Single-copy Transgene Research Articles

A site-specific, single-copy transgenesis strategy to identify 5' regulatory sequences of the mouse testis-determining gene Sry.

The Y-chromosomal gene SRY acts as the primary trigger for male sex determination in mammalian embryos. Correct regulation of SRY is critical: aberrant timing or level of Sry expression is known to disrupt testis development in mice and we hypothesize that mutations that affect regulation of human SRY may account for some of the many cases of XY gonadal dysgenesis that currently remain unexplained. However, the cis-sequences involved in regulation of Sry have not been identified, precluding a test of this hypothesis. Here, we used a transgenic mouse approach aimed at identifying mouse Sry 5′ flanking regulatory sequences within 8 kb of the Sry transcription start site (TSS). To avoid problems associated with conventional pronuclear injection of transgenes, we used a published strategy designed to yield single-copy transgene integration at a defined, transcriptionally open, autosomal locus, Col1a1. None of the Sry transgenes tested was expressed at levels compatible with activation of Sox9 or XX sex reversal. Our findings indicate either that the Col1a1 locus does not provide an appropriate context for the correct expression of Sry transgenes, or that the cis-sequences required for Sry expression in the developing gonads lie beyond 8 kb 5′ of the TSS.

Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster

The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters.

Development of pronuclear injection-based targeted transgenesis in mice through Cre-loxP site-specific recombination.

Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre-loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.

Abstract 4713: A Kampo (Japanese herbal) medicine Juzentaihoto may suppress the expression of indoleamine 2, 3-dioxygenase on macrophages and prolong the survival of transgenic mice bearing lens-epithelial cell carcinoma.

Abstract Kampo medicine (the traditional Chinese medicine) is a pharmaceutical grade multi-herbal medicine widely used for immunocompromized patient in Japan. Juzentaihoto is a well known Kampo medicine that comprises 10 different herbs. The effect of a Kampo medicine Juzentaihoto on survival of tumor-bearing mice was measured in this study. We previously reported αT3 transgenic mice carrying αA-SV40Tag transgene (αA denotes murine αA-crystallin promoter). The αT3 mice carry a single copy of transgene, which have been generated from αA-loxP-SV40Tag/Ella-cre double transgenics. The αT3 lens tumors show multi-step carcinogenesis(J Cancer Res Clin Oncol 135:1521-1532 2009). At birth the αT3 lens tumors demonstrate moderate∼severe dysplasia, and in 2-3 month-old they become carcinoma in situ and start to invade lens capsule, and in 4-6 month-old they proliferate in the eyeball and finally they enlarge and destroy eyeballs. The αT3 lens tumor is growing very slow and finally invading and destroying surrounding brain tissue, and some of the αT3 lens tumor, metastasizing to cervical lymph node and lung. Finally αT3 mice died due to cancer cachexia. Carcinogenesis of the αT3 lens tumor induced by SV40 T antigens is pathologically very similar to uterine cervical cancer, which is induced by high-risk human papillomavirus E6 and E7 oncoprotein(Cancer Lett 179:165-173 2002, Oncol Rep 12:253-258 2004, Oncogene 25:3670-3679 2006). We also reported that expression of indoleamine 2, 3-dioxygenase(IDO)increases as tumors progress from carcinoma in situ to microinvasive and invasive cancer, and that tumor associated macrophages(TAMs) localized at the invasive front express IDO in patients with cervical cancer(Cancer Sci 98:874-881 2007). In this study, the αT3 mice were fed bait containing 1.5% Juzentaihoto for more than one year and the survival rate was compared with the untreated control. We also analyzed various biological activities, such as phagocytosis, IL-10 and IL-12 induction, especially IDO-expression on macrophages. Oral administration of Juzentaihoto activated peritoneal exudate macrophages and recruited the IDO-negative macrophages. The number of TAMs also increased as compared with the untreated control. These results suggest that oral administration of Juzentaihoto affect macrophage expression of IDO, possibly through a mechanism mediated by the activation of M1 macrophages and modulate local innate immunity. Citation Format: Takafumi Nakamura. A Kampo (Japanese herbal) medicine Juzentaihoto may suppress the expression of indoleamine 2, 3-dioxygenase on macrophages and prolong the survival of transgenic mice bearing lens-epithelial cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4713. doi:10.1158/1538-7445.AM2013-4713

