Transgene Fragments Research Articles

Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice.

Genomic imprinting at the mouse Igf2/H19 locus is controlled by the H19 ICR, within which paternal allele-specific DNA methylation originating in sperm is maintained throughout development in offspring. We previously found that a 2.9 kb transgenic H19 ICR fragment in mice can be methylated de novo after fertilization only when paternally inherited, despite its unmethylated state in sperm. When the 118 bp sequence responsible for this methylation in transgenic mice was deleted from the endogenous H19 ICR, the methylation level of its paternal allele was significantly reduced after fertilization, suggesting the activity involving this 118 bp sequence is required for methylation maintenance at the endogenous locus. Here, we determined protein binding to the 118 bp sequence using an in vitro binding assay and inferred the binding motif to be RCTG by using a series of mutant competitors. Furthermore, we generated H19 ICR transgenic mice with a 5-bp substitution mutation that disrupts the RCTG motifs within the 118 bp sequence, and observed loss of methylation from the paternally inherited transgene. These results indicate that imprinted methylation of the H19 ICR established de novo during the post-fertilization period involves binding of specific factors to distinct sequence motifs within the 118 bp sequence.

The transgenic IG-DMR sequence of the mouse Dlk1-Dio3 domain acquired imprinted DNA methylation during the post-fertilization period

BackgroundAllele-specific methylation of the imprinting control region (ICR) is the molecular basis for the genomic imprinting phenomenon that is unique to placental mammals. We previously showed that the ICR at the mouse H19 gene locus (H19 ICR) was unexpectedly established after fertilization and not during spermatogenesis in transgenic mice (TgM), and that the same activity was essential for the maintenance of paternal methylation of the H19 ICR at the endogenous locus in pre-implantation embryos. To examine the universality of post-fertilization imprinted methylation across animal species or imprinted loci, we generated TgM with two additional sequences.ResultsThe rat H19 ICR, which is very similar in structure to the mouse H19 ICR, unexpectedly did not acquire imprinted methylation even after fertilization, suggesting a lack of essential sequences in the transgene fragment. In contrast, the mouse IG-DMR, the methylation of which is acquired during spermatogenesis at the endogenous locus, did not acquire methylation in the sperm of TgM, yet became highly methylated in blastocysts after fertilization, but only when the transgene was paternally inherited. Since these two sequences were evaluated at the same genomic site by employing the transgene co-placement strategy, it is likely that the phenotype reflects the intrinsic activity of these fragments rather than position-effect variegation.ConclusionsOur results suggested that post-fertilization imprinted methylation is a versatile mechanism for protecting paternal imprinted methylation from reprogramming during the pre-implantation period.

Improvement of Bacterial Blight Resistance in Two Conventionally Cultivated Rice Varieties by Editing the Noncoding Region.

xa13 is a recessive pleiotropic gene that positively regulates rice disease resistance and negatively regulates rice fertility; thus, seriously restricting its rice breeding application. In this study, CRISPR/Cas9 gene-editing technology was used to delete the Xa13 gene promoter partial sequence, including the pathogenic bacteria-inducible expression element. Rice with the edited promoter region lost the ability for pathogen-induced gene expression without affecting background gene expression in leaves and anthers, resulting in disease resistance and normal yield. The study also screened a family of disease-resistant and normal fertile plants in which the target sequence was deleted and the exogenous transgene fragment isolated in the T1 generation (transgene-free line). Important agronomic traits of the T2 generation rice were examined. T2 generation rice with/without exogenous DNA showed no statistical differences compared to the wild type in heading stage, plant height, panicles per plant, panicle length, or seed setting rate in the field. Two important conventional rice varieties, namely Kongyu131 (KY131, Geng/japonica) and Huanghuazhan (HHZ, Xian/indica), were successfully transformed, and disease-resistant and fertile materials were obtained. Currently, these are the two important conventional rice varieties in China that can be used directly for production after improvement. Expression of the Xa13 gene in the leaves of transgenic rice (KY-PD and HHZ-PD) was not induced after pathogen infection, indicating that this method can be used universally and effectively to promote the practical application of xa13, a recessive disease-resistant pleiotropic gene, for rice bacterial blight resistance. Our study on the regulation of gene expression by editing noncoding regions of the genes provides a new idea for the development of molecular design breeding in the future.

