外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Short Communication: Increased gene dosage augments antifreeze protein levels in transgenic Drosophila melanogaster

One of the principal environmental adaptations of certain fishes inhabiting polar and northern coastal waters is the synthesis of antifreeze proteins (AFPs). AFPs bind to and prevent the growth of nascent ice crystals, thus depressing the serum freezing point. The transgenic expression of AFP holds great promise for conferring freeze resistance to commercially important plant and animal species. Since fish at the greatest risk of freezing have multiple AFP gene copies in order to synthesize higher levels of this protein, we have evaluated this evolutionary strategy as a way to maximize AFP expression in a model transgenic host, the fruit fly Drosophila melanogaster. A construct in which AFP genes of the Atlantic wolffish are fused to the Drosophila yolk protein 1,2 promoter/enhancer region was transferred to flies through P-element mediated transformation. Several independent transgenic fly lines were used in genetic crosses to obtain multi-insert lines. Haemolymph freezing point depression (thermal hysteresis) was greater in homozygotes relative to heterozygotes for a given insert. Similarly, multi-insert lines consistently displayed greater haemolymph AFP activity than the single insert lines from which they were derived. The thermal hysteresis value obtained with a fly line harboring 8 AFP gene copies, 0.43 °C, represents the highest such value to date recorded in a transgenic host, and is even higher than the levels found in some AFP-producing fish.     

Theoretical study of interaction of winter flounder antifreeze protein with ice

Antifreeze proteins (AFPs) are synthesized by various organisms to enable their cells to survive subzero environment. These proteins bind to small ice crystals and inhibit their growth, which if left uncontrolled would be fatal to cells. The crystal structures of a number of AFPs have been determined; however, crystallographic analysis of AFP-ice complex is nearly impossible. Molecular modeling studies of AFPs' interaction with ice surface is therefore invaluable. Early models of AFP-ice interaction suggested H-bond as the primary driving force behind such interaction. Recent experimental evidence, however, suggested that hydrophobic interactions could be the main contributor to AFP-ice association. All computational studies published to date were carried out to verify the H-bond model, and no works attempting to verify the hydrophobic interaction model have been published. In this work, we Monte Carlo-minimized complexes of several AFPs with ice taking into account nonbonded interactions, H-bonds, and the hydration potential for proteins. Parameters of the hydration potential for ice were developed with the assumption that the free energy of the water-ice association should be close to zero at equilibrium melting temperature. Our calculations demonstrate that desolvation of hydrophobic groups in the AFPs upon their binding to the grooves at the ice surface is indeed the major stabilizing contributor to the free energy of AFP-ice binding. This study is consistent with available structural and mutation data on AFPs. In particular, it explains the paradoxical finding that substitution of Thr residues with Val does not affect the potency of winter flounder AFP whereas substitution with Ser abolished its antifreeze activity.     

昆虫抗冻蛋白基因的克隆与表达研究进展

产生抗冻蛋白(antifreeze protein,AFP)是许多昆虫抵御寒冷的一种重要机制。昆虫抗冻蛋白基因的克隆和表达是研究抗冻蛋白活性和功能的主要途径。文章归纳GenBank所登录的昆虫抗冻蛋白基因及其特点,总结昆虫抗冻蛋白基因的天然表达和基因工程表达方面尚未明确或需要克服的一些问题。目前在GenBank注册的昆虫抗冻蛋白基因约100个,集中于9种昆虫隶属鞘翅目3个科和鳞翅目1个科。昆虫抗冻蛋白基因具有多拷贝和多同种型(isoforms)的特点。昆虫抗冻蛋白的天然表达具有物种间和同种型间的多样性。基因工程表达昆虫抗冻蛋白需要克服表达量低活性不高的问题。对昆虫抗冻蛋白表达规律的研究有助于全面认识其功能。     

Protein slot blotting: An easy,rapid and reliable technique to identify the expression of a protein in transgenic plants

A technique based on immunological recognition of a foreign protein in transgenic plants has been developed. It allows a quick and reliable screening of many plant samples, improves the accuracy of the results compared to ELISA and is easier to carry out and more sensitive than a western immunoblot. This technique has also been tested to recognize foreign proteins in rice and tobacco leaf extracts.     

