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Results for “binary vectorization”

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PubMed2003

Enhanced recombinant adeno-associated virus-mediated vascular endothelial growth factor expression in the adult mouse retina: a potential model for diabetic retinopathy.

Diabetic retinopathy, one of the most serious complications of long-term diabetes, could clinically be divided into two stages: 1) background retinopathy that does not cause visual impairment and 2) proliferative retinopathy, which is a potentially blinding condition. This study aims to investigate the correlation between enhancement of vascular endothelial growth factor (VEGF) expression and neovascular changes. A binary recombinant adeno-associated virus construct producing green fluorescent protein (GFP) and VEGF under the control of the human cytomegalovirus promoter, recombinant adeno-associated virus (rAAV).VEGF.GFP, was produced and injected into the subretinal space of C57BL mice. GFP expression was tracked by fluorescence fundus photography, and VEGF expression was confirmed by immunohistochemistry and enzyme-linked immunoassay. Neovascular changes were monitored by fluorescein angiography and histology and by quantifying the number of inner retinal vessels. GFP expression was found in 100% of injected eyes, and vascular changes were detected in 9 of 10 rAAV.VEGF.GFP-injected eyes. Of these, four demonstrated microaneurysms and five showed moderate to severe leakage. There was a statistically significant increase in blood vessel number in the inner nuclear layer (P < 0.03) and dilatation of retinal veins (P < or = 0.05). This work has demonstrated that the development of different stages of diabetic retinopathy is closely correlated with an increased VEGF level in the retina.

Animals↗

PubMed1998

A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.

The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).

Agrobacterium tumefaciens↗

PubMed2005

A novel binary adenovirus-based dual-regulated expression system for independent transcription control of two different transgenes.

BACKGROUND: Stringent multitransgene control is a prerequisite for future gene-therapy and tissue-engineering scenarios and requires constant improvements in design to achieve optimal conditional transcription profiles. METHODS: We have pioneered a variety of recombinant adenoviruses which (i) enable streptogramin-responsive transgene transduction in a compact autoregulated one-virus format, (ii) manage independent streptogramin- and tetracycline-responsive control of two different transgenes from a single divergent expression unit, and (iii) control sense and antisense expression of the human cyclin-dependent kinase inhibitor p27(Kip1) to engineer conditional positive (enforced S-phase entry, p27(Kip1)-antisense expression) or negative (G1-phase-specific growth arrest, p27(Kip1)-sense expression) growth control in mammalian cell lines and human primary cells. RESULTS: The transgene control performance of all adenoviral expression configurations has been rigorously optimized for tight, balanced and maximum expression levels and was validated for intracellular as well as for secreted product in a variety of biotechnologically relevant cell lines (Chinese hamster ovary cells [CHO-K1], baby hamster kidney cells [BHK-21]) as well as in human cell lines (human fibrosarcoma cells [HT-1080]) and primary cells (human aortic fibroblasts [HAFs]). CONCLUSIONS: We believe that multiregulated multigene-controlled adenoviruses are important assets for successful therapeutic reprogramming of mammalian cells in clinically relevant scenarios.

Adenoviridae↗

PubMed2005

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.

Amino Acid Sequence↗

PubMed1991

Amblyospora sp. (Microspora, Amblyosporidae) infecting nerve ganglia of Culex pipiens (Diptera, Culicidae) from Egypt.

A species of Amblyospora-infecting neurones of Culex pipiens is described. Diplokaryotic meronts, which divided by binary fission, were distinguished at the electron microscope level by their unthickened plasma membranes. Sporonts with an electron-dense surface coat gave rise to eight uninucleate sporoblasts within a sporophorous vesicle, cytoplasmic division occurring at the quadrinucleate or octonucleate stages. Indications that nuclear fusion and chromosome reorganization occurred in merogony and sporogony were obtained by light microscopy but meiosis was not detected at the ultrastructural level. Spores were typical of Amblyospora, being ovoid when fresh, truncate when stained, and having an exospore of two membranous layers subtended by a thick amorphous layer, an electron-lucent endospore, an anisofilar polar filament, and a polaroplast comprised of an anterior region of close-packed lamellae and a posterior region of expanded sacs. The metabolic products in the sporophorous vesicle took the form of large globules, small globules with electron-dense borders, and fine granules. These were depleted in mature sporophorous vesicles, though a surface layer of fine granules on the spores may have been derived from them. Many stages were degenerate and it is suggested that C. pipiens may be an accidental host in which the parasite could develop suboptimally in nervous tissue only. Infections in larvae hatched from eggs in the laboratory indicate that vertical transmission occurs.

