Reverse transfection using antibodies against a cell surface antigen in mammalian adherent cell lines

Reverse transfection from a solid surface has the potential to deliver genes to various cells more efficiently than conventional methods. However, the effective gene delivery from a solid surface requires an optimized extracellular matrix (ECM) for the coating of glass slides, dependent on the nature of the cells. In a search for an appropriate substrate for the universal application to multiple types of cell, we focused on cell surface antigens and examined the effects of antibodies raised against them on gene transfer from an antibody-coated surface. We found that a coating of CD29-specific antibody allowed the most effective delivery of genes by reverse transfection in every type of cell that we examined. Our results suggest that reverse transfection with antibodies against CD29 might provide a universal tool for gene delivery and cell array-based analyses.     

Novel in vitro screening system for monoclonal antibodies using hypermutating chicken B cell library

Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.     

Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera

In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.     

转基因大米及其安全性评价研究进展

近年来,转基因稻米的迅速发展引起了广泛关注,尤其是与食品密切相关的营养与安全问题。分析转基因大米的基因来源及对大米品质的影响,总结转基因大米可能存在的危害及其安全性评价的方法,可使人们科学的对待转基因大米,并为研究与开发新型转基因大米提供参考。     

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.     

Stem cell factor affects fate determination of human gonocytes in vitro

The stem cell factor (SCF), binding its tyrosine kinase receptor c-Kit, has been shown to play essential roles in the proliferation, differentiation, and survival of germline cells. However, few reports are available about the effect of SCF on the development of human gonocytes within the fetal testis. The objective of this study was to investigate whether SCF affects the biological behaviors of human gonocytes before or after they enter the mitotic arrest stage. Employing an organ culture system, we observed that addition of exogenous SCF could influence the morphology of human gonocytes in vitro. Moreover, SCF was able to trigger the colony formation of round gonocytes, which were characterized positive for alkaline phosphatase activity, Oct-4, SSEA-4, and c-Kit as well. We found that SCF exerted actions in a dose- and age-dependent manner, although the stimulatory effect lasted no more than 14 days. We also showed that SCF played a role in suppressing the apoptosis of human gonocytes. Blocking of SCF signaling with either phosphatidylinositol 3-kinase or mitogen-activated protein kinase inhibitor resulted in similar apoptotic features as well as the SCF-withdrawal cultures. Taken together, we report that SCF acts as a potent regulator in the fate determination of human gonocytes. Our studies should form the basis for in vitro studies and facilitate investigation of the molecular mechanisms underlying this unique stage.     

玉米及其制品中转基因成分的单一 PCR及多重PCR检测

采用CTAB法提取玉米及其制品的总DNA，用PCR方法检测其中的转基因成分如花椰菜花叶病毒（Cauliflower mosaic virus，CaMV）35S启动子、根癌农杆菌（Agrobacterium tumefaciens）胭脂碱合成酶甚因（nos）终止子、根癌农杆菌CP4菌株的EPSPS基因、吸水链霉菌（Treptomyces hygroscopicus）bar基因及苏云金芽孢杆菌库尔斯塔克亚种（Bacillus thuringiensis subsp．kurstaki）crylA（b）基因，筛选到阳性样品，并建立了几组玉米内源zein基因和转基因成分之间的多重PCR检测方法。结果表明，建立的多重PCR方法用于同时检测玉米内源基因和转基因成分是可行的，值得推广；虽然我国还未有己获准商品化生产的转基因玉米，但国外转基因玉米已流入福建省。     

Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens

We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens.     

Genetically modified Lactococcus lactis producing a green fluorescent protein–bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.     

环介导等温扩增技术检测转基因组分的研究进展

随着越来越多的转基因作物获得商业化生产和种植,转基因农产品的安全问题一直都有异议,因此,无论对消费人群还是政府监管部门,快速检测转基因组分都是非常必要。转基因组分的检测通常以核酸为基础进行检测,而环介导等温扩增技术(LAMP)是一种等温条件下核酸扩增技术,具有高特异性、高灵敏度、操作简单、成本低廉、结果可视化及不需要专用设备的特点,已逐渐用于转基因组分的检测中。本文针对LAMP技术的原理、优点、关键技术及其在转基因大豆、玉米、水稻等农产品中的应用做一综述,从而为其更广泛的用于转基因组分的检测提供理论依据。     

转基因食用农产品的快速检测方法

商业化转基因农作物广泛种植,转基因农产品被大量用作食品和动物饲料。我国和全球大部分国家都实行转基因生物强制标识制度。转基因农产品的现场监督检测急需稳定可靠的快速检测手段。本文论述了主要的基因水平检测方法包括PCR、基因芯片、环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)和蛋白质水平检测方法(蛋白质免疫印迹法、酶联免疫吸附法、胶体金免疫层析试纸条法)。通过比较分析上述各检测方法的优缺点,LAMP和胶体金试纸条法是当前最理想的现场快速检测方法。     

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.     

