Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression

Background  

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation.     

Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event

Background  

Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage λ red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step.     

<Emphasis Type="Italic">In utero</Emphasis>recombinant adeno-associated virus gene transfer in mice,rats, and primates

Background  

Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes.     

Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

Background  

The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.     

High-level gene expression in Aedes albopictus cells using a baculovirus Hr3 enhancer and IE1 transactivator

Background  

Aedes aegypti is the key vector of both the Yellow Fever and Dengue Fever viruses throughout many parts of the world. Low and variable transgene expression levels due to position effect and position effect variegation are problematic to efforts to create transgenic laboratory strains refractory to these viruses. Transformation efficiencies are also less than optimal, likely due to failure to detect expression from all integrated transgenes and potentially due to limited expression of the transposase required for transgene integration.     

Identification of genes associated with regenerative success of <Emphasis Type="Italic">Xenopus laevis</Emphasis>hindlimbs

Background  

Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.     

The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms

Background  

The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms.     

Cre-LoxP-regulated expression of monoclonal antibodies driven by an ovalbumin promoter in primary oviduct cells

Background  

A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Ovalbumin promoters can be used as oviduct-specific regulatory sequences in transgenic chickens, but their promoter activities are not high, according to previous reports.     

Homologous illegitimate random integration of foreign DNA into the X chromosome of a transgenic mouse line

Background  

It is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration.     

Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress

Background  

Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc ⁺) gene and low temperature-responsive luciferase activation was monitored in real time.     

Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo

Background

Gene delivery vectors based on poly(L ‐lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non‐specifically. In this study we have attempted to form folate‐targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers.

Methods

pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da.

Results

Polymer‐coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000‐fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA.

Conclusions

We show increased systemic circulation, decreased blood cell and protein binding, and folate‐targeted transgene expression using pLL/DNA complexes surface‐modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma‐circulating targeted vectors for in vivo applications. Copyright © 2002 John Wiley & Sons, Ltd.

     

Optimisation of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes

Background  

Genetic engineering of parthenocarpy confers to horticultural plants the ability to produce fruits under environmental conditions that curtail fruit productivity and quality. The DefH9-iaaM transgene, whose predicted action is to confer auxin synthesis specifically in the placenta, ovules and derived tissues, has been shown to confer parthenocarpy to several plant species (tobacco, eggplant, tomato) and varieties.     

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

Background  

The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites.     

An efficient <Emphasis Type="Italic">Foxtail mosaic virus</Emphasis> vector system with reduced environmental risk

Background  

Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment.     

Novel system uses probasin-based promoter,transcriptional silencers and amplification loop to induce high-level prostate expression

Background  

Despite several effective treatment options available for prostate cancer, it remains the second leading cause of cancer death in American men. Thus, there is a great need for new treatments to improve outcomes. One such strategy is to eliminate cancer through the expression of cytotoxic genes specifically in prostate cells by gene therapy vectored delivery. To prevent systemic toxicity, tissue- and/or cancer-specific gene expression is required. However, the use of tissue- or cancer-specific promoters to target transgene expression has been hampered by their weak activity.     

Introgression potential between safflower (Carthamus tinctorius) and wild relatives of the genus Carthamus

Background  

Safflower, Carthamus tinctorius, is a thistle that is grown commercially for the production of oil and birdseed and recently, as a host for the production of transgenic pharmaceutical proteins. C. tinctorius can cross with a number of its wild relatives, creating the possibility of gene flow from safflower to weedy species. In this study we looked at the introgression potential between different members of the genus Carthamus, measured the fitness of the parents versus the F1 hybrids, followed the segregation of a specific transgene in the progeny and tried to identify traits important for adaptation to different environments.     

Stable expression and phenotypic impact of <Emphasis Type="Italic">attacin E</Emphasis>transgene in orchard grown apple trees over a 12 year period

Background  

Transgenic trees currently are being produced by Agrobacterium -mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years.