High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.     

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 × B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by thebar orpat genes, was more efficient than selection on kanamycin, mediated by thenptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants-transgene silencing, as well as poor transgene transmission to progeny, was observed in some plant lines in which the parent plants had expressed the transgene.     

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.     

Heterochromatic Trans-Inactivation of Drosophila White Transgenes

Position effect variegation of most Drosophila melanogaster genes, including the white eye pigment gene, is recessive. We find that this is not always the case for white transgenes. Three examples are described in which a lesion causing variegation is capable of silencing the white transgene on the paired homologue (trans-inactivation). These examples include two different transgene constructs inserted at three distinct genomic locations. The lesions that cause variegation of white minimally disrupt the linear order of genes on the chromosomes, permitting close homologous pairing. At one of these sites, trans-inactivation has also been extended to include a vital gene in the vicinity of the white transgene insertion. These findings suggest that many Drosophila genes, in many positions in the genome, can sense the heterochromatic state of a paired homologue.     

Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing

Tobacco plants transformed with the RNA polymerase (RdRp) gene of potato virus X (PVX) that are extremely resistant to infection by potato virus X have previously been described. The PVX-resistant plants accumulated the RdRp protein at a lower level than fully susceptible plants transformed with the same RdRp construct. In this paper the difference between the PVX-resistant and susceptible transformed plants is investigated and it is demonstrated that there are three associated phenotypes of the RdRp transgene that vary in parallel between transformed lines. These phenotypes are: accumulation of the transgenic RdRp RNA at a low level; strain-specific resistance to PVX; and the ability of the transgene to trans -inactivate homologous transgenes. This gene-silencing potential of the transgenes conferring PVX resistance was illustrated by analysis of progeny from a cross between a transformant that was extremely resistant to PVX and a second PVX-susceptible transformant. In other transformants, in which the resistance was less extreme, the same three phenotypes were associated but in a transgene dosage-dependent manner. The same association of strain-specific resistance and low-level accumulation of the transgenic RdRp RNA was observed with plants that were transformed with mutant or wild-type versions of the RdRp gene. Strain-specific resistance was also produced in plants transformed with untranslatable versions of the RdRp transgene. Based on these data it is proposed that homology-dependent gene silencing and transgenic resistance to PVX may be due to the same RNA-based mechanism. An undefined genomic feature is proposed to account for the variation in the resistance and trans -inactivation phenotypes of different transformants. It is further proposed that this genome feature influences a cytoplasmic mechanism that degrades RNA with sequence homology to the silencing transgene.     

Intronic sequences modulate the sensitivity of β-lactoglobulin transgenes to position effects

We have analysed the expression of β-lactoglobulin (BLG) gene constructs with combinations of introns deleted to further define the role of intronic regions in directing position-independent mammary expression of BLG transgenes. Intron removal had no obvious effect on hormonal induction of BLG expression in vitro but dramatically reduced expression in vivo, in that removal of intron pairs always resulted in a proportion of the transgenic lines generated failing to express the transgene in the mammary gland. Position-dependent expression was seen for all intron-deleted transgenes regardless of which introns were removed and the ability of the intron-deleted transgenes to be expressed bore no relationship to transgene copy number. Thus, intron removal per se increases the sensitivity of BLG transgenes to position effects.     

Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants

Silencing ofNia host genes and transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobaccoNia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whetherNii host genes and transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a transgene consisting of the tobaccoNii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of theNii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing ofNii host genes and transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of transgene loci and/or on certain allelic or ectopic combinations of transgene loci. In one transformant carrying a single transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this transgene locus is brought into the presence of a non-allelic transgene locus by crossing, suggesting that a homozygous state is not absolutely required.     