Animals↗

PubMed2005

Molecular characterization of marker-free transgenic lines of indica rice that accumulate carotenoids in seed endosperm.

A single Agrobacterium strain harbouring two binary plasmids was successfully used for the first time to develop a marker-free transgenic rice of improved nutritional value. Sixty-eight T0 co-transformants were obtained in three indica rice cultivars--two popular high-yielding Bangladeshi varieties (BR28 and BR29), and one high-iron rice cultivar (IR68144). Marker-free lines were obtained from 14 out of 24 selected co-transformants screened in the T1 generation. The accumulation of total carotenoids in polished T2 rice seeds of the primary transgenic VPBR29-17-37 reached levels of up to 3.0 microg/g, with the level of beta-carotene reaching 1.8 microg/g. In the cultivars BR28 and IR68144, total carotenoid levels in the transformants reached 2.0 microg/g of polished rice seeds. The levels of lutein and other carotenoids in the seeds were also significantly enhanced. T1 plants obtained from primary transgenics with simple gene-integration patterns tended to have a lower carotenoid content than the original parental lines. This study describes the development of marker-free transgenic rice lines containing high levels of carotenoids, and addresses the relationship between the rearrangement of transgenes and the presence of metabolic end products in transgenic rice.

Blotting, Southern↗

PubMed2002

Depletion of collagen II-reactive T cells and blocking of B cell activation prevents collagen II-induced arthritis in DBA/1j mice.

Collagen II (CII)-induced arthritis in DBA/1j mice is mediated by both CII-reactive T cells and anti-CII Ab-producing B cells. To determine the relative role of these processes in the development of arthritis, we specifically eliminated CII-reactive T cells by treating the mice with CII-pulsed syngeneic macrophages that had been transfected with a binary adenovirus system. These macrophages express murine Fas ligand in a doxycycline-inducible manner with autocrine suicide inhibited by concomitant expression of p35. The mice were treated i.v. with four doses of CII-APC-AdFasLp35Tet or a single dose of AdCMVsTACI (5 x 10(9) PFU), or both simultaneously, beginning 2 wk after priming with CII in CFA. Treatment with CII-APC-AdFasLp35Tet alone or in combination with a single dose of AdCMVsTACI prevented the development of CII-induced arthritis and T cell infiltration in the joint. The elimination of T cells was specific in that a normal T cell response was observed on stimulation with OVA after treatment with CII-APC-AdFasLp35Tet. Treatment with AdCMVsTACI alone prevented production of detectable levels of circulating anti-CII autoantibodies and reduced the severity of arthritis but did not prevent its development. These results indicate that the CII-reactive T cells play a crucial role in the development of CII-induced arthritis and that the anti-CII Abs act to enhance the development of CII-induced arthritis.

Adenoviridae↗

PubMed1999

Serine 48 in initiation factor 2 alpha (eIF2 alpha) is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.

Phosphorylation of the serine 51 residue in the alpha-subunit of translational initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 alpha monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2 alpha kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alpha subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2 alpha compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.

Amino Acid Substitution↗

PubMed1999

Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch.

The objective of this work was to further develop a tetracycline repressor (TetR) protein system that allows control of transgene expression. First, to circumvent the need for a binary approach, a single plasmid design was constructed and tested in tissue culture. To indirectly assay integrations that express the synthetic transcription factor (rtTA), a bicistronic gene was built which included an internal ribosome entry site (IRES) and a green fluorescent protein coding region (GFP) on the same expression cassette as the coding region of rtTA (pTetGREEN). This construct did not produce fluorescent colonies when stably integrated and provided minimal expression of GFP in the face of adequate expression of rtTA. The coding region for TetR was then altered by introducing 156 silent point mutations to simulate mammalian genes. Replacement of wild-type TetR gene (tetR) in pTetGREEN with 'mammalianized' tetR provided GFP expression. Adjustment of codon usage in the tetR region of rtTA nearly doubled the expression level of functional rtTA. To increase the number of rtTA expressing lines, the chicken egg-white lysozyme matrix attachment region (MAR) was introduced into the single plasmid design just upstream of the tetracycline operators (tetO). Inclusion of the MAR doubled the number of colonies that expressed rtTA (44% vs 88%). With the modifications described here, the number of lines that express rtTA and provide induction from a single plasmid design can be increased by the inclusion of a MAR and the level of rtTA expression can be further increased by adjusting the base composition of the TetR coding region. The MAR also insulates the inducible gene from the promoter driving rtTA.

Amino Acid Sequence↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