Intermolecular interaction studies of winter flounder antifreeze protein reveal the existence of thermally accessible binding state

The physical nature underlying intermolecular interactions between two rod-like winter flounder antifreeze protein (AFP) molecules and their implication for the mechanism of antifreeze function are examined in this work using molecular dynamics simulations, augmented with free energy calculations employing a continuum solvation model. The energetics for different modes of interactions of two AFP molecules is examined in both vacuum and aqueous phases along with the water distribution in the region encapsulated by two antiparallel AFP backbones. The results show that in a vacuum two AFP molecules intrinsically attract each other in the antiparallel fashion, where their complementary charge side chains face each other directly. In the aqueous environment, this attraction is counteracted by both screening and entropic effects. Therefore, two nearly energetically degenerate states, an aggregated state and a dissociated state, result as a new aspect of intermolecular interaction in the paradigm for the mechanism of action of AFP. The relevance of these findings to the mechanism of function of freezing inhibition in the context of our work on Antarctic cod antifreeze glycoprotein (Nguyen et al., Biophysical Journal, 2002, Vol. 82, pp. 2892-2905) is discussed.     

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.     

赤翅甲抗冻蛋白基因的原核表达及蛋白生物活性检测

根据GenBank中序列人工合成赤翅甲Dendroides canadensis的抗冻蛋白基因（afp）,将其克隆到载体pGEX-4T-1上,构建融合表达的重组质粒,转化大肠杆菌 BL21并进行原核表达。通过优化表达的诱导条件和SDS-PAGE检测,证明人工合成的赤翅甲抗冻蛋白基因能够特异性地表达,并以可溶性融合蛋白形式存在,相对分子质量约为40 kD。抗冻蛋白的生物活性检测表明,赤翅甲的抗冻融合蛋白能够提高细菌的耐寒能力。     

黄粉甲抗冻蛋白AFP84a在大肠杆菌中的高效表达及活性检测

为表达和纯化黄粉甲Tenebrio molitor抗冻蛋白, 采用RT-PCR方法扩增得到黄粉甲抗冻蛋白基因afp84a cDNA, 将其连接到pMAL-p2X质粒上, 构建分泌型融合表达载体pMAL-p2X-afp84a, 并在大肠杆菌Escherichia coli TBI中表达; 进一步利用Amylose柱亲和纯化出该重组蛋白, 后利用细菌抗寒性检测重组蛋白的生物活性。结果显示: 融合蛋白含量占总可溶蛋白的40%。SDS-PAGE分析表明, 用MgSO4处理法与超声波细胞破碎法均可使融合蛋白从细胞中释放; 融合蛋白经Amylose柱亲和纯化, Factor Xa因子酶切, 电泳显示获得的目的蛋白呈单一条带。细菌抗寒性检测表明该重组蛋白具有较高的抗冻活性。黄粉甲抗冻蛋白基因afp84a cDNA的克隆、 原核表达为进一步研究抗冻蛋白的性质和应用提供了有用的实验材料。     

异源表达蒙古沙冬青AmDREB1F基因提高转基因拟南芥的耐逆性

脱水应答元件结合蛋白(Dehydration-responsive element binding proteins,DREBs)是一类重要的植物耐逆相关转录因子.蒙古沙冬青Ammopiptanthus mongolicus是中国西北荒漠区特有的强耐逆常绿阔叶灌木.为探明其AmDREB1F基因在耐受非生物逆境中的功能和...     

Transgenic salmon: tailoring the genome for food production

The production of transgenic salmon using gene transfer technology is described. Both antifreeze proteins and growth hormone genes have been successfully transferred. The expression, inheritance and phenotypes are examined using a wide variety of techniques. The development of new transgenics will be beneficial to aquaculture.     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Allelic composition and genetic background effects on transgene expression and inheritance in white clover

Allelic composition and genetic background effects on GUS expression and inheritance using a chimeric (cauliflower mosaic virus 35Sp:uidA) transgene were investigated in white clover as a prelude to transgenic cultivar development. Stable expression and Mendelian inheritance of the uidA transgene was observed over two generations when the uidA transgene was maintained in a heterozygous state. Transgenic backcross progeny (BC1) were intercrossed to produce segregating F2 populations. GUS-positive F2 plants were test-crossed with a non-transgenic control plant to determine whether individuals were heterozygous or homozygous for the transgene. Both expected and distorted segregation ratios were observed. Distortion of the segregation ratio was not caused by transgene inactivation or rearrangement, but was influenced by genetic background. BC1, BC2 and F2 populations were found to have similar levels of uidA gene expression. Quantification of GUS expression from progeny of high and low GUS expressing plants indicate that it is possible to alter transgene expression through selection. No difference was found between the level of expression for F2 plants homozygous or heterozygous for the transgene. These results indicate that F2 plants, homozygous for a transgene, might be used to develop a transgenic cultivar. However, progeny testing to determine the influence of genetic background is a prerequisite to such a development.