转基因大豆DNA检测芯片的研究

为提高对转基因大豆的监督检测能力，研制了转基因大豆DNA检测芯片。根据转基因大豆(Roundup Ready)中所转入的外源基因，选择CaMV35S启动子、NOS终止子、NOSIEPSPE基因和内源Lectin基因设计特异性引物，采用多重PCR法对待测样品进行扩增，通过缺口平移法合成DIGdUTP标记杂交探针，并制备基因芯片。在对PCR反应和扩增产物与芯片杂交条件进行优化的同时，比较了芯片检测的特异性和重复性，并对检测的灵敏度进行测试。结果表明，该方法具有较好的特异性和重复性，检测灵敏度可达0．5％，由于采用了多重PCR技术，一次可同时检测多个基因，提高了检测的准确性和效率。     

转基因食品安全性评价与管理体系的基本框架

本文阐述建立转基因食品安全性评价与管理体系的意义，从转基因食品安全性的内涵和外延出发，重点论述转基因食品安全性评价与管理体系的基本框架及其运作机制。     

代谢组学在转基因作物非预期效应评价中的应用

外源基因的非预期效应可能形成新的代谢产物或改变代谢模式,也可能引起转基因作物的营养成分发生改变,甚至可能会产生一些新的有毒物质,是转基因食品安全性评价的重要内容之一。本文主要对近年来代谢组学技术在转基因作物非预期效应评价中的最新应用做了总结,并阐述了对该研究领域的预期,以期促进转基因食品安全性评价体系的发展和完善。     

Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro

Angiotensin II type 1 receptors have been identified in Fallopian tube epithelia. Polarized confluent human Fallopian tube epithelial cell cultures were used under short-circuit conditions to study the actions of angiotensin II on electrogenic ion transport. The results demonstrate that angiotensin II increases baseline short-circuit current, implying a net transport of negatively charged ions from a basal to apical direction. This effect was inhibited by the selective angiotensin II type 1 receptor antagonist losartan. The effects of angiotensin II on short-circuit current were rapid in onset, brief in duration, and although less than those achieved with ATP, similar in amplitude to those described for other epithelia with angiotensin II. These findings reflect a significant retention of function for these cells in monolayer culture. Immunohistochemistry using the antibody 6313/G2, which is directed against a specific sequence in the extracellular domain of the angiotensin II type 1 receptor, confirmed that the receptor was retained in cultured cells. The results indicate that angiotensin II plays a role in regulating the composition of Fallopian tube secretions.     

Extracellular Expression of a Recombinant Ganoderma lucidium Immunomodulatory Protein by Food-Grade Lactococcus lactis System

Lactococcus lactis is a food-grade microorganism of major commercial importance in food industry. For commercial application of genetically modified L. lactis, a food-grade expression system is strongly recommended. In this study, two food-grade selection markers, nisin immunity gene nisI and nisin resistance gene nsr, were evaluated as dominant markers for the L. lactis food-grade expression system. By using an efficient P_SlpAl promoter fused to a signal peptide from subtilisin YaB (SP_YAB), a functional recombinant Ganoderma lucidium immunomodulatory protein rLZ8 was expressed extracellularly in L. lactis. Replacing the antibiotic marker gene into the proper food-grade selection marker nsr gene, the rLZ8 was expressed extracellularly in the food-grade L. lactis system. This study provides a rationale basis for a food-grade system to express functional peptides extracellularly as an important tool for oral administration of genetically modified L. lactis.     

Transcriptional profiling in rats and an ex vivo analysis implicate novel beneficial function of egg shell membrane in liver fibrosis

Egg shell membrane (ESM) is a natural and safe food by-product from egg processing whilst there is little information about the role of ESM as a food component. Effects of dietary ESM on gene expression in rat liver were investigated through DNA microarray comprehensive analysis. The expression of smooth muscle-α-actin, which is a marker of hepatic stellate cells (HSCs) activation, and integrin beta-like 1, decorin, asporin, lumican and collagen type 1 alpha 1, which are components of extracellular matrix (ECM) and involved in the up-regulation of HSCs activation and fibrosis, was found to be significantly down-regulated after 14 days of ESM treatment. Subsequently, serum obtained from rats given ESM diet also suppressed the expression of COL1A1 and COL1A2 in human hepatoma C3A cells. Our in vivo and ex vivo findings demonstrate that these gene alterations may contribute to the beneficial effect of ESM partially through down-regulation of c-jun and c-fos signal transduction thereby blunting HSCs activation and eventually preventing liver fibrosis. These outcomes not only provide novel information about the functional and nutritional availability of ESM, but also might contribute to the field of environmental protection.