Improvement of pronuclear injection-based targeted transgenesis (PITT) by iCre mRNA-mediated site-specific recombination

We recently reported a novel transgenic (Tg) technique named pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010; Ohtsuka et al. 2012a). The PITT technique enables targeted integration of a single-copy transgene into a specified locus by Cre-loxP mediated recombination and it guarantees faithful and reproducible transgene expression. As described in the first report, the PITT technique requires co-injection of a Cre expression plasmid and a donor vector containing loxP sequences into pronuclei of zygotes obtained from the seed mice that carry loxP sequences at the Rosa26 or H2-Tw3 loci. This method has a (targeted) integration efficiency of 26/585 (4.4 %; 95 % CI 2.9–6.4 %) (Ohtsuka et al. 2012b). Although PITT has a lower transgenic rate (4.4 %) when compared to traditional transgenesis (*10–20 % (Fielder and Montoliu 2011)), it is superior to the latter method since just obtaining one transgenic founder is sufficient in case of PITT. With an aim to improve the PITT technique further we attempted to co-inject iCre mRNA [codon improved Cre recombinase; (Shimshek et al. 2002)] into zygotes with the expectation that the Cre protein would be available more readily in the zygotes from injected iCre mRNA compared to when the Creexpression plasmid DNA was co-injected. We coinjected 10 ng/ll of pAOM donor vector (Ohtsuka et al. 2010) along with various concentrations of iCre mRNA (1–45 ng/ll) into zygotes obtained from the Electronic supplementary material The online version of this article (doi:10.1007/s11248-013-9703-x) contains supplementary material, which is available to authorized users.

Long-term assessment of transgene behavior in barley: Ds-mediated delivery of bar results in robust, stable, and heritable expression

The utility of transgenic plants for both experimental and practical agronomic purposes is highly dependent on stable, predictable, and heritable expression of the introduced genes. This requirement is frequently unfulfilled, and transgenes often are completely silenced. Studies of transgenic loci have shown that rearrangements of transgenes occur during the integration process, some of which are potent cues that induce silencing. Conversely, intact, single-copy transgenes produced via transposon-mediated gene delivery have shown relatively stable expression, at least in early-generation progeny. To examine the long-term expression stability of a bar expression cassette delivered via Dissociation (Ds)-mediated transposition, we examined qualitative and quantitative expression in barley (Hordeum vulgare L.) populations developed for transposon tagging. Qualitative assessments of herbicide resistance among 106 lines showed bar expression to be stable for at least five generations of advance via self-pollination. Similarly, qualitatively stable expression was observed among 31 near-isogenic lines derived from at least seven backcrosses to the cultivar Garnet. Quantitative RT-PCR measurements of bar expression were conducted for eight near-isogenic lines and their donor parents. The expression of bar was highly correlated in parent and progeny near-isogenic lines, showing high heritability of bar expression. These data demonstrate stable, predictable transgene expression following Ds-mediated delivery.

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri TL-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.

Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase

Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.

Extremely stable Piwi-induced gene silencing inCaenorhabditis elegans

In recent years, the Piwi pathway has been shown to regulate the silencing of mobile genetic elements. However, we know little about how Piwi pathways impose silencing and even less about trans-generational stability of Piwi-induced silencing. We demonstrate that the Caenorhabditis elegans Piwi protein PRG-1 can initiate an extremely stable form of gene silencing on a transgenic, single-copy target. This type of silencing is faithfully maintained over tens of generations in the absence of a functional Piwi pathway. Interestingly, RNAi can also trigger permanent gene silencing of a single-copy transgene and the phenomenon will be collectively referred to as RNA-induced epigenetic silencing (RNAe). RNAe can act in trans and is dependent on endogenous RNAi factors. The involvement of factors known to act in nuclear RNAi and the fact that RNAe is accompanied by repressive chromatin marks indicate that RNAe includes a transcriptional silencing component. Our results demonstrate that, at least in C. elegans, the Piwi pathway can impose a state of gene silencing that borders on 'permanently silent'. Such a property may be more widely conserved among Piwi pathways in different animals.

PiRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline

Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.

A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists because monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particle-mediated transformation, result in poor transgene expression because of integration of truncated DNA molecules. We developed a method of transgene delivery into monocots. This method relies on the use of an in vitro-prepared nano-complex consisting of transferred DNA, virulence protein D2, and recombination protein A delivered to triticale microspores with the help of a Tat2 cell-penetrating peptide. We showed that this approach allowed for single transgene copy integration events and prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with the full transferred DNA/protein complex. This approach can easily substitute the bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters.

