Multiple Hairpin Research Articles

Enzyme-Powered, Label-Free DNA Walker for Uracil-DNA Glycosylase Detection at Single-Cell Level.

Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered, label-free DNA walker was devised for UDG activity detection. Initially, a label-free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G-quadruplex sequence, enabling the generation of a label-free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme-powered walking process and generating significant dissociative G-quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label-free fluorescence response was achieved. The proposed DNA walker presented a direct and label-free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single-cell level was accomplished. Consequently, the developed DNA walker serves as a label-free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.

Identification and Functional Analysis of Novel Long Intergenic RNA in Chicken Macrophages Infected with Avian Pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC), a widespread bacterium, results in serious economic losses to the poultry industry annually, and it poses a threat to human health due to the contaminated retail poultry meat and eggs. Recently, it has been demonstrated that long non-coding RNAs played important roles in regulating gene expression and the animal immune response. This study aimed to systematically explore the function of the novel long intergenic non-coding transcript, lincRNA-73240, upon APEC infection. A bioinformatics analysis indicated that lincRNA-73240 had no coding ability and a relative stable secondary structure with multiple hairpin rings. Moreover, the RT-qPCR results showed that lincRNA-73240 was highly expressed in lungs, heart, liver, spleen, cecum tonsils, thymus, ileum, bursa of Fabricius, harderian gland, and muscles in comparison to the cerebrum. Additionally, overexpression of lincRNA-73240 can promote the expression levels of inflammation, apoptosis, autophagy, and oxidative stress-related genes, as well as the production of reactive oxygen species (ROS), malondialdehyde (MDA), and nitric oxide (NO) upon APEC infection, which lead to cellular injury and apoptosis. These findings collectively establish a foundation for the study of the biological function of chicken lincRNA-73240 and provide a theoretical basis for further research on the molecular mechanisms of the chicken immune response.

Using supramolecular polymerization of DNA to produce tandem repeat proteins

AbstractTandem repeat (TR) proteins are abundant in nature and show diverse biochemical and mechanical properties. However, these proteins can be difficult to synthesize in a precise way because of their repetitive nature. Using a supramolecular polymerization scheme known as hybridization chain reaction, we show how to generate the DNA sequences encoding such TR proteins. The polymerization mechanism allows for identical “monomers” to selectively create TR proteins of different molecular weight. We demonstrate this strategy with examples from elastin‐like polypeptides (ELPs) and mussel foot proteins (mfps). Moreover, the scheme can be adapted to create protein libraries through the use of multiple DNA hairpin “monomers.”

Quantitative experimental research on vortex generation and self-maintenance mechanisms in turbulence

Turbulence is not completely random, but contains organized and multi-scale structures. Vortices have always been recognized as the most important coherent structure in turbulent flow, playing a significant role in the generation, evolution, and maintenance of turbulence. In the present work, the vortex formation and evolution process of a fully developed turbulent boundary layer in a rectangular channel flow is experimentally studied by moving-single frame and long exposure and moving-particle image velocimetry measurements. The Rortex integral (RI) method is proposed for quantitative statistics on the critical vortex core size as well as the accurate rotation strength during the evolution process. In this paper, the vortex regeneration and self-maintenance mechanisms in near-wall turbulent flow are experimentally revealed and quantified by the RI method, to give some revelations for the future research on the turbulence theory. On the one hand, three behaviors of the hairpin vortex regeneration are discovered to play a significant role in turbulence development: (A) hairpin regeneration induced by the interaction between hairpins and packets; (B) auto-generation of multiple hairpin vortex in one packet (secondary and tertiary hairpins); and (C) the merging of hairpin vortices in the packets. On the other hand, the circulation process, which contains a mass of young vortex growth with the parent self-decaying, is verified to sustain and promote the development of turbulence. In consequence, the self-sustaining turbulence theory based on mother–child hairpins generation mechanism is supported by the experimental results.

CRISPR-Cas12a coupled with cyclic reverse transcription for amplified detection of miRNA.

