Gene expression in mice after high efficiency retroviral-mediated gene transfer

A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression.     

Gene transfer in cereals

Until recently, gene transfer in plants was achieved only by sexual hybridization. Now, in addition, plant genetic manipulation, with the use of both recombinant DNA and protoplast fusion technology, is being applied to an increasing range of plants. The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. Instead, the direct uptake of plasmid DNA into cereal protoplasts is being used for the transformation of cells in rice, wheat, and maize. Transformation efficiencies, in some cases, are becoming comparable to those obtained in dicotyledons with Agrobacterium. In rice it is now possible to regenerate efficiently whole plants from protoplasts, and this capability may soon be extended to the other cereals. By means of direct interaction of cereal protoplasts with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.     

Gene transfer in crop improvement

Transfer of genes between plant species has played an important role in crop improvement for many decades. Useful traits such as resistance to disease, insects, and stress have been transferred to crop varieties from noncultivated plants. Recombinant DNA methods greatly extend (even outside the plant kingdom) the sources from which genetic information can be obtained for crop improvement. Gene transfer systems based on recombinant DNA are available for several crop species and are under development for others. The concerted use of traditional and more recent methods for plant genetic manipulation will contribute to crop improvement.     

Gene transfer and expression of human phenylalanine hydroxylase

Phenylketonuria (PKU) is caused by a genetic deficiency of the enzyme phenylalanine hydroxylase (PAH). A full-length complementary DNA clone of human PAH was inserted into a eukaryotic expression vector and transferred into mouse NIH3T3 cells which do not normally express PAH. The transformed mouse cells expressed PAH messenger RNA, immunoreactive protein, and enzymatic activity that are characteristic of the normal human liver products, demonstrating that a single gene contains all of the necessary genetic information to code for functional PAH. These results support the use of the human PAH probe in prenatal diagnosis and detection of carriers, to provide new opportunities for the biochemical characterization of normal and mutant enzymes, and in the investigation of alternative genetic therapies for PKU.     

Gene transfer and molecular cloning of the human NGF receptor

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.     

山羊胎儿成纤维细胞转染人t-PA指形区缺失基因及其核移植研究

采用阳离子脂质体法将人t-PA指形区缺失基因乳腺特异性表达载体(pEBT)导入山羊胎儿成纤维细胞,以山羊胎儿成纤维细胞和转染的山羊胎儿成纤维细胞作供体,构建核移植胚,对其体外发育情况进行了研究,比较了2种供体细胞(山羊胎儿成纤维细胞和转人t-PA指形区缺失基因的山羊胎儿成纤维细胞)及转人t-PA指形区缺失基因的山羊胎儿成纤维细胞饥饿处理与否对核移植胚胎体外发育的影响。结果表明,早期核移植胚有荧光蛋白(GFP)的表达;以山羊胎儿成纤维细胞作供体细胞时,核移植胚的桑葚胚率(50.3%)及囊胚率(16.0%)均高于以转人t-PA指形区缺失基因胎儿成纤维细胞为供体时的桑葚胚率(48.4%)和囊胚率(10.9%),但差异不显著(P>0.05);转人t-PA指形区缺失基因的山羊胎儿成纤维细胞经饥饿处理后,其核移植胚胎的卵裂率(73.6%)与不饥饿时的卵裂率(73.9%)差异不显著;饥饿处理后核移植胚胎的桑葚胚率(48.5%)和囊胚率(11.2%)均高于不饥饿处理的桑葚胚率(39.2%)和囊胚率(9.2%),但差异不显著(P>0.05)。本研究成功地构建了转人t-PA指形区缺失基因的体细胞核移植胚胎,体外囊胚率为11.2%。     

Chromosomal gene transfer in Spiroplasma citri

The study of resistance marker rearrangement in Spiroplasma citri mutants provides evidence of transfer of chromosomal information followed by recombination. This is the first report of such a transfer in vivo in the mollicutes--that is, in the smallest self-replicating organisms. The double-resistant phenotypes obtained are stable even without selection pressure. The mechanism of gene transfer is insensitive to deoxyribonuclease, requires contact, and possibly, areas of fusion of the cell membranes; it shares properties with the transfer by protoplast fusion in Gram-positive bacteria. The extensive degenerative evolution of mollicutes has retained, in S. citri, bacterial functions of chromosomal transfer and recombination.     

Direct visualization of horizontal gene transfer

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.     

鱼类基因转移研究中存在的问题与对策

转基因动物技术是20世纪80年代初发展起来的一项生物领域高新技术[1]。转基因鱼模型的构建为鱼类基因工程育种奠定了理论基础,基因导入方法的成熟、胚胎干细胞技术的发展以及基因组学理论的应用为鱼类基因工程育种提供了操作平台。目前,转基因技术在外源基因片段长度、外源基因定点整合率、外源基因表达率、转基因动物存活率等技术方面都有了很大提高。转基因动物的研究内容也非常广泛,从基础理论到应用技术,从实验室到生产也都有了较大发展,如基因表达的调控、基因产品的制备、动物品种的培育等[2]。以下就鱼类基因转移研究中转基因技…     

鱼类基因转移研究中存在的问题与对策

转基因动物技术是20世纪80年代初发展起来的一项生物领域高新技术[1]。转基因鱼模型的构建为鱼类基因工程育种奠定了理论基础,基因导入方法的成熟、胚胎干细胞技术的发展以及基因组学理论的应用为鱼类基因工程育种提供了操作平台。目前,转基因技术在外源基因片段长度、外源基因定点整合率、外源基因表达率、转基因动物存活率等技术方面都有了很大提高。转基因动物的研究内容也非常广泛,从基础理论到应用技术,从实验室到生产也都有了较大发展,如基因表达的调控、基因产品的制备、动物品种的培育等[2]。以下就鱼类基因转移研究中转基因技…     

农杆菌介导phyB基因转化枳壳研究

以枳壳(Poncirus trifoliate (L. ) Raf.)实生苗上胚轴为试材,利用农杆菌介导法将光敏色素基因phyB导入其中,建立了枳壳实生苗的遗传转化体系,共获得27株抗性植株,PCR检测结果表明其中有5株扩增出phyB基因的特异目的带;RT-PCR分析结果表明 phyB基因在phyB3,phyB4株系枳壳基因组中能表达;Li-6400光合测定仪对转基因枳壳进行的光合特性测定结果显示,转基因枳壳光合效率高于对照;转phyB基因枳壳植株在形态上与对照也存在差异,表现出植株矮化、节间缩短、叶面积变小.     

小麦外源基因导入的研究进展

远缘杂交是创造新物种、新种质以及转移异源遗传物质的有效途径,在理论和实践上都具有重要意义。本文综述了近年来小麦外源基因导入的研究进展概况,并根据我们多年的远缘杂交育种实践,综合分析了当地的生态气候条件,提出了生态环境与糖蛋白互作诱导远缘杂种育性假说。