A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).     

Characterization of a deep-coverage carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) BAC library and initial analysis of BAC-end sequences

Carrot is the most economically important member of the Apiaceae family and a major source of provitamin A carotenoids in the human diet. However, carrot molecular resources are relatively underdeveloped, hampering a number of genetic studies. Here, we report on the synthesis and characterization of a bacterial artificial chromosome (BAC) library of carrot. The library is 17.3-fold redundant and consists of 92,160 clones with an average insert size of 121 kb. To provide an overview of the composition and organization of the carrot nuclear genome we generated and analyzed 2,696 BAC-end sequences (BES) from nearly 2,000 BACs, totaling 1.74 Mb of BES. This analysis revealed that 14% of the BES consists of known repetitive elements, with transposable elements representing more than 80% of this fraction. Eleven novel carrot repetitive elements were identified, covering 8.5% of the BES. Analysis of microsatellites showed a comparably low frequency for these elements in the carrot BES. Comparisons of the translated BES with protein databases indicated that approximately 10% of the carrot genome represents coding sequences. Moreover, among eight dicot species used for comparison purposes, carrot BES had highest homology to protein-coding sequences from tomato. This deep-coverage library will aid carrot breeding and genetics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ147695–FJ150390.     

A baculovirus expression system for insect cells

The review considers the biology of baculoviruses, construction of transfer vectors for the baculovirus expression system, selection of recombinant baculoviruses, approaches to expression of multimeric proteins, and the potentialities and prospects of the system.     

The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences

Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of 10.9% of the Brachypodium genome based on 64,696 bacterial artificial chromosome (BAC) end sequences (BES). Analysis of repeat DNA content in BES revealed that approximately 11.0% of the genome consists of known repetitive DNA. The vast majority of the Brachypodium repetitive elements are LTR retrotransposons. While Bare-1 retrotransposons are common to wheat and barley, Brachypodium repetitive element sequence-1 (BRES-1), closely related to Bare-1, is also abundant in Brachypodium. Moreover, unique Brachypodium repetitive element sequences identified constitute approximately 7.4% of its genome. Simple sequence repeats from BES were analyzed, and flanking primer sequences for SSR detection potentially useful for genetic mapping are available at . Sequence analyses of BES indicated that approximately 21.2% of the Brachypodium genome represents coding sequence. Furthermore, Brachypodium BES have more significant matches to ESTs from wheat than rice or maize, although these species have similar sizes of EST collections. A phylogenetic analysis based on 335 sequences shared among seven grass species further revealed a closer relationship between Brachypodium and Triticeae than Brachypodium and rice or maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. Huo and G.R. Lazo contributed equally to this work.     

Expression profiling of herpesvirus and vaccinia virus proteins using a high-throughput baculovirus screening system

We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337 proteins from three different herpesviruses (HSV-1, EBV, and CMV) and vaccinia virus. Subsets of these proteins were also tested for expression and solubility in E. coli. Comparisons of the results in the two systems are presented for each virus.     

杆状病毒作为基因治疗载体的研究进展

杆状病毒是昆虫专一性的病原病毒.近来的研究表明杆状病毒能进入哺乳动物细胞, 但病毒自身不能在哺乳动物细胞中复制, 感染也不引起细胞病变.另外,已经证明杆状病毒能在体外或体内高效地转导许多类型哺乳动物细胞,并且能得到固定表达细胞系,显示了杆状病毒作为基因治疗载体有着良好的应用前景.综述了该领域的最新研究进展并探讨了其发展趋势.     

Baculoviruses: diversity,evolution and manipulation of insects

Baculoviruses, members of the family Baculoviridae, are large, enveloped viruses that contain a double‐stranded circular DNA genome of 80–180 kbp, encoding 90–180 putative proteins. These viruses are exclusively pathogenic for arthropods, particularly insects, and have been developed, or are being developed, as environmentally sound pesticides and eukaryotic vectors for foreign protein expression, surface display, gene delivery for gene therapy, vaccine production and drug screening. The baculoviruses contain a set of approximately 30 core genes that are conserved among all baculovirus genomes sequenced to date. Individual baculoviruses also contain a number of lineage‐ or species‐specific genes that have greatly impacted the diversification and evolution of baculoviruses. In this review, we first describe the general properties and biology of baculoviruses and then focus on the baculovirus genes and mechanisms involved in the replication, spread and survival of baculoviruses within the context of their diversity, evolution and insect manipulation.     

Baculoviruses as Vectors in Mammalian Cells

The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.