Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize <Emphasis Type="Italic">adh-1</Emphasis> matrix attachment regions: tissue and developmental specificity in maize transgenic plants

Matrix attachment regions (MARs) have been used to enhance transgene expression and to reduce transgene expression instability in various organisms. In plants, contradictory data question the role of MAR sequences. To assess the use of MAR sequences in maize, we have used two well-characterized MARs from the maize adh-1 region. The MARs have been cloned either 5 to or at both sides of a reporter gene expression cassette to reconstitute a MAR-based domain. Histochemical staining revealed a new transgene expression pattern in roots of regenerated plants and their progeny. Furthermore, MARs systematically induced variegation. We show here that maize adh-1 MARs are able to modify transgene expression patterns as a heritable trait, giving a new and complementary outcome following use of MARs in genetic transformation.Abbreviations adh-1 Alcohol dehydrogenase 1 - GUS -Glucuronidase - HSC80 Heat shock cognate 80 gene - MAR Matrix attachment regions - Rsyn-7 Root specific synthetic promoter     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells

Low‐level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR‐6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR‐6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP‐B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP‐B, DHFR intron MAR element and MAR‐6. Additionally, as expected, the three MAR‐containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non‐MAR‐containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP‐B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.     

A transformation booster sequence (<Emphasis Type="Italic">TBS</Emphasis>) from <Emphasis Type="Italic">Petunia hybrida</Emphasis> functions as an enhancer-blocking insulator in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Several matrix-attachment regions (MARs) from animals have been shown to block interactions between an enhancer and promoter when situated between the two. Since a similar function for plant MARs has not been discerned, we tested the Zea mays ADH1 5′ MAR, Nicotiana tabacum Rb7 3′ MAR and a transformation booster sequence (TBS) MAR from Petunia hybrida for their ability to impede enhancer–promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP)::β-glucuronidase (GUS) fusion were assayed for GUS expression in vegetative tissues. We demonstrate that the TBS MAR element, but not the ADH1 or Rb7 MARs, is able to block interactions between the 35S enhancer and AGIP without compromising the function of either with elements from which they are not insulated. Accession numbers: TBS from Petunia hybrida cultivar V26, GenBank accession number EU864306.     

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese‐hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human β‐globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 5′ or 3′ site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 5′ site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 3′ site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.     

Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region

Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5' MAR had the effect of localizing beta-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5' MAR is discussed.     

A short synthetic MAR positively affects transgene expression in rice and Arabidopsis

Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number of sequence features and DNA elements in common, and using different subsets of these sequence elements, three independent synthetic MARs were created. Although short, these MARs were able to bind nuclear scaffold preparations with an affinity equal to or greater than naturally occurring plant MARs. One synthetic MAR was extensively tested for its effect on transgene expression, using different MAR orientations, plant promoters, transformation methods and plant species. This MAR was able to increase average transgene expression and produced integration patterns of lower complexity. These data show the potential of making well defined synthetic MARs and using them to improve transgene expression.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

Increased expression of transgene in stably transformed cells of <Emphasis Type="Italic">Dunaliella salina</Emphasis> by matrix attachment regions

Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated from Dunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects of MARs on expression of transgenes in the stably transformed cells of D. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven by D. salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells of D. salina by electroporation. Twenty stably transformed colonies of D. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies of D. salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression may not be through increasing the transgene copy numbers.     

A tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants

The RB7 matrix attachment region (MAR), when flanking a uidA (GUS) reporter gene, has been previously shown to increase uidA gene expression by 60-fold in stably transformed tobacco suspension cell lines. We have now used the same co-transformation procedure to determine the effect of flanking MARs on uidA gene expression in tobacco plants. The neomycin phosphotransferase selection gene and uidA reporter gene on separate plasmids were co-transformed into seedlings by microprojectile bombardment. In primary transgenic plants, the average uidA expression in plants with MARs was twofold greater than in control plants without MARs, but there was no effect on variation of expression. GUS activity was not proportional to the number of integrated uidA transgenes over the entire range of copy numbers. However, in the lower part of the copy number range, MAR lines show a tendency for expression to increase with copy number. Transgene expression in backcross progenies of the MAR-containing lines averaged threefold higher than in control progenies. MARs also reduced the loss of transgene expression in the BC₁ generation. Sixty-three per cent of the 21 MAR-containing primary transformants, but only 20% of the 14 control primary transformants, produced backcross progenies in which no loss of transgene expression was observed. These observations are discussed in the context of homology-dependent gene silencing.     

