Determination of Transgene Copy Number by Real-Time Quantitative PCR

Efficient methods to characterize transgenic plants are important to quickly understand the state of the transformant. Determining transgene copy number is an important step in transformant characterization and can differentiate between complex and simple transformation events. This knowledge can be extremely useful when determining what future experiments and uses the transgenic lines can be utilized for. The method described here uses real-time quantitative PCR to determine the transgene copy number present in the genome of the transformant. Specifically, this method measures the relative transgene copy number by comparing it with an endogenous gene with a known copy number. This method is a quick alternative to the Southern blot, a method that is commonly used to determine gene copy number, and is effective when screening large numbers of transformants

Species identification of European forest pathogens of the genus Milesina (Pucciniales) using urediniospore morphology and molecular barcoding including M. woodwardiana sp. nov

Species of rust fungi of the genus Milesina (Pucciniastraceae, Pucciniales) are distributed mainly in northern temperate regions. They host-alternate between needles of fir (Abies spp.) and fronds of ferns (species of Polypodiales). Milesina species are distinguished based on host taxonomy and urediniospore morphology. In this study, 12 species of Milesina from Europe were revised. Specimens were examined by light and scanning electron microscopy for urediniospore morphology with a focus on visualising germ pores (number, size and position) and echinulation. In addition, barcode loci (ITS, nad6, 28S) were used for species delimitation and for molecular phylogenetic analyses. Barcodes of 72 Milesina specimens were provided, including 11 of the 12 species. Whereas urediniospore morphology features were sufficient to distinguish all 12 Milesina species except for 2 (M. blechni and M. kriegeriana), ITS sequences separated only 4 of 11 species. Sequencing with 28S and nad6 did not improve species resolution. Phylogenetic analysis, however, revealed four phylogenetic groups within Milesina that also correlate with specific urediniospore characters (germ pore number and position and echinulation). These groups are proposed as new sections within Milesina (sections Milesina, Vogesiacae M. Scholler & Bubner, sect. nov., Scolopendriorum M. Scholler & Bubner, sect. nov. and Carpaticae M. Scholler & Bubner, sect. nov.). In addition, Milesina woodwardiana Buchheit & M. Scholler, sp. nov. on Woodwardia radicans, a member of the type section Milesina, is newly described. An identification key for European Milesina species, based on urediniospore features, is provided

Statistical tools for transgene copy number estimation based on real-time PCR

Background As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Results Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. Conclusion These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification

Random and systematic sampling error when hooking fish to monitor skin fluke (Benedenia seriolae) and gill fluke (Zeuxapta seriolae) burden in Australian farmed yellowtail kingfish (Seriola lalandi)

© 2018 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license:http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (February 2018) in accordance with the publisher’s archiving policyThe Australian farmed yellowtail kingfish (Seriola lalandi, YTK) industry monitor skin fluke (Benedenia seriolae) and gill fluke (Zeuxapta seriolae) burden by pooling the fluke count of 10 hooked YTK. The random and systematic error of this sampling strategy was evaluated to assess potential impact on treatment decisions. Fluke abundance (fluke count per fish) in a study cage (estimated 30,502 fish) was assessed five times using the current sampling protocol and its repeatability was estimated the repeatability coefficient (CR) and the coefficient of variation (CV). Individual body weight, fork length, fluke abundance, prevalence, intensity (fluke count per infested fish) and density (fluke count per Kg of fish) were compared between 100 hooked and 100 seined YTK (assumed representative of the entire population) to estimate potential selection bias. Depending on the fluke species and age category, CR (expected difference in parasite count between 2 sampling iterations) ranged from 0.78 to 114 flukes per fish. Capturing YTK by hooking increased the selection of fish of a weight and length in the lowest 5th percentile of the cage (RR = 5.75, 95% CI: 2.06–16.03, P-value = 0.0001). These lower end YTK had on average an extra 31 juveniles and 6 adults Z. seriolae per Kg of fish and an extra 3 juvenile and 0.4 adult B. seriolae per Kg of fish, compared to the rest of the cage population (P-value < 0.05). Hooking YTK on the edge of the study cage biases sampling towards the smallest and most heavily infested fish in the population, resulting in poor repeatability (more variability amongst sampled fish) and an overestimation of parasite burden in the population. In this particular commercial situation these finding supported that health management program, where the finding of an underestimation of parasite burden could provide a production impact on the study population. In instances where fish populations and parasite burdens are more homogenous, sampling error may be less severe. Sampling error when capturing fish from sea cage is difficult to predict. The amplitude and direction of this error should be investigated for a given cultured fish species across a range of parasite burden and fish profile scenarios

ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Reverse Genetics in Ecological Research

By precisely manipulating the expression of individual genetic elements thought to be important for ecological performance, reverse genetics has the potential to revolutionize plant ecology. However, untested concerns about possible side-effects of the transformation technique, caused by Agrobacterium infection and tissue culture, on plant performance have stymied research by requiring onerous sample sizes. We compare 5 independently transformed Nicotiana attenuata lines harboring empty vector control (EVC) T-DNA lacking silencing information with isogenic wild types (WT), and measured a battery of ecologically relevant traits, known to be important in plant-herbivore interactions: phytohormones, secondary metabolites, growth and fitness parameters under stringent competitive conditions, and transcriptional regulation with microarrays. As a positive control, we included a line silenced in trypsin proteinase inhibitor gene (TPI) expression, a potent anti-herbivore defense known to exact fitness costs in its expression, in the analysis. The experiment was conducted twice, with 10 and 20 biological replicates per genotype. For all parameters, we detected no difference between any EVC and WT lines, but could readily detect a fitness benefit of silencing TPI production. A statistical power analyses revealed that the minimum sample sizes required for detecting significant fitness differences between EVC and WT was 2–3 orders of magnitude larger than the 10 replicates required to detect a fitness effect of TPI silencing. We conclude that possible side-effects of transformation are far too low to obfuscate the study of ecologically relevant phenotypes

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for tw

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca2+. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca2+ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae

Aesthetic Freedom and Democratic Ethical Life: A Hegelian Account of the Relationship between Aesthetics and Democratic Politics

This paper presents a novel Hegelian view of the relationship between aesthetics and democratic politics. My account avoids the drawbacks associated with approaches that (like Rancière’s) reconceive all of the political in aesthetic terms or (like Rockhill’s) reduce the aesthetic to art. Instead, I maintain that the aesthetic is best understood as a distinct relationship of individual freedom. My argument proceeds by highlighting shortcomings of Honneth’s account of democratic Sittlichkeit and then addressing these impasses by integrating aesthetic freedom into the picture. The first two steps of my argument concern the fact that the form of life outlined by Honneth aspires to be a form of free life, yet his account of democratic Sittlichkeit gives rise to two dimensions of unfreedom. The first problem of unfreedom pertains to the scope of freedom. The relationships of freedom incorporated into Honneth’s account fail to turn given social roles into the subject matter of a sufficiently unrestricted practice of freedom. The second problem of freedom concerns conformism. In a final step, I complete my argument that Honneth’s account is unsatisfactory and incomplete by showing that aesthetic freedom is socially valid and thus ought to form part of our accounts of democratic ethical life