In this work, we present a highly sensitive, specific, and versatile method to quantify miRNA expression by coupling CRISPR-Cas12a with cyclic reverse transcription (CRT), termed as CRISPR-CRT. Each miRNA target was first converted and amplified into multiple hairpin RT products via CRT. Afterward, the hairpin RT products could serve as activators to initiate the collateral cleavage activity of CRISPR-Cas12a. Due to the above two-stage amplification, this assay could detect miRNA at sub-femtomolar level (LOD, 0.201 fM). Since the sequence of target miRNA is double checked: first in the CRT and then in the CRISPR system, the proposed assay also shows an excellent specificity in detecting miR-21. Finally, with the usage of this assay, the sensitive assessment of miR-21 levels in human serum samples has been achieved and the disease human serum has been detected. Conclusively, CRISPR-CRT holds a great application prospective in the field of clinical molecular diagnosis.

Three-Dimensional Surface-Enhanced Raman Scattering Platform with Hotspots Built by a Nano-mower for Rapid Detection of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the greatest threats to human health due to its strong drug resistance, wide distribution range, and high infection rate. Rapid identification of MRSA strains is very important for accurate diagnosis and early treatment of MRSA infections. Here, we introduced an Exo III-assisted nanomotor mower to build 3D hotspots for rapid detection of MRSA by surface-enhanced Raman scattering (SERS). As the bacteria bound to the aptamer, two trigger chains were released from the double-stranded structure, and the nano-mowers were activated by opening a hairpin probe on gold nanoparticles (AuNPs). With the continued cleavage of Exo III and cyclic release of the trigger chain, multiple hairpin DNAs on the AuNPs were cleaved to increase the motor power. The resulting nano-mower continued slicing protective DNA from larger AuNPs, exposing the AuNPs. Without the protection of DNA, Mg2+ in the buffer induced spontaneous aggregation of the AuNPs, and a large number of hotspots were formed for SERS measurements. Under optimal conditions, MRSA can be detected within 40 min, and the concentration of MRSA showed a good linear relationship with the SERS intensity at 1342 cm-1, with a limit of detection as low as 1 CFU/mL and a wide linear range (100 to 107 CFU/mL). This strategy creates a rapid bacterial detection method that performs well on actual samples utilizing portable Raman spectroscopy instruments, with potential applications in food detection, water detection, clinical treatment, and diagnosis.

Highly sensitive electrochemical aptasensor for SARS-CoV-2 antigen detection based on aptamer-binding induced multiple hairpin assembly signal amplification

In this work, a brief electrochemical aptasensor was developed for highly sensitive detection of SARS-CoV-2 antigen utilizing an aptamer-binding induced multiple hairpin assembly strategy for signal amplification. In the presence of SARS-CoV-2, a pair of aptamers was brought in a close proximity according to the aptamer-protein antigen binding, which initiated strand displacement reaction thereby triggering a multiple hairpin assembly to obtain long linear DNA concatemers on the electrode surface. As the fabricated hairpin probes were labeled with biotin, massive streptavidin-alkaline phosphatases (ST-ALP) could be further introduced on the electrode interface via biotin-streptavidin interaction thus generating strong electrochemical signal in electrolyte solution containing 1-naphthol phosphate. Benefiting from the non-enzymatic multiple hairpin assembly signal amplification strategy, the designed aptasensor for SARS-CoV-2 spike protein detection exhibited the wide linear range from 50 fg·mL−1 to 50 ng·mL−1 and low detection limit of 9.79 fg·mL−1. Meaningfully, this proposed electrochemical assay provided a potential application for the point of care analysis of viral diseases under ambient temperature.

A ratiometric fluorescent nanoprobe for signal amplification monitoring of intracellular telomerase activity.

