Transgene Locus Research Articles

Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco.

Transgene integration into plant genomes is a complex process accompanied by molecular rearrangements. Classic methods that are normally used to study transgenic population genetics are generally inadequate for assessing such integration. Two major characteristics of transgenic populations are that a transgenic genome may harbor many copies of the transgene and that molecular rearrangements can create an unstable transgenic locus. In this work, we examined the segregation of T1, T2 and T3 transgenic tobacco progenies. Since transfer DNA (T-DNA) contains the NptII selectable marker gene that confers resistance to kanamycin, we used this characteristic in developing a method to estimate the number of functional inserts integrated into the genome. This approach was based on calculation of the theoretical segregation ratios in successive generations. Mendelian ratios of 3:1, 15:1 and 63:1 were confirmed for five transformation events whereas six transformation events yielded non-segregating progenies, a finding that raised questions about causal factors. A second approach based on a maximum likelihood method was performed to estimate recombination frequencies between linked inserts. Recombination estimates varied among transformation events and over generations. Some transgenic loci were unstable and evolved continuously to segregate independently in the T3 generation. Recombination and amplification of the transgene and filler DNA yielded additional transformed genotypes.

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.

Genetic and Epigenetic Effects of Plant–Pathogen Interactions: An Evolutionary Perspective

Recent reports suggest that exposure to stress is capable of influencing the frequency and pattern of inherited changes in various parts of the genome. In this review, we will discuss the influence of viral pathogens on somatic and meiotic genome stability of Nicotiana tabacum and Arabidopsis thaliana. Plants infected with a compatible pathogen generate a systemic recombination signal that precedes the spread of pathogens and results in changes in the somatic and meiotic recombination frequency. The progeny of infected plants exhibit changes in global and locus-specific DNA methylation patterns, genomic rearrangements at transgenic reporter loci and resistance gene-like-loci, and even tolerance to pathogen infection and abiotic stress. Here, we will discuss the contribution of environmental stresses to genome evolution and will focus on the role of heritable epigenetic changes in response to pathogen infection.

Var Gene Promoter Activation in Clonal Plasmodium falciparum Isolates Follows a Hierarchy and Suggests a Conserved Switching Program that Is Independent of Genetic Background

Antigenic variation of Plasmodium falciparum is mediated by a mutually exclusive expression mechanism that limits expression to an individual member of the multicopy var gene family. This process determines the antigenic and adhesive phenotype of the infected red blood cell. Previously, we showed that var gene switching is influenced by chromosomal position. Here, we address whether var gene transcription follows a general conserved pattern in long-term laboratory parasites and in recently culture-adapted field parasites. Activation of the var gene family was monitored in biological replicates in each parasite isolate every 3-5 generations for up to 3 months. We used transgenic parasites carrying a drug-selectable marker at a defined var locus to characterize var gene activation after the exclusive expression of the transgene. Transgenic parasites exhibited a repeatable hierarchy of var gene activation and a fluctuating transcriptional activity of the transgenic var locus. Transcriptional profiling of wild-type laboratory and field parasites showed a universal bias toward transcription of UpsC var genes and a fluctuating transcriptional activity of the dominant var promoter. The data suggest the existence of an intrinsic var gene transcription program that is independent of genetic background.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Recombinase‐mediated Gene Stacking as a Transformation Operating SystemF

The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If re-engineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site-specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.

Identification of Tobacco Haploids on the Basis of Transgenic Overexpression of PAP1 from Arabidopsis thaliana

ABSTRACTHaploid plants have utility in plant breeding for several purposes. In tobacco (Nicotiana tabacum L.) gynogenic haploids can be produced from seed due to parthenogenesis. For practical use, a system is needed to identify infrequent haploid plants at the seed or seedling stage. Interspecific hybridization with N. africana is presently used to isolate gynogenic tobacco haploids because a suitable dominant seedling marker does not currently exist for N. tabacum. Here we investigated the utility of a purple seedling trait conferred by overexpression of the Arabidopsis gene, PAP1, to identify gynogenic haploids produced from seed. Two tobacco cultivars were crossed as females with a genetic stock homozygous at two 35S:PAP1 transgene loci. Gynogenic haploids were recognized as green seedlings among purple F1 hybrid seedlings. The average frequency of gynogenic haploidy using this system was f = 0.00027. For comparison, the same cultivars were also hybridized as females with N. africana. The average frequency of gynogenic haploidy using this system was approximately seven times higher than that observed for the 35S:PAP1 system. Having a dominant seedling marker for N. tabacum may permit development of genetic stocks that contribute to an increased predisposition for haploid formation via parthenogenesis. In addition, the 35S:PAP1 genetic marker may have utility for identifying androgenic haploids from seed for the purpose of rapidly generating alloplasmic lines of tobacco.

Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing

It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele.

An Antisense CAG Repeat Transcript at JPH3 Locus Mediates Expanded Polyglutamine Protein Toxicity in Huntington's Disease-like 2 Mice

Huntington's disease-like-2 (HDL2) is a phenocopy of Huntington's disease caused by CTG/CAG repeat expansion at the Junctophilin-3 (JPH3) locus. The mechanisms underlying HDL2 pathogenesis remain unclear. Here we developed a BAC transgenic mouse model of HDL2 (BAC-HDL2) that exhibits progressive motor deficits, selective neurodegenerative pathology, and ubiquitin-positive nuclear inclusions (NIs). Molecular analyses reveal a promoter at the transgene locus driving the expression of a CAG repeat transcript (HDL2-CAG) from the strand antisense to JPH3, which encodes an expanded polyglutamine (polyQ) protein. Importantly, BAC-HDL2 mice, but not control BAC mice, accumulate polyQ-containing NIs in a pattern strikingly similar to those in the patients. Furthermore, BAC mice with genetic silencing of the expanded CUG transcript still express HDL2-CAG transcript and manifest polyQ pathogenesis. Finally, studies of HDL2 mice and patients revealed CBP sequestration into NIs and evidence for interference of CBP-mediated transcriptional activation. These results suggest overlapping polyQ-mediated pathogenic mechanisms in HD and HDL2.

Identification of EMS-Induced Causal Mutations in a Non-Reference Arabidopsis thaliana Accession by Whole Genome Sequencing

The most frequently used method to identify mutations induced by a commonly used mutagen, EMS (ethyl methane sulfonate), in Arabidopsis thaliana has been map-based cloning. The first step of this method is crossing a mutant with a plant of another accession as it requires polymorphisms between accessions for linkage analysis. Therefore, to perform the method routinely, it is greatly preferred to use accession combinations between which enough polymorphisms are already known. Further, it requires laborious examination of a large number of F₂ recombinants using many markers to detect each polymorphism. After linkage analysis narrows down the chromosomal region containing the causal mutation, sequencing candidate genes one by one within the region is necessary until the mutation is finally identified. Overall, this method is generally time-consuming and labor intensive, and it becomes harder when multiple loci are involved in phenotypes. A few recent reports showed that causal mutations induced by EMS could be identified by deep-sequencing technologies with less labor compared with the conventional method when mutants were generated in the Arabidopsis reference Columbia background whose genome organization is well known. Here we report that we succeeded in rapid identification of EMS-induced causal mutations in a non-reference accession background, whose whole genome sequence is not publicly available, using one round of whole genome sequencing. Moreover, in our case, we could monitor the causal locus and the transgenic reporter locus simultaneously, implying that this methodology could theoretically be applicable to analyzing even complex traits. We describe the pipeline of this methodology and discuss its characteristics.

Improved FLP Recombinase, FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–lox Mediated Site-Specific Gene Integration in Rice

Site-specific recombination systems, such as FLP-FRT and Cre-lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP-FRT system for excising marker gene from the transgene locus developed by Cre-lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP-FRT-based biotechnology applications in plants.

Langerhans cells are not required for graft-versus-host disease

AbstractGraft-versus-host disease (GVHD) is initiated and maintained by antigen-presenting cells (APCs) that prime alloreactive donor T cells. APCs are therefore attractive targets for GVHD prevention and treatment. APCs are diverse in phenotype and function, making understanding how APC subsets contribute to GVHD necessary for the development of APC-targeted therapies. Langerhans cells (LCs) have been shown to be sufficient to initiate skin GVHD in a major histocompatibility complex–mismatched model; however, their role when other host APC subsets are intact is unknown. To address this question, we used mice genetically engineered to be deficient in LCs by virtue of expression of diphtheria toxin A under the control of a BAC (bacterial artificial chromosome) transgenic hu-man Langerin locus. Neither CD8- nor CD4-mediated GVHD was diminished in recipients lacking LCs. Similarly, CD8- and CD4-mediated GVHD, including that in the skin, was unaffected if bone marrow came from donors that could not generate LCs, even though donor LCs engrafted in control mice. Engraftment of donor LCs after irradiation in wild-type hosts required donor T cells, with immunofluorescence revealing patches of donor and residual host LCs. Surprisingly, donor LC engraftment in Langerin-diphtheria toxin A (DTA) transgenic hosts was independent of donor T cells, suggesting that a Langerin+ cell regulates repopulation of the LC compartment.