Temporal Phenotypic Changes in Huntington's Disease Models for Preclinical Studies.

Background:Mouse models bearing genetic disease mutations are instrumental in the development of therapies for genetic disorders. Huntington’s disease (HD) is a late-onset lethal dominant genetic disorder due to a CAG repeat within exon 1 of the Huntingtin (Htt) gene. Several mice were developed to model HD through the expression of a transgenic fragment (exon 1 of the human HTT), the knock-in mutation of the CAG repeat in the context of the mouse Htt gene, or the full-length HTT human gene. The different mouse models present distinct onset, symptoms, and progression of the disease.Objective:The objective of this study is to advise on the best behavioral tests to assess disease progression in three HD mouse models.Methods:We tested N171-82Q transgenic mice, zQ175 knock-in mice, and BACHD full-length mice in a comprehensive behavior test battery in early, mid-, and late disease stages.Results:We contrast and compare the models and the emerging phenotypes with the available literature. These results suggest the most effective behavioral tests and appropriate sample sizes to detect treatment efficacy in each model at the different ages. We provide options for early detection of motor deficits while minimizing testing time and training.Conclusion:This information will inform researchers in the HD field as to which mouse model, tests and sample sizes can accurately and sensitively detect treatment efficacy in preclinical HD research.

Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice.

BackgroundGene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV<hFST>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<hFST>.MethodsFirst, we generated rAdV<hFST> and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<hFST> was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing.ResultsThe expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest.ConclusionsThese results indicate the possibility of detecting gene doping using rAdV<hFST> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<hFST>.

A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging.

The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.

Transgenes of genetically modified animals detected non-invasively via environmental DNA.

We demonstrate that simple, non-invasive environmental DNA (eDNA) methods can detect transgenes of genetically modified (GM) animals from terrestrial and aquatic sources in invertebrate and vertebrate systems. We detected transgenic fragments between 82–234 bp through targeted PCR amplification of environmental DNA extracted from food media of GM fruit flies (Drosophila melanogaster), feces, urine, and saliva of GM laboratory mice (Mus musculus), and aquarium water of GM tetra fish (Gymnocorymbus ternetzi). With rapidly growing accessibility of genome-editing technologies such as CRISPR, the prevalence and diversity of GM animals will increase dramatically. GM animals have already been released into the wild with more releases planned in the future. eDNA methods have the potential to address the critical need for sensitive, accurate, and cost-effective detection and monitoring of GM animals and their transgenes in nature.

Novel tool to quantify with single-cell resolution the number of incoming AAV genomes co-expressed in the mouse nervous system

Adeno-associated viral (AAV) vectors are an established and safe gene delivery tool to target the nervous system. However, the payload capacity of <4.9 kb limits the transfer of large or multiple genes. Oversized payloads could be delivered by fragmenting the transgenes into separate AAV capsids that are then mixed. This strategy could increase the AAV cargo capacity to treat monogenic, polygenic diseases and comorbidities only if controlled co-expression of multiple AAV capsids is achieved on each transduced cell. We developed a tool to quantify the number of incoming AAV genomes that are co-expressed in the nervous system with single-cell resolution. By using an isogenic mix of three AAVs each expressing single fluorescent reporters, we determined that expression of much greater than 31 AAV genomes per neuron in vitro and 20 genomes per neuron in vivo is obtained across different brain regions including anterior cingulate, prefrontal, somatomotor and somatosensory cortex areas, and cerebellar lobule VI. Our results demonstrate that multiple AAV vectors containing different transgenes or transgene fragments, can efficiently co-express in the same neuron. This tool can be used to design and improve AAV-based interrogation of neuronal circuits, map brain connectivity, and treat genetic diseases affecting the nervous system.