Telomerase is considered a valuable diagnostic and prognostic cancer biomarker. Accurate and reliable detection of telomerase activity is of great value in clinical diagnosis, screening of inhibitors, and therapeutics. Here, we developed a novel amplified fluorescence resonance energy transfer (FRET) nanoprobe for highly sensitive and reliable monitoring of intracellular telomerase activity. The nanoprobe (QDSA@DNA) was composed of a streptavidin-modified quantum dot (QDSA) which was functionalized with a telomerase primer sequence (TP) and Cy5-tagged signal switching sequence (SS) through biotin-streptavidin interaction. When the nanoprobe was assembled, the Cy5 was in close proximity to the QDSA, resulting in high FRET efficiency from the QDSA to Cy5. In the presence of telomerase, the TP could be extended to produce telomeric repeat units, which was complementary to the loop of SS. Thus, the SS could hybridize with elongated sequences to form a rigid double-stranded structure, which forced the Cy5 away from the surface of the QDSA, causing low FRET efficiency. Furthermore, due to the production of multiple repeat units by telomerase, multiple hairpin structures could be opened, yielding significant fluorescence ratio (FQDsa/FCy5) enhancement for sensing of telomerase activity. In this way, the combination of a FRET and target-assisted strategy in a nanoprobe improved the detection accuracy and amplified the detection signal, respectively. The QDSA@DNA nanoprobe also showed high selectivity, excellent nuclease stability, and good biocompatibility. More importantly, this nanoprobe was found to be an excellent platform for efficient monitoring of intracellular telomerase activity, providing a potential platform in tumor diagnosis and screening of telomerase-related inhibitors.

DNA nanolantern-mediated catalytic hairpin assembly nanoamplifiers for simultaneous detection of multiple microRNAs

Simultaneous detection of multiple microRNAs (miRNAs) with high sensitivity can give accurate and reliable information for clinical applications. By uniformly anchoring hairpin probes on the surface of DNA nanolantern, a three-dimensional DNA nanostructure contains abundant and adjustable modification sites, highly integrated DNA nanoprobes were designed and developed as catalytic hairpin assembly (CHA)-based signal amplifiers for enzyme-free signal amplification detection of target miRNAs. The nanolantern-based CHA (NLC) amplifiers, which were facilely prepared via a simple “one-pot” annealing method, showed enhanced biostability, improved cell internalization efficiency, accelerated CHA reaction kinetics, and increased signal amplification capability compared to the single-stranded DNA hairpin probes used in traditional CHA reaction. By co-assembling multiple hairpin probes on a DNA nanolantern surface, as-prepared NLC amplifiers were demonstrated to work well for highly sensitive and specific imaging, expression level fluctuation analysis of two miRNAs in living cells, and miRNAs-guided tumor imaging in living mice. The proposed DNA nanolantern-based nanoamplifier strategy might provide a feasible way to promote the cellular and in vivo applications of nucleic acid probes.

Multifunctional electrochemical biosensor with "tetrahedral tripods" assisted multiple tandem hairpins assembly for ultra-sensitive detection of target DNA.

Nucleic acids are genetic materials in the human body that play important roles in storing, copying, and transmitting genetic information. Abnormal nucleic acid sequences, base mutations, and genetic changes often lead to cancer and other diseases. Meanwhile, methylated DNA is one of the main epigenetic modifications, which is considered to be an excellent biomarker in the early detection, prognosis, and treatment of cancers. Therefore, a multifunctional electrochemical biosensor was constructed with sturdy tetrahedral tripods, which assisted multiple tandem hairpins through base complementary pairing and effective ultra-sensitive detection of targets (DNA, microRNA, and methylated DNA). In the experiments, experimental conditions were optimized, and different DNA concentrations in serum were detected to verify the sensitivity of the biosensor and the feasibility of this protocol. In addition, microRNA and DNA methylation were detected through different designs of tetrahedral tripods (TTs) that capture probes to prove the superiority of this scheme. A sturdy pyramid structure of TTs extremely enhanced the capture efficiency of targets. The targets triggered the one-step isothermal multi-tandem amplification reaction by incubating multiple hairpin assemblies. To our knowledge, a combination of two parts, which greatly reduced background interference and decreased non-specific substance interference, has appeared for the first time in this paper. Moreover, the load area of electrochemical substances was significantly increased than that in previous studies. This greatly increased the detection range and detection limit of targets. The electrochemical signal responses were generated in freely diffusing hexaammineruthenium(iii) chloride (RuHex). RuHex could adhere to the DNA phosphate backbone by a powerful electrostatic attraction, causing increased current responses.