Interrogating the transgenic genome: development of an interspecies tiling array

A single expressing copy of the human protamine domain was randomly inserted into an intron of Cyp2c38. The transgenic locus was shown to recapitulate the level of expression observed in normal human testis while not perturbing endogenous protamine expression. The development of an interspecies tiling array was pursued to enable direct comparison of the orthologous protamine domains in a single experiment. Probe design was adapted to generate species-specific high resolution probe sets that would tolerate repetitive elements. Results from competitive hybridizations demonstrate that interspecies tiling arrays are a valuable tool for parallel analysis of highly similar DNA sequences. This approach provides a rapid and reliable means of interrogating samples prior to deep sequencing analysis. These arrays should readily compliment most DNA isolation and analysis techniques such as ChIP, nuclease sensitivity and nuclear matrix association assays.

Real-time PCR for the detection of precise transgene copy number in durum wheat.

Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis

Telomerase is tightly regulated in humans relative to mice, owing to the differential regulation of TERT genes. To explore hTERT regulation in vivo, we engineered mice with a 160-kb transgenic bacterial artificial chromosome (BAC) spanning the hTERT locus with a Renilla luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter locus reproduced that of the native hTERT gene in all tissues and organs examined, demonstrating that genetic sequence determined the species-specific developmental regulation of the hTERT gene and that mouse epigenetic and transcription machineries faithfully regulated hTERT transcription. Thus, these mice allowed detailed analyses of developmental hTERT regulation. Both the transgenic hTERT reporter and the endogenous mTERT locus were expressed in early embryonic stages, and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT transcription was much lower than mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. In testis, the Rluc mRNA was enriched in elongating spermatids of seminiferous tubules. In addition, the transcription of transgenic hTERT reporter, but surprisingly not the endogenous mTERT gene, was activated during Wnt1-induced mammary tumorigenesis, allowing the monitoring of tumor development via noninvasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT transgenic reporter system recapitulates the developmental regulation of the hTERT gene in a chromosomal position-independent manner and serves as a legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo.

Inhibiting Virus Infection by RNA Interference of the Eight Functional Genes of the Potato Virus Y Genome

AbstractThe genome of Potato virus Y is a single‐stranded, positive‐sense RNA molecule that encodes a single large polypeptide that is cleaved by self‐encoded proteases into 10 functional proteins. Using specific primers designed based on the cloned genome sequence of the necrotic strain of Potato virus Y (PVYN), we amplified 400 bp and 500 bp cDNA fragments from the 3′ ends of P1, HC‐Pro, P3, CI, Vpg, NIa, NIb and CP genes. These cDNA fragments were then inserted into the binary vector pROKII to construct the vectors pROK‐P1, pROK‐HC‐Pro, pROK‐P3, pROK‐CI, pROK‐NIa‐Vpg, pROK‐NIa‐Pro, pROK‐NIb and pROK‐CP, all of which contained hairpin RNAs (hpRNAs). These recombinant binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404 and then into Nicotiana tabacum L. var. NC89 plants, respectively. Virus challenge inoculation indicated that the plants transformed with each construct were resistant to PVY infection, and the percentage of resistant plants obtained ranged from 33–64%. Northern blot showed an inverse correlation between transgenic transcript accumulation and virus resistance. siRNAs could be detected in all the resistant plants. We also studied the relationship between the secondary structure and the gene‐silencing efficiency and found a positive correlation between local free energies (ΔGloc) and the virus‐resistance ratio. For each construct, one line of progeny T1 was randomly selected to analyse the inheritance of the transgene and the resistance. All transgenic single locus lines were completely resistant, and this resistance could be stably inherited.