Impact of Bioflavonoid Exposure on DNA Damage Response and of Non-Homologous End Joining (NHEJ) Pathway Repair Proteins in Stem Cells

DNA double-strand breaks (DSBs) activate DNA damage response signaling pathways and multiple repair pathways depending on cell cycle stage and differentiation state. An array of recurrent, non-random chromosomal translocations are associated with hematologic malignancies, and we are interested in determining the role of specific DNA repair proteins or pathway choice in the etiology of these rearrangements. Classic non-homologous end joining (NHEJ) utilizes LIG4, XRCC4, and XLF during the final steps; LIG4 and XRCC4 deficiency is implicated in both leukemais and lymphomas but the role of XLF deficiency in leukemia etiology is not clear. Activation of alternative NHEJ (Alt-NHEJ) is largely dependent on CtIP that promotes the resection of DNA ends to uncover micro-homologies promoting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in one NHEJ repair pathway may promote use of the other more error-prone pathway. The topoisomerase II poison etoposide is a cytotoxic chemotherapeutic agent associated with therapy-related leukemias characterized by translocations involving the MLL gene at 11q23. Leukemias with MLL gene fusions have been detected in newborns indicating that these translocations can occur in utero, and additional evidence from our lab and others suggests maternal exposure to environmental topoisomerase II inhibitors such as bioflavonoids have the potential to promote these events. We are interested in understanding the crosstalk involved in bioflavonoid exposure, DNA damage response signaling, and repair pathway choice as a model system to understand the underlying mechanisms of oncogenic transformation in infant leukemias.Using established in vitro differentiation protocols, we expose embryonic stem (ES) cells and hematopoietic stem cells (HSC) derived from them to a panel of bioflavonoids alone or in combination for one hour at increasing doses ranging from 0μM-200μM based on determined LD50 values. As a control, cells are exposed to etoposide at the same doses. Following exposure, cells recovered for a period of 0-24hrs, followed by protein extraction and Western blotting. Quantitation of increases in total gH2AX confirm DNA damage, presence of Ku70 and XLF to support classical NHEJ, and CtIP to support Alt-NHEJ. Cells exposed to bioflavonoids genistein and quercetin as well as etoposide show elevated γH2AX levels by 4 hrs post-treatment compared to untreated cells. Parallel studies show dose-dependent increase in the presence of γH2AX foci in these cells. These results confirm that although bioflavonoids have pleiotropic effects, they directly promote DSBs in a short period of time post-exposure. Protein levels of Ku70 and XLF in cells exposed to genistein, quercetin, and etoposide at doses of 25, 50, 75 and 100μM are not significantly different from untreated cells. However, CtIP levels are elevated in cells exposed to quercetin compared to all other groups suggesting a specific potential for this bioflavonoid to promote Alt-NHEJ repair and genome instability.To correlate the activity of these DNA repair protein activities with downstream repair products, we use a model cell system in which parental ES cells were engineered to contain two constructs: GFP exons inserted into MLL and AF9 bcr transgene fragments. Aberrant repair resulting in chromosomal translocations are scored by GFP+ fluorescence. In both ES and derived HSC populations from them translocations can be scored following exposure to bioflavonoids and etoposide. Ongoing experiments include downregulation of XLF or CtIP by siRNA in cells prior to exposure to these compounds. Results will be presented on impact of downregulation of these two NHEJ proteins on 1) amount and persistence of γH2AX; 2) protein levels of other DNA repair pathway proteins, and 3) frequency of generating chromosomal translocations.Understanding the integrated activity and role of these proteins in preventing or promoting translocations, particularly in the context of exposure to a wide range of environmental and dietary compounds, will be critical to understanding the potential for specific isoforms or mutants to increase leukemia risk. DisclosuresNo relevant conflicts of interest to declare.

Elucidation of genomic organizations of transgenic soybean plants through de novo genome assembly with short paired-end reads.

Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay.

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay.

