A set of modular binary vectors for transformation of cereals

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.     

Multi-Locus Assortment (MLA) for Transgene Dispersal and Elimination in Mosquito Populations

Background

Replacement of wild-type mosquito populations with genetically modified versions is being explored as a potential strategy to control vector-borne diseases. Due to lower expected relative fitness of transgenic individuals, transgenes must be driven into populations for these scenarios to be successful. Several gene drive mechanisms exist in a theoretical sense but none are currently workable in mosquitoes. Even if strategies were workable, it would be very difficult to recall released transgenes in the event of unforeseen consequences. What is needed is a way to test transgenes in the field for feasibility, efficacy and safety prior to releasing an active drive mechanism.

Methodology/Principal Findings

We outline a method, termed Multi-locus assortment (MLA), to spread transgenes into vector populations by the release of genetically-modified mosquitoes carrying multiple stable transgene inserts. Simulations indicate that [1] insects do not have to carry transgenes at more than 4 loci, [2] transgenes can be maintained at high levels by sequential small releases, the frequency of which depends on the construct fitness cost, and [3] in the case of unforeseen negative non-target effects, transgenes can be eliminated from the population by halting transgenic releases and/or mass releases of wild-type insects. We also discuss potential methods to create MLA mosquito strains in the laboratory.

Conclusions/Significance

While not as efficient as active drive mechanisms, MLA has other advantages: [1] MLA strains can be constructed for some mosquito species with currently-available technology, [2] MLA will allow the ecological components of transgenic mosquito releases to be tested before actual gene drive mechanisms are ready to be deployed, [3] since MLA is not self-propagating, the risk of an accidental premature release into nature is minimized, and [4] in the case that active gene drive mechanisms prove impossible to develop, the MLA approach can be used as a back-up transgene dispersal mechanism for disease control efforts in some systems.     

Integration and inheritance of transgenes in crop plants and trees

Transgene integration and inheritance have been investigated in a number of crop plants and few tree species. Transgene integration is predominantly a random process, whether mediated by Agrobacterium or particle bombardment. Depending on the genomic position of the integrated transgene and structure of the integration site as well as copy number of the transgene in the genome, its expression may be stable or variable. Therefore, integration patterns would affect the mode of transgene inheritance in plants, regardless of the method of gene transfer. So far, both Mendelian and non-Mendelian inheritance of transgenes has been reported across several generations (T1–T3) of crop plants. In few tree species (apple, poplar, plum, and American chestnut), mostly Mendelian inheritance of the transgenes has been observed in the T1 or BC1 generations. However, detailed studies in the transgenic papaya trees showed Mendelian segregation of the transgene in the T1 generation but non-Mendelian inheritance in the T2 generation. Variation in transgene inheritance was also detected in transgenic apple and plum trees. Long generation cycles in many economically important tree species preclude investigation of inheritance of transgenes in the tree progeny. Production of early flowering trees, either by genetic modification or by environmental modulation, would facilitate the study of transgene inheritance across generations of transgenic trees. In order to overcome problems of randomness of transgene integration, targeted transgene insertions by homologous or site-specific recombination or by designer recombinases or nucleases offer prospects for stable integration of transgenes in predetermined locations in the plant genome. And perhaps, that might provide a platform for stable expression and Mendelian inheritance of transgenes in plants.     

Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.     

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Mouse telokin and SM22 promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22 promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22 transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22 transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells. visceral smooth muscle; development; myosin light chain kinase; embryos; CArG element     

Prospects and implications of using chromatin insulators in gene therapy and transgenesis

Gene therapy has emerged from the idea of inserting a wild-type copy of a gene in order to restore the proper expression and function of a damaged gene. Initial efforts have focused on finding the proper vector and delivery method to introduce a corrected gene to the affected tissue or cell type. Even though these first attempts are clearly promising, several problems remain unsolved. A major problem is the influence of chromatin structure on transgene expression. To overcome chromatin-dependent repressive transgenic states, researchers have begun to use chromatin regulatory elements to drive transgene expression. Insulators or chromatin boundaries are able to protect a transgene against chromatin position effects at their genomic integration sites, and they are able to maintain transgene expression for long periods of time. Therefore, these elements may be very useful tools in gene therapy applications for ensuring high-level and stable expression of transgenes.     

New Genes in Traditional Seed Systems: Diffusion,Detectability and Persistence of Transgenes in a Maize Metapopulation

Gene flow of transgenes into non-target populations is an important biosafety concern. The case of genetically modified (GM) maize in Mexico has been of particular interest because of the country’s status as center of origin and landrace diversity. In contrast to maize in the U.S. and Europe, Mexican landraces form part of an evolving metapopulation in which new genes are subject to evolutionary processes of drift, gene flow and selection. Although these processes are affected by seed management and particularly seed flow, there has been little study into the population genetics of transgenes under traditional seed management. Here, we combine recently compiled data on seed management practices with a spatially explicit population genetic model to evaluate the importance of seed flow as a determinant of the long-term fate of transgenes in traditional seed systems. Seed flow between farmers leads to a much wider diffusion of transgenes than expected by pollen movement alone, but a predominance of seed replacement over seed mixing lowers the probability of detection due to a relative lack of homogenization in spatial frequencies. We find that in spite of the spatial complexities of the modeled system, persistence probabilities under positive selection are estimated quite well by existing theory. Our results have important implications concerning the feasibility of long term transgene monitoring and control in traditional seed systems.     

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

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

Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either the selectable marker gene or of all introduced transgenes during microsporogenesis. This way, transgene removal becomes an integral part of the biology of pollen maturation, not requiring any external stimulus such as chemical induction by spraying. We here show the feasibility of engineering transgenic plants to produce pollen devoid of any transgene. Highly efficient excision of transgenes from tobacco pollen was achieved with a potential failure rate of at most two out of 16 800 seeds (0.024%). No evidence for either premature activation or absence of activation of the recombinase system was observed under stress conditions in the laboratory. This approach can prevent adventitious presence of transgenes in non-GM crops or related wild species by gene flow. Such biological containment may help the deployment and management of coexistence practices to support consumer choice and will promote clean molecular farming for the production of high-value compounds 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.     

Introducing desirable transgenes into insect populations using Y-linked meiotic drive - a theoretical assessment

The use of genetic drive mechanisms to replace native mosquito genotypes with individuals bearing antipathogen transgenes is a potential strategy for repressing insect transmission of human diseases such as malaria and dengue. Antipathogen transgenes have been developed and tested, but efficient gene drive mechanisms are lacking. Here we theoretically assess the feasibility of introducing antipathogen genes into wild Aedes aegypti populations by using a naturally occurring meiotic drive system. We consider the release of males having both a Y-linked meiotic drive gene and an X-linked drive-insensitive response allele to which an antipathogen gene is linked. We use mathematical models and computer simulations to determine how the post-introduction dynamics of the antipathogen gene are affected by specific genetic characteristics of the system. The results show that when the natural population is uniformly sensitive to the meiotic drive gene, the antipathogen gene may be driven close to fixation if the fitness costs of the drive gene, the insensitive response allele, and the antipathogen gene are low. However, when the natural population has a small proportion of an X-linked insensitive response allele or an autosomal gene that strongly reduces the effect of the drive gene, the antipathogen gene does not spread if it has an associated fitness cost. Our modeling results provide a theoretical foundation for further experimental tests.