Establishment of homozygous transgenic rice lines after elimination of unlinked albino traits from transgenic events through segregation

In an analysis of 339 independent T 0 transgenic rice lines generated by Agrobacterium-mediated transformation, albino plants appeared in the T 1 generation in two single-copy transgenic lines, O54 and O36 and in one double-copy transgenic line, C18. While the T 0 plants of these three lines were green, albino and green plants emerged in a 1:3 ratio in the T 1 generation. The albino phenotype segregated as a monogenic recessive trait. Southern blot analysis of the green and albino plants in the T 1 generation confirmed that the albino trait and the T-DNA insertion events were unlinked. Segregation of the albino trait from the transgenic trait in the lines O54 and O36 was confirmed in T 2 and T 3 generations, respectively. Homozygous transgenic plants free from the albino trait were also identified. In the double-copy transgenic line C18, we genetically separated the two transgenic loci, out-segregated the albino locus from both transgene loci, and identified homozygous plants for each of the transgenic events by Southern blot analysis in the T 1 generation itself. Thus, we demonstrate that when an albino trait appears in the T 1 generation and is unlinked to a transgene locus, the albino locus can be segregated from the transgene locus and homozygous transgenic lines free from albinos can be established.

Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis

RNA silencing in plants and some animals has a non-cell-autonomous effect due to an RNA signal that moves between cells or organs. To identify unique factors involved in this process, we analyzed a group of Arabidopsis mutants with defective spread of RNA silencing from a transgene expressed specifically in the phloem. These mutants accumulated reduced amounts of small interfering (si)RNA from the transgene locus and from endogenous loci TAS1, TAS2, and an inverted repeat locus IR71. The defect in TAS1 and TAS2 siRNA biogenesis is in the processing of a long siRNA precursor. We mapped the mutations to a gene encoding the Arabidopsis homolog of a protein, TEX1, which is involved in intracellular transport of RNA in animals. TEX1 is a component of the THO/TREX complex, and we show that the Arabidopsis TEX1 interacts with other predicted components of a THO/TREX complex. Correspondingly, we found at least two other components of the Arabidopsis THO core complex that are involved in RNA silencing. To reconcile the effect of these mutations on transgene and endogenous gene siRNA, we propose a mechanism in which THO/TREX processes or transports a long RNA molecule so that it can be a template for secondary siRNA production.

Molecular characterization of T-DNA integration sites in transgenic birch

The integration and structure of a transgene locus can have profound effects on the level and stability of transgene expression. We screened 28 transgenic birch (Betula platyphylla Suk.) lines transformed with an insect-resistance gene (bgt) using Agrobacterium tumefaciens. Among the transgenic plants, the copy number of transgene varied from one to four. A rearrangement or partial deletion had occurred in the process of T-DNA integration. T-DNA repeat formation, detected by reverse primer PCR, was found among randomly screened transgenic lines. Sequencing of the junctions between the T-DNA inserts revealed deletions of 19–589 bp and an additional 45 bp filler DNA sequence was inserted between the T-DNA repeats at one junction. Micro-homologous sequences (1–6 bp) were observed in the junctions between the T-DNA inserts. Using SiteFinding-PCR, a relatively high percentage of AT value was found for the flanking regions. Deletion of the right border repeat was observed in 12/18 of the T-DNA/plant junctions analyzed. The number of nucleotides deleted varied from 3 to 712. Deletions of 17–89 bp were observed in all left T-DNA/plant junctions analyzed. A vector backbone DNA sequence in the transgene loci was also detected using primer pairs outside the left and right T-DNA borders. Approximately 89.3% of the lines contained some vector backbone DNA. These observations revealed that it is important to check the specificity of the integration. A mechanism of T-DNA transport and integration is proposed for this long-lived tree species.

An Intron Sense Suppression Construct Targeting Soybean FAD2-1 Requires a Double-Stranded RNA-Producing Inverted Repeat T-DNA Insert

We demonstrate that the transformation of soybean (Glycine max) with sense suppression constructs using intron sequences from the fatty acid oleyl Delta12 desaturase gene FAD2-1A leads to efficient and specific reduction of FAD2-1 transcripts in developing seeds, increased oleic acid, and decreased polyunsaturated fatty acids. The related FAD2-2 transcripts are only marginally affected. Despite screening a large number of independent transformants, no single-copy efficacious transformants could be found. Invariably, all the least complex transgenic loci have two T-DNA copies in an inverted repeat configuration, centered at the right borders. We show that this T-DNA configuration produces an inverted repeat transcript and that small interfering RNAs accumulate against the target sequence.