Pre-selection of integration sites imparts repeatable transgene expression

Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for expression of a randomly integrated HPRT marker lying 5′ to an Oct4/lacZ transgene. 794 clones were assessed in vitro for appropriate down-regulation of lacZ following differentiation. Two clones were chosen for further analysis which displayed appropriate and inappropriate gene regulation (clones 710 and 91, respectively). Three developmental promoters (thyroglobulin, Hox2.6 and Myf5) were then sequentially introduced into the original insertion sites in each clone (710 and 91) by homologous recombination, to drive expression of lacZ. Transgenic embryos were assessed for their ability to direct lacZ expression to tissues in which the respective promoter sequences are normally active. The site which appropriately down-regulated lacZ in vitro (710) also showed appropriate in vivo regulation of lacZ from the three developmental promoters. Site 91, however, directed an additional pattern of ectopic expression, which was common to all four promoters. Pre-selection of genomic sites for the introduction of transgenes by gene targeting improves the repeatability of transgene expression and provides an efficient means of single copy transgene introduction by homologous recombination.     

The physiological functions of prion protein

Foreign DNA can be readily integrated into the genomes of mammalian embryonic cells by retroviral infection, DNA microinjection, and transfection protocols. However, the transgenic DNA is frequently not expressed or is expressed at levels far below expectation. In a number of organisms such as yeast, plants, Drosophila, and nematodes, silencing of transfected genes is triggered by the interaction between adjacent or dispersed copies of genes of identical sequence. We set out to determine whether a mechanism similar to repeat-induced gene silencing (RIGS) is responsible for the silencing of transgenes in murine embryonal carcinoma stem cells. We compared the expression of identical reporter gene constructs in cells carrying single or multiple copies and found that the level of expression per integrated copy was more than 10-fold higher in single-copy integrants. In cells carrying tandem copies of the transgene, many copies were methylated and clones frequently failed to express both copies of near-identical integrated alleles. Addition of extra copies of the reporter gene coding sequence reduced the level of expression from the same reporter driven by a eukaryotic promoter. We also found that inhibitors of histone deacetylase such as trichostatin A forestall the silencing of multicopy transgenes, suggesting that chromatin mediates the silencing of transfected genes. This evidence is consistent with the idea that RIGS does occur in mammalian embryonic stem cells although silencing of single-copy transgenes also occurs, suggesting that RIGS is only one of the mechanisms responsible for triggering transgene silencing.     

Influence of the nature of the T-DNA insertion region on transgene expression in Arabidopsis thaliana

In the experiment reported here, effect of the nature of T-DNA integration region on the activity of the transgenes was studied by using a colour marker gene in Arabidopsis thaliana. For this purpose a pale homozygous ch-42 mutant was transformed with the wild-type copy of the gene (CH-42) using kanamycin resistance gene as a selectable marker. Two independent lines were identified in which CH-42 transgene was inactive. The T-DNA flanking sequences were recovered from these inactive and two active lines. These flanking sequences were used to examine copy number and DNA methylation of the T-DNA insertion site in active and inactive lines. Southern blots produced by using MspI/HpaII digested genomic DNA showed signs of methylation in both inactive lines. Furthermore, in one of the inactive line the T-DNA flanking sequence probe hybridized to highly repetitive sequence. The results suggest some correlation between silencing of the transgene and methylation of its insertion region.     

Approaches to Minimize Variation of Transgene Expression in Plants

Genetic transformation of plants has become a widely used technology that serves multiple purposes in plant biology research. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct. This inter-transformant variation of transgene expression hampers proper evaluation of transgenes and might be most undesirable when high-throughput transgene screening is intended. The general plant transformation strategy today is to generate a sufficiently high number of transgenic plants to find some transformants with the desired level of expression. To reduce cost, labor and interpretational flaws, multiple efforts are being directed toward achieving stable expression of transgenes with an expected level of expression. Various factors are thought to contribute to transgene expression variation including the transgene copy number, RNA silencing, transgene insertion site and the employment of certain regulatory sequences to drive transgene expression. This review provides an update on current methodologies to minimize inter-individual variation of transgene expression in nuclear transformed plants.