件下比较）时增殖能力（P〈0.01）,缺氧时增殖能力显著低于常氧时（P〈0.01）;3.Akt基因显著增高常氧时MSCsAkt mRNA（P〈0.01）和蛋白（P〈0.01）表达,而不增高p-Akt蛋白（P〉0.05）表达;Akt基因显著提高缺氧时p-Akt蛋白（P〈0.01）表达,而不提高常氧时p-Akt蛋白（P〉0.05）表达;4.Akt基因显著增高MSCs常氧和缺氧（与未转染Akt基因MSCs同等条件下比较）时^3H-G的摄取（P〈0.01）,缺氧时^3H-G的摄取显著性低于常氧培养时（P〈0.01）;^3H-G的摄取与细胞增殖显著正相关（r=0.79,P=0.015）而与细胞凋亡显著负相关（r=-1.47,P=0.023）。结论Akt基因转染可显著提高MSCs耐缺氧能力,此可能与缺氧时改善MSCs葡萄糖摄取等有关。     

新基因BRD7对鼻咽癌蛋白质表达谱影响的初步研究

BRD7基因是与鼻咽癌相关的候选抑瘤基因.为了进一步研究该基因的作用机制,将pcDNA3.1(+)/BRD7的表达质粒经脂质体导入HNE1细胞,RNA斑点杂交筛选出阳性克隆,通过双向电泳分离过表达BRD7的HNE1细胞内蛋白质,筛选出差异表达的蛋白质点,并进行质谱分析鉴定.鉴定出10种表达上调的蛋白质点,包括精氨(基)琥珀酸裂解酶,TSA(thio-specific antioxdant), Proteaseome activator28 beta subunit(PA28),金属蛋白酶抑制因子-2前体等,这些蛋白质涉及细胞生长,细胞代谢等很多相关事件.这些结果说明BRD7基因可能通过多种途径影响鼻咽癌的发生发展.     

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.