Self-assembly of DNA nanoparticles through multiple catalyzed hairpin assembly for enzyme-free nucleic acid amplified detection

It is known that DNA molecules can be used to build a various of complicated geometrical DNA nanostructures with programmable sequence design, and these DNA nanomaterials show a promising application in biotechnology and biomedicine. However, the construction of large-sized three dimensional DNA-based nanomaterials still remains a challenge. In this work, we propose a new strategy that only employs one target DNA to trigger multiple catalyzed hairpin assembly (CHA) reactions and sticky ends self-assembly to prepare hundreds of nanometer-sized DNA nanoparticles. Moreover, the obtained DNA nanoparticles can be served as efficient biosensors for sensitive colorimetric nucleic acids detection with a detection limit of 7.7pM

Forced convection in the wakes of impacting and sliding bubbles

Both vapour and gas bubbles are known to significantly increase heat transfer rates between a heated surface and the surrounding fluid, even with no phase change. The cooling structures observed are highly temporal, intricate and complex, with a full description of the surface cooling phenomena not yet available. The current study uses high speed infrared thermography to measure the surface temperature and determine the convective heat flux enhancement associated with the interaction of a single air bubble with a heated, inclined surface. This process can be discretised into the initial impact, in which enhancement levels in excess of 20 times natural convection are observed, and the subsequent sliding behaviour, with more moderate maximum enhancement levels of 8 times natural convection. In both cases, localised regions of suppressed heat transfer are also observed due to the recirculation of warm fluid displaced from the thermal boundary layer with the surface. The cooling patterns observed herein are consistent with the interaction between an undulating wake containing multiple hairpin vortex loops and the thermal boundary layer that exists under the surface, with the initial nature of this enhancement and suppression dependent on the particular point on its rising path at which the bubble impacts the surface.

A novel surface plasmon resonance biosensor for enzyme-free and highly sensitive detection of microRNA based on multi component nucleic acid enzyme (MNAzyme)-mediated catalyzed hairpin assembly

MicroRNAs (miRNAs) are potentially useful biomarkers for early diagnosis of human diseases. Here, a simple surface plasmon resonance (SPR) biosensor has been developed for highly sensitive detection of miRNA by designing a new enzyme-free and isothermal amplification strategy, named multi component nucleic acid enzyme-mediated mismatched catalyzed hairpin assembly (MNAzyme-CHA). The partial MNAzymes co-recognized the target to form a stable active MNAzyme, which continued to digest multiple hairpin H0 substrates, concomitantly generating a lot of fragments. The H0 fragments could initiate the mismatched CHA cycles, resulting in the generation of massive hairpin H1–H2 complexes. As a result, the H1–H2 complexes and streptavidin were attached to the sensor surface, leading to a significantly amplified SPR signal readout. The established biosensor showed high sensitivity and selectivity with a wide dynamic range from 1pM to 100nM. It was also successfully applied to the determination of target miRNA spiked into human total RNA samples. Thus, this developed biosensing strategy presents a simple and stable platform toward sensitive and convenient miRNA detection, and has great potential in assays of many other nucleic acids analytes for biomedical research and early clinical diagnosis.

Physics of multiple level hairpin vortex structures in turbulence

Previous experimental and numerical studies have revealed that the hairpin vortex is a basic flow element of transitional boundary layer. The hairpin vortex is believed to have legs, necks and a ring head. Based on our DNS study, the legs and the ring head are generated separately by different mechanisms. The legs function like an engine to generate low speed zones by rotation, create shear layers with surrounding high speed neighbor fluids, and further cause vortex ring formation through shear layer instability. In addition, the ring head is Ω-shaped and separated from quasi-streamwise legs from the beginning. Contrary to the classical concept of “vortex breakdown”, we believe transition from laminar flow to turbulence is a “buildup” process of multiple level vortical structures. The vortex rings of first level hairpins are mostly responsible for positive spikes, which cause new vorticity rollup, second level vortex leg formation and finally smaller second level vortex ring generation. The third and lower level vortices are generated following the same mechanism. In this paper, the physical process from Λ-vortex to multi-level hairpin vortices is described in detail.

Superconducting Ultra-Wideband