Comparative analysis of transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and particle bombardment

Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.     

High frequency <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated plant transformation induced by ammonium nitrate

Success in plant genetic transformation depends on the efficiency of explant regeneration and transgene integration. Whereas the former one depends on explant totipotency, the latter depends on the activity of host DNA repair and chromatin organisation factors. We analyzed whether factors that result in an increase in recombination frequency can also increase transformation efficiency. Here, we report that a threefold increase in the concentration of NH₄NO₃ in the growth medium results in more than a threefold increase in the Agrobacterium tumefaciens-mediated transformation frequency of Nicotiana tabacum plants. Regeneration of calli without selection showed that the increase in transformation frequency was primarily due to the increase in transgene integration efficiency rather than in tissue regeneration efficiency. PCR analysis of insertion sites showed a decrease in the frequency of truncations of the T-DNA right border and an increase on the left border. We hypothesize that exposure to ammonium nitrate modifies the activity of host factors leading to higher frequency of transgene integrations and possibly to the shift in the mechanism of transgene integrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Elevation of Transgene Expression Level by Flanking Matrix Attachment Regions (MAR) is Promoter Dependent: A Study of the Interactions of Six Promoters with the RB7 3′ MAR

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.     

The potato Lhca3.St.1 promoter confers high and stable transgene expression in chrysanthemum,in contrast to CaMV-based promoters

Theenhanced cauliflower mosaic virus 35S (dCaMV) promoter and the potatoLhca3.St.1 promoter were evaluated for their expressionabilities in chrysanthemum. The promoters were fused to the-glucuronidase(GUS) reporter gene with and without flanking matrix-associated regions (MARs).They were transferred into chrysanthemum viaAgrobacterium-mediated transformation. The quantitativeevaluation of GUS activity in a total of 127 independently derivedtransformantsestablished that in chrysanthemum the Lhca3.St.1 promoterwas 175 fold more active in the leaves than the dCaMV promoter was. The latterwas as poor in expression as the single CaMV promoter. The use of suchCaMV-based promoters in the genetic engineering of chrysanthemum should bediscouraged when high levels of transgene expression are desired. No clearinfluence of the presence of MARs was observed on the variability of GUS geneexpression, in contrast to earlier studies in tobacco. This may indicate apossible plant species dependent activity of MAR elements.Lhca3.St.1 promoter-driven GUS activity was relativelyhigher in the stem of chrysanthemum and proved stable over extensive timeperiods. Therefore this potato promoter is attractive to obtain high expressionlevels in chrysanthemum.     

Functional Analysis of Two Matrix Attachment Region (MAR) Elements in Transgenic Maize Plants

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.     

核基质结合区的位置对转基因表达的影响

为研究核基质结合区 (MAR）序列不同插入位置对转基因表达作用的影响,PCR扩增人β 珠蛋白MAR分别插入到含氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因真核表达载体pCATG表达盒两侧、5′端及3′端.酶切鉴定后,用阳离子聚合物转染CHO细胞,G418筛选出阳性细胞克隆,ELISA分析CAT基因的表达水平,半定量PCR分析CAT基因相对拷贝数.结果表明,表达盒两侧含MAR序列的载体能提高介导的转基因表达水平平均提高10.4倍,5′端含MAR序列的载体表达水平平均提高3.9倍,3′端含MAR序列的载体反而降低转基因表达水平.5′端含MAR序列的表达载体其转基因相对拷贝数高于其它两组载体的基因拷贝数,转基因表达量与基因拷贝数不成正比.