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.

PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.

Targeted insertion of two Mthfr promoters in mice reveals temporal- and tissue-specific regulation

Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, synthesizes 5-methyltetrahydrofolate, the main circulatory form of folate which is required for maintaining nontoxic levels of homocysteine and providing one-carbon units for methylation. A common 677C→T variant in MTHFR confers mild MTHFR deficiency and has been associated with a number of human disorders, including neural tube defects and vascular disease. Two promoters of Mthfr, designated as upstream and downstream promoters, are located upstream of a transcription start site cluster and have previously demonstrated cell-specific activities. In this study we used a unique approach for targeted, single-copy transgene insertion to generate transgenic mice carrying a Mthfr upstream or Mthfr downstream promoter-reporter construct located 5' to the endogenous Hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The Mthfr downstream promoter demonstrated activity in the neural tube, neural crest cells, dorsal root ganglia, heart, and endothelial cells of blood vessels in 10.5-days post coitum embryos and placentas. Upstream promoter activity was absent at this developmental stage. Postnatally, both promoters demonstrated activity in the brain stem, hippocampus, and thalamus of 1-week-old brain that became stronger in the adult. The Mthfr upstream promoter also showed activity in the cerebellum and cerebral cortex. Both promoters were active in male reproductive tissues, including 1-week-old epididymides, and there was upstream promoter-specific activity in the adult testis. Our investigation of Mthfr regulation in an in vivo mouse model revealed temporal- and tissue-specific regulation that supports important roles for MTHFR in the developing embryo, and in postnatal brain and male reproductive tissues.

Engineering the embryo

Generation of transgenic mice by pronuclear DNA injection into fertilized eggs has been well-established for over 30 y (1). However, it remains a rather crude approach: injected sequences randomly insert as concatamers, they can be mutagenic, and they have variable expression depending on the site of integration, a phenomenon called position effects. In PNAS, Tasic et al. (2) report that pronuclear injection of PhiC31 integrase can catalyze site-specific integration of single-copy transgenes in up to 40% of live pups. This approach can reduce variability because of position effects and provide improved control over transgene expression. This and other recent reports of site-specific integration in zygotes (2–6) reveal additional possibilities for targeted transgenesis in mice and species where ES cells are not available.

Efficient screening of transgenic plant lines for ecological research

Plants stably transformed to manipulate the expression of genes mediating ecological performance have profoundly altered research in plant ecology. Agrobacterium-mediated transformation remains the most effective method of creating plants harbouring a limited number of transgene integrations of low complexity. For ecological/physiological research, the following requirements must be met: (i) the regenerated plants should have the same ploidy level as the corresponding wild-type plant and (ii) contain a single transgene copy in a homozygous state; (iii) the T-DNA must be completely inserted without vector backbone sequence and all its elements functional; and (iv) the integration should not change the phenotype of the plant by interrupting chromosomal genes or by mutations occurring during the regeneration procedure. The screening process to obtain transformed plants that meet the above criteria is costly and time-consuming, and an optimized screening procedure is presented. We developed a flow chart that optimizes the screening process to efficiently select transformed plants for ecological research. It consists of segregational analyses, which select transgenic T₁ and T₂ generation plants with single T-DNA copies that are homozygous. Indispensable molecular genetic tests (flow cytometry, diagnostic PCRs and Southern blotting) are performed at the earliest and most effective times in the screening process. qPCR to quantify changes in transcript accumulation to confirm gene silencing or overexpression is the last step in the selection process. Because we routinely transform the wild tobacco, Nicotiana attenuata, with constructs that silence or ectopically overexpress ecologically relevant genes, the proposed protocol is supported by examples from this system.

Site-specific integrase-mediated transgenesis in mice via pronuclear injection

Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system

Transgene escape, a major environmental and regulatory concern in transgenic crop cultivation, could be alleviated by removing transgenes from pollen, the most frequent vector for transgene flow. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). CinH recombinase recognized 119bp of nucleic acid sequences, RS2, in pollen and excised the transgene flanked by the RS2 sites. In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment.

Overexpression of Sinapine Esterase BnSCE3 in Oilseed Rape Seeds Triggers Global Changes in Seed Metabolism

Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism.