Variable Transgene Expression Research Articles

Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced.

In an effort to control the variability of transgene expression in plants, we used Cre-lox mediated recombination to insert a gus reporter gene precisely and reproducibly into different target loci. Each integrant line chosen for analysis harbors a single copy of the transgene at the designated target site. At any given target site, nearly half of the insertions give a full spatial pattern of transgene expression. The absolute level of expression, however, showed target site dependency that varied up to 10-fold. This substantiates the view that the chromosome position can affect the level of gene expression. An unexpected finding was that nearly half of the insertions at any given target site failed to give a full spatial pattern of transgene expression. These partial patterns of expression appear to be attributable to gene silencing, as low gus expression correlates with DNA methylation and low transcription. The methylation is specific for the newly integrated DNA. Methylation changes are not found outside of the newly inserted DNA. Both the full and the partial expression states are meiotically heritable. The silencing of the introduced transgenes may be a stochastic event that occurs during transformation.

Hierarchical patterns of transgene expression indicate involvement of developmental mechanisms in the regulation of the maize P1-rr promoter.

The maize P1-rr gene encodes a Myb-homologous transcription factor that regulates the synthesis of red flavonoid pigments. Maize plants transformed with segments of the P1-rr promoter driving a GUS reporter gene exhibit significant variation in transgene expression, both between independent transformation events and among sibling plants derived from a single event. Interestingly, variability in spatial expression is not random; rather, transgene activity occurs predominantly in five patterns that fit a hierarchy: expression is most common in kernel pericarp, with sequential addition of expression in cob glumes, husk, silk, and tassel. The hierarchical expression pattern of P-rr::GUS transgenes suggests a possible model for developmental regulation of the P1-rr gene. Our results demonstrate that variability in transgene expression, a common occurrence in transgenic plant studies, can be informative if adequately analyzed to uncover underlying patterns of gene expression.

Variegated expression of the endogenous immunoglobulin heavy-chain gene in the absence of the intronic locus control region.

The expression of chromosomally integrated transgenes usually varies greatly among independent transfectants. This variability in transgene expression has led to the definition of locus control regions (LCRs) as elements which render expression consistent. Analyses of expression in single cells revealed that the expression of transgenes which lack an LCR is often variegated, i.e., on in some cells and off in others. In many cases, transgenes which show variegated expression were found to have inserted near the centromere. These observations have suggested that the LCR prevents variegation by blocking the inhibitory effect of heterochromatin and other repetitive-DNA-containing structures at the insertion site and have raised the question of whether the LCR plays a similar role in endogenous genes. To address this question, we have examined the effects of deleting the LCR from the immunoglobulin heavy-chain locus of a mouse hybridoma cell line in which expression of the immunoglobulin mu heavy-chain gene is normally highly stable. Our analysis of mu expression in single cells shows that deletion of this LCR resulted in variegated expression of the mu gene. That is, in the absence of the LCR, expression of the mu gene in the recombinant locus could be found in either of two epigenetically maintained, metastable states, in which transcription occurred either at the normal rate or not at all. In the absence of the LCR, the on state had a half-life of approximately 100 cell divisions, while the half-life of the off state was approximately 40,000 cell divisions. For recombinants with an intact LCR, the half-life of the on state exceeded 50,000 cell divisions. Our results thus indicate that the LCR increased the stability of the on state by at least 500-fold.

Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj-globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.

Fast forward genetics based on virus-induced gene silencing.

Gene expression in plants can be suppressed in a sequence-specific manner by infection with virus vectors carrying fragments of host genes. Recent developments have revealed that the mechanism of this gene silencing is based on an RNA-mediated defence against viruses. It has also emerged that a related mechanism is involved in the post-transcriptional silencing that accounts for between line variation in transgene expression and cosuppresion of transgenes and endogenous genes. The technology of virus-induced gene silencing is being refined and adapted as a high throughput procedure for functional genomics in plants.

Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster.

Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and alpha1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.

The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgenic plants.

Nuclear matrix attachment regions (MARs) are thought to influence gene expression by anchoring active chromatin to the proteinaceous nuclear matrix. In this study, two plant DNA fragments with strong MAR activity were selected and tested for their effects on expression of a linked reporter gene in transgenic tobacco. One MAR was isolated from the 5' flanking region of a pea vicilin gene previously reported to be expressed in a copy number-dependent manner in transgenic tobacco. A second MAR was isolated from the genome of Arabidopsis thaliana by preselection for autonomously replicating sequence (ARS) activity in yeast. Flanking copies of the A. thaliana MAR stimulated median reporter gene expression in transgenic plants by five to ten fold. Neither MAR significantly reduced the variation in transgene expression between individual transformants, or conferred copy number-dependence in gene expression.

Phenotypic variation in a genetically identical population of mice.

The parental alleles of an imprinted gene acquire their distinctive methylation patterns at different times in development. For the imprinted RSVIgmyc transgene, methylation of the maternal allele is established in the oocyte and invariably transmitted to the embryo. In contrast, the methylation of the paternal allele originates during embryogenesis. Here, we show that the paternal methylation pattern among mice with identical genetic backgrounds is subject to extensive variation. In addition to this nongenetic variation, the process underlying RSVIgmyc methylation in the embryo is also subject to considerable genetic regulation. The paternal transgene allele is highly methylated in an inbred C57BL/6J strain, whereas it is relatively undermethylated in an inbred FVB/N strain. Individual methylation patterns of paternal alleles, and therefore all of the variation (nongenetic and genetic) in methylation patterns within an RSVIgmyc transgenic line, are established in early embryogenesis. For each mouse, the paternal RSVIgmyc allele is unmethylated at the day-3.5 blastocyst stage, and the final, adult methylation pattern is found no later than day 8.5 of embryogenesis. Because of the strong relationship between RSVIgmyc methylation and expression, the variation in methylation is also manifest as variation in transgene expression. These results identify embryonic de novo methylation as an important source of both genetic and nongenetic contributions to phenotypic variation and, as such, further our understanding of the developmental origin of imprinted genes.

From transgene expression to public acceptance of transgenic plants: a matter of predictability

A good strategy for acceptable legislation of transgenic plants can be thought to be composed of several stacked levels of decision-making. These levels range from global to individual to cellular to nuclear and beyond. Any decision will depend on decisions made on the level below. Various examples are given, with emphasis on the most basal level, the level of transgene expression. Plant transformation suffers from a huge variability in transgene expression. In addition, in recent years a variety of (epi)genetic transgene instabilities have been described. It is demonstrated that the addition of so-called matrix-associated regions (MARs) around transgenes before Agrobacterium-mediated transformation improves the predictability of transgene expression significantly. MARs are thought to function as boundary elements that shield the enclosed transgenes from influences of the surrounding chromatin. Assuming that such boundary elements will make transgene expression overall more predictable, this approach is then likely to contribute to a more straight-forward assessment of the biosafety of transgenic plants. This will enhance the social acceptance, hence more successful market introduction of transgenic plants.

Variability of transgene expression in clonal cell lines of wheat

A method for polyethyleneglycol (PEG) mediated direct DNA transfer into protoplasts was successfully established for transient and stable transformation of Triticum aestivum L. cell cultures. Transgenic cell lines, which had been transformed with the neomycin phosphotransferase II gene ( nptII) fused to different promoters, were selected and integration and expression of the marker gene was shown by Southern analysis and enzyme activity test. For investigation of expression stability, five nptII positive cell lines maintained under selection were protoplasted and clonal callus lines were cultivated from the genetically identical single cells without selection pressure. Marker gene activity of 271 clonal callus lines was determined and compared with the corresponding parental line. A reduction or loss of marker gene expression in up to 50% of the clonal cell lines was observed. Detailed analysis of randomly selected clones showed that the observed variability in marker gene expression occurred due to a reduction in the nptII transcript level and was associated with hypermethylation of the integrated DNA. The silencing effect was reversible by a 4 week culture phase on media supplemented with the demethylation agent 5-azacytidin. These differences in marker gene expression could be observed regardless of copy number and position of the integrated nptII gene. The significance of such observations for a stable expression of foreign genes in plant cells is discussed.

