Characterization of transgene loci in plants using FISH: A picture is worth a thousand words

Transgenic plants are traditionally characterized using phenotypic and Southern analyses. Over the last several years, fluorescence in situ hybridization (FISH) has been efficiently used for visualization, chromosomal localization and estimation of numbers of transgene loci in transgenic plants. Recent results obtained in different plant species transformed using either Agrobacterium tumefaciens or microprojectile bombardment indicate that FISH is also a powerful tool for characterization of transgene locus structure that significantly augments conventional Southern analysis. The aim of this review is to document the usefulness of FISH for characterization of transgene loci in plants.     

Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status.

There are now many mammalian examples in which single cell assays of transgene activity have revealed variegated patterns of expression. We have previously reported that transgenes in which globin regulatory elements drive the lacZ reporter gene exhibit variegated expression patterns in mouse erythrocytes, with transgene activity detectable in only a sub-population of circulating erythroid cells. In order to elucidate the molecular mechanism responsible for variegated expression in this system, we have compared the chromatin structure and methylation status of the transgene locus in expressing and non-expressing populations of erythrocytes. We find that there is a difference in the chromatin conformation of the transgene locus between the two states. Relative to active transgenes, transgene loci which have been silenced exhibit a reduced sensitivity to general digestion by DNase I, as well as a failure to establish a transgene-specific DNase I hypersensitive site, suggesting that silenced transgenes are situated within less accessible chromatin structures. Surprisingly, the restrictive chromatin structure observed at silenced transgene loci did not correlate with increased methylation, with transgenes from both active and inactive loci appearing largely unmethylated following analysis with methylation-sensitive restriction enzymes and by sequencing PCR products derived from bisulphite-converted genomic DNA.     

Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.     

Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement

To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of the delivered DNA, and inverted and imperfect direct repeats were detected in the same transgene locus indicating that homologous recombination and synthesis-dependent mechanism(s), respectively, were also involved in transgene locus rearrangement. The most unexpected result was the small size of the fragments of delivered and genomic DNA incorporated into the transgene loci via illegitimate recombination; 50 of the 82 delivered DNA fragments were shorter than 200 bp. Eleven transgene and genomic fragments were shorter than the DNA lengths required for Ku-mediated non-homologous end joining. Detection of these small fragments provided evidence that illegitimate recombination was most likely mediated by a synthesis-dependent strand-annealing mechanism that resulted in transgene scrambling. Taken together, these results indicate that transgene locus formation involves the concerted action of several DNA break-repair mechanisms.     

Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by epigene conversion involving DNA-DNA pairing.     

Field trial analysis of nitrate reductase co-suppression: a comparative study of 38 combinations of transgene loci

Co-syppression of host genes and 35S transgenes encoding nitrate reductase was previously reported in transgenic tobacco plants (Nicotiana tabacum cv. Paraguay or Burley) using either a full-length cDNA or fragments devoid of the 3 and/or 5 UTR. Co-suppression was previously shown to affect a limited fraction of the progeny of one transgenic tobacco line homozygous for a single transgene locus, and the phenomenon occurred at each generation. In this work, 38 combinations of transgene loci derived from 13 independent transgenic lines homozygous for a single transgene locus were field-tested under two different conditions in an attempt to determine the corresponding frequencies of co-suppression, i.e. the percentage of plants showing co-suppression.Each of the 13 homozygous lines exhibited a different frequency of co-suppression, ranging from 0% to 57%. High frequencies were found to be associated with transgene loci carrying a high number of copy of the transgene, suggesting a transgene dose effect. Combinations carrying 2 non-allelic transgene loci in a hemizygous state exhibited frequencies of co-suppression between those of each of the 2 transgene loci in a homozygous state, while combinations carrying 2 non-allelic transgene loci in a homozygous state exhibited frequencies of co-suppression higher than the sum of those of the 2 transgene loci alone in a homozygous state, clearly confirming a transgene dose effect.Co-suppression frequencies were increased when the plants were grown initially in vitro, suggesting some environmental effect. The roles of transgene copy number, number of transgene loci and environmental factors are discussed in the light of a threshold hypothesis.     

基因枪转化获得的转基因水稻中外源基因整合与表达规律研究

通过基因枪法将cecropin B和bar基因共转化水稻,获得多个水稻优良品系的转化材料,对转基因的结构和表达的系统分析,发现外源基因的整合模式多种多样,有简单也有复杂,其中插入位点数的变化范围为1－7个,拷贝数的变化范围为1－10个,转基因拷贝数的多少与其表达和沉默不存在必然的联系,相同整合模式的转基因事件中基因的表达存在较大的差异,选择标记基因bar比非选择标记基因cecropin B的表达框的完整性和转录概率要高,但是发现大部分表达框完整的bar基因发生基因沉默,而在终止子发生序列丢失的cecropin B基因的表达则明显提高。     

转基因的分子生物学特性

现代分子生物学研究中,转基因概念的出现越来越频繁,它渐渐被用来表示所有的用基因工程手段构建、导入高等真核生物细胞,并稳定地整合入受体基因组的外源DNA．文章较系统地总结了已见报道的转基因的结构类型、它在受体细胞内的遗传学行为、表达特性和影响因素及其相应的生物学效应;分析了转基因研究中尚待研究的问题,提出了系统地进行转基因本身的分子生物学特性研究的必要性.     

Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge)

Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern United States and other subtropical regions. Biolistic co-transfer of two unlinked, minimal, linear transgene expression cassettes (MCs) into the apomictic bahiagrass cv. Argentine was carried out to evaluate co-integration, quantify co-expression and analyze inheritance to apomictic seed progeny. Gold projectiles were coated with minimal unlinked nptII and bar expression cassettes in a 1:2 molar ratio. Complexity of transgene loci correlated with the amount of DNA used during gene transfer. Transgenic plants displayed a simple nptII integration pattern with 1–4 hybridization signals compared to the non-selected bar gene with 2 to more than 5 hybridization signals per transgenic line. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transformed lines (95% co-expression frequency). Protein quantification revealed that several lines with complex integration patterns displayed a higher transgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplification and sequence analysis of transgene loci. The obligate apomictic bahiagrass cv. Argentine produced uniform seed progeny without segregation of simple or complex transgene loci. NPTII- and PAT-ELISA, as well as herbicide application, confirmed stable expression of the nptII and bar gene at levels similar to the primary transformants. These results demonstrate that biolistic transfer of MCs support stable and high level co-expression of transgenes in bahiagrass.     

Molecular Characteristics of Transgenic Wheat and the Effect on Transgene Expression

A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study.     

2004 SIVB Congress Symposium Proceeding: Transgene Management via Multiple Site-specific Recombination Systems

Summary Current methods for creating transgenic varieties are labor and time intensive, comprised of the generation of hundreds of plants with random DNA insertions, screening for the few individuals with appropriate transgene expression and simple integration structure, and followed by a lengthy breeding process to introgress the engineered trait into cultivated varieties. Various modifications of existing methods have been proposed to speed up the different steps involved in plant transformation, as well as a few add-on technologies that seek to address issues related to biosafety or intellectual property. The problem with an assortment of independently developed improvements is that they do not integrate seamlessly into a single transformation system. This paper presents an integrated strategy for plant transformation, where the introduced DNA will be inserted precisely into the genome, the transgenic locus will be introgressed rapidly into field varieties, the extraneous transgenic DNA will be removed, the transgenic plants will be molecularly tagged, and the transgenic locus may be excised from pollen and/or seed.     

Characterization of three loci for homologous gene targeting and transgene expression

Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare‐cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site‐specific transgene integration, suitable for a variety of biotechnological applications. Biotechnol. Bioeng. 2013; 110: 2225–2235. © 2013 Wiley Periodicals, Inc.     

Genomic organization and characterization of the white locus of the Mediterranean fruitfly, Ceratitis capitata

An approximately 14-kb region of genomic DNA encoding the wild-type white eye (w+) color gene from the medfly, Ceratitis capitata has been cloned and characterized at the molecular level. Comparison of the intron-exon organization of this locus among several dipteran insects reveals distinct organizational patterns that are consistent with the phylogenetic relationships of these flies and the dendrogram of the predicted primary amino acid sequence of the white loci. An examination of w+ expression during medfly development has been carried out, displaying overall similarity to corresponding studies for white gene homologues in Drosophila melanogaster and other insects. Interestingly, we have detected two phenotypically neutral allelic forms of the locus that have arisen as the result of an apparently novel insertion or deletion event located in the large first intron of the medfly white locus. Cloning and sequencing of two mutant white alleles, w1 and w2, from the we,wp and M245 strains, respectively, indicate that the mutant conditions in these strains are the result of independent events--a frameshift mutation in exon 6 for w1 and a deletion including a large part of exon 2 in the case of w2.     

Perinatal lethality (ple): a mutation caused by integration of a transgene into distal mouse chromosome 15

We have used cytogenetic and recombinational analysis to determine the position of a transgene integrated into the mouse genome. The transgene maps to band F on the physical map of mouse chromosome 15 by in situ analysis and is tightly linked genetically to a cluster of loci that include the mutations caracul (Ca) and microcytic anemia (mk). Genetic analysis of the offspring of noninbred animals carrying the transgene and marker loci demonstrates a significant deficiency of homozygous progeny at weaning. When inbred mice heterozygous for the transgene are mated, about one-quarter of their offspring are homozygous; none of these animals survives more than 1 day after birth. It appears likely that a recessive insertional mutation has occurred as a result of transgene integration into a locus required for postnatal viability. We call this mutation transgenic perinatal lethality (Tg.ple).     

Genetic and molecular analysis of the 87A7 and 87C1 heat-inducible loci of D. melanogaster.

Two different types of heat-inducible sequences are found at the cytogenetic loci 87A7 and 87C1 of D. melanogaster. One of these codes for the 70,000 dalton heat shock protein (hsp 70) and is found at both loci. The other type of sequence (alpha beta) codes for an RNA of unknown function and is found only at 87C1. We have completed a study of the organization of the two loci, using deficiencies that delete one or other locus, and have estimated the number of the hsp 70 genes at each locus. Thus in at least three strains of files there are a total of five coding sequences, three at 87C1 and two at 87A7. Restriction mapping of the coding regions at the two loci reveals that each of the two cytogenetic loci has its own characteristic coding sequence. The overall organization of the two loci appears to differ considerably. The alpha beta and hsp 70 heat-induced sequences at 87C1 are closely linked and are contained within two Eco RI restriction fragments.