BackgroundWith the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping.MethodsWhole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR.ResultsIn whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected.ConclusionsThese results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

The NIN-like protein 5 (ZmNLP5) transcription factor is involved in modulating the nitrogen response in maize.

SummaryMaize exhibits marked growth and yield response to supplemental nitrogen (N). Here, we report the functional characterization of a maize NIN‐like protein ZmNLP5 as a central hub in a molecular network associated with N metabolism. Predominantly expressed and accumulated in roots and vascular tissues, ZmNLP5 was shown to rapidly respond to nitrate treatment. Under limited N supply, compared with that of wild‐type (WT) seedlings, the zmnlp5 mutant seedlings accumulated less nitrate and nitrite in the root tissues and ammonium in the shoot tissues. The zmnlp5 mutant plants accumulated less nitrogen than the WT plants in the ear leaves and seed kernels. Furthermore, the mutants carrying the transgenic ZmNLP5 cDNA fragment significantly increased the nitrate content in the root tissues compared with that of the zmnlp5 mutants. In the zmnlp5 mutant plants, loss of the ZmNLP5 function led to changes in expression for a significant number of genes involved in N signalling and metabolism. We further show that ZmNLP5 directly regulates the expression of nitrite reductase 1.1 (ZmNIR1.1) by binding to the nitrate‐responsive cis‐element at the 5′ UTR of the gene. Interestingly, a natural loss‐of‐function allele of ZmNLP5 in Mo17 conferred less N accumulation in the ear leaves and seed kernels resembling that of the zmnlp5 mutant plants. Our findings show that ZmNLP5 is involved in mediating the plant response to N in maize.

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR.

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

Genetic Rodent Models of Huntington Disease.

The monogenic nature of Huntington disease (HD) has led to the development of a spectrum of useful genetically modified models. In particular, rodents have pioneered as the first HD model being generated and have since been the most widely used animal model for HD in both basic research and preclinical therapeutic studies. Based on the generation strategies, these rodent models can be classified into 3 major groups, the transgenic fragment models, the transgenic full-length models and the knock-in models. These models display a range of HD-like characteristics which resemble the clinical symptoms of HD patients. Their applications in research are thus regarded as an invaluable approach to speeding up the unraveling of the underlying pathological mechanisms of HD and for finding a disease-modifying treatment for this devastating disease. In this chapter, the similarities and differences of the most commonly used rodent HD models and their relevance to human HD will be compared and discussed. This also serves to guide the selection of an appropriate rodent HD model according to the nature of investigation.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance

BackgroundThe mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential.ResultsA BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth.ConclusionsA TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

Ratiometric fluorescence sensor for the sensitive detection of Bacillus thuringiensis transgenic sequence based on silica coated supermagnetic nanoparticles and quantum dots

Improving the accuracy and sensitivity of fluorescence analysis is of great importance in clinical diagnosis, environmental and food monitoring. Herein, based on fluorescence resonance energy transfer (FRET), a facile and effective ratiometric fluorescence sensor was constructed for the detection of Bacillus thuringiensis (Bt) special gene fragment. In this work, green quantum dots (gQDs) decorated Fe3O4 magnetic beads (MBs) with streptavidin (SA) were acted as donor, and gold nanoparticles (GN) modified red quantum dots (rQDs@SiO2) with hairpin DNA as receptor. When the target sequences exist, the hairpin DNA were unfolded and subsequently captured by MBs@SiO2@gQD-SA via biotin–SA specific interaction. By using the magnetic separation method, the hybridized composites could be easily purified for florescence tests. By this means, rQDs@SiO2@GN could enhance the fluorescence intensity of rQDs (I620) and simultaneously quench the fluorescence response of gQDs (I540) via FRET. Under optimal conditions, the ratio of fluorescence intensity at 620nm and 540nm (I620/I540) showed that Bt transgene fragment detection owning a good linearity from 5.0pM to 10nM with a detection limit of 0.10pM (S/N=3). The high selectivity of the probes was also demonstrated using the single-base and three-base mismatch method.

Ultrasensitive SERS detection of Bacillus thuringiensis special gene based on Au@Ag NRs and magnetic beads

Highly sensitive and selective detection of specific DNA sequences is of great importance in clinical diagnosis, environmental and food monitoring, but it still remains challenges to develop a facile method for real sample detection in aqueous solution. Here, a simple and recyclable surface enhanced Raman scattering (SERS) sensor was constructed for Bacillus thuringiensis (Bt) special gene fragment detection by Fe3O4 magnetic beads (MBs) and Au-Ag core-shell nanorods (Au@Ag NRs). A hairpin DNA with sulfhydryl and biotin was attached to Au@Ag NRs as indicator, and MBs with streptavidin (SA) were acted as the capture probe. On the basis of the biotin-SA specific interaction, target sequences were first hybridized with the hairpin DNA and exposed the biotin. Subsequently, the Au@Ag NRs were captured by the streptavidin modified MBs, which reduced the suspended NRs and led to the change of Raman intensity. Under the optimal conditions, the SERS intensity revealed a good linearity with Bt transgene fragment ranging from 0.1pM to 1nM with a detection limit of 0.14pM (S/N=3). To demonstrate the specificity of the strategy, the single-base mismatch in DNA was discussed in the SERS assay. The results showed that the sensitivity and accuracy of the proposed method was acceptable in DNA detection, revealing a great potential in special gene detection.

Delayed Onset and Reduced Cognitive Deficits through Pre-Conditioning with 3-Nitropropionic Acid is Dependent on Sex and CAG Repeat Length in the R6/2 Mouse Model of Huntington's Disease.

Impairments in energy metabolism are implicated in Huntington's disease (HD) pathogenesis. Reduced levels of the mitochondrial enzyme succinate dehydrogenase (SDH), the main element of complex II, are observed post mortem in the brains of HD patients, and energy metabolism defects have been identified in both presymptomatic and symptomatic HD patients. Chemical preconditioning with 3-nitropropionic acid (3-NP), an irreversible inhibitor of SDH, has been shown to increase tolerance against experimental hypoxia in both heart and brain. Here we studied the effect of chronic preconditioning in the R6/2 mouse model of HD using mice carrying CAG repeat lengths of either 250 or 400 repeats. Both are transgenic fragment models, with 250CAG mice having a more rapid disease progression than 400CAG mice. Low doses of 3-NP (24 mg/kg) were administered via the drinking water and the effect on phenotype progression and cognition function assessed. After 3-NP treatment there were significant improvements in all aspects of the behavioural phenotype, apart from body weight, with timing and magnitude of improvements dependent on both CAG repeat length and sex. Specifically, a delay in the deterioration of general health (as shown by delayed onset of glycosuria and increased survival) was seen in both male and female 400CAG mice and in female 250CAG mice and was consistent with improved appearance of 3-NP treated R6/2 mice. Male 250CAG mice showed improvements but these were short term, and 3-NP treatment eventually had deleterious effects on their survival rate. When cognitive performance of 250CAG mice was assessed using a two-choice discrimination touchscreen task, we found that female mice showed significant improvements. Together, our results support the idea that energy metabolism contributes to the pathogenesis of HD, and suggest that improving energy deficits might be a therapeutically useful target.

SYBR® Green qPCR Screening Methods for Detection of Anti-herbicide Genes in Genetically Modified Processed Products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of GMOs are necessary in order to verify compliance with labelling requirements. There are few effective screening methods for processed GM (genetically modified) products. Three anti-herbicide genes (CP4-EPSPS, BAR and PAT) are common exogenous genes used in commercialized transgenic soybean, maize and rice. In the present study, a new SYBR® Green qPCR screening method was developed to simultaneously detect the three exogenous anti-herbicide genes and one endogenous gene in a run. We tested seven samples of representative processed products (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, maize protein powder, maize starch, and maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products, and the sensitivity was 0.1%. These results indicated that SYBR® Green qPCR screening method was appropriate for qualitative detection of transgenic soybean, maize and rice in processed products.