Nuclear Matrix Attachment Regions and Transgene Expression in Plants.

DNA sequences called matrix attachment regions (MARs) or scaffold attachment regions (SARs) have recently attracted much attention because of their perceived capacity to increase levels of transgene expression and reduce transformant-to-transformant variation of transgene expression in both plants and animals. Work with these sequences is in its early stages and data that seem to be contradictory have been presented. We do not intend to resolve these controversies here (this will be accomplished by further research). Rather, we will discuss the hypothesized role of MARs in chromatin structure, how MARs are isolated and characterized, what effects MARs have had on the expression of transgenes and the models that have been evoked to explain those effects.

Stability of a Transgene in Potato Depends on Endogenous Plant Tissue Factors

Transformation of potato cultivar (Desiree) has been achieved using different types of explant tissue (minituber, stem and leaf discs) via infection with an Agrobacterium tumefaciens harboring a kanamycin resistance gene. The product of it, an aminoglycoside phosphotransferase, has been determined in the protein extracts of the transformants in addition to the common rooting tests. Certain influences of the type of the explants upon the transformation efficiency and the morphology of the regenerated transgenic plantlets have been noticed. A selection and regeneration protocol for the potato transformants is described. A periodical study of the foreign gene stability and reversibility tests with demethylating agent of the relevant lines have been performed. A possible involvement of DNA methylation processes in the variable transgene expression in potato plantlets is discussed.

Variation of transgene expression in plants

Variation of transgene expression in plants

Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T‐DNA borders

SummaryA new binary vector for Agrobacterium‐mediated plant transformation was constructed, in which two selectable markers, for kanamycin and hygromycin resistance, were placed next to the right and left T‐DNA borders, respectively, and a CaMV 35S promoter‐driven β‐glucuronidase (GUS) gene was placed between these markers as a reporter gene (transgene). Using double antibiotic selection, all transgenic tobacco plants carrying at least one intact copy of the T‐DNA expressed the transgene, and this population exhibited reduced variability in transgene expression as compared with that obtained from the parent vector pBI121. Absence of the intact transgene was the major reason for transgenic plants with little or no transgene expression. Integration of truncated T‐DNAs was also observed among transgenic plants that expressed the transgene and carried multiple T‐DNA inserts. The copy number of fully integrated T‐DNAs was positively associated with transgene expression levels in R0 plants and R1 progeny populations. Variability due to position effect was determined among 17 plants carrying a single T‐DNA insert. The coefficient of variability among these plants was only 35.5%, indicating a minor role for position effects in causing transgene variability. The new binary vector reported here can therefore be used to obtain transgenic populations with reduced variability in transgene expression.

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region.

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.

Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters

Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters

Reconstitution of tracheal grafts with a genetically modified epithelium.

A rational approach to the development of gene therapies for cystic fibrosis requires a better understanding of the cellular targets for gene transfer in the airway epithelium. We have used recombinant retroviruses to study the dynamics and lineage relationships of a regenerating rat tracheal epithelium. Primary cultures of tracheal epithelial cells were exposed to lacZ-transducing retroviruses and subsequently seeded into denuded trachea that were implanted into BALB/c (nu/nu) mice. The grafts developed a fully differentiated mucociliary epithelium containing large clones of lacZ-expressing cells with virtually all cell types represented within each clone. These data are most consistent with gene transfer into a putative progenitor cell that is capable of extensive self renewal and pleuripotent development. Vector-specific variation in transgene expression was noted in the various cell types.

Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters

Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants. Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold. The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure. There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49. Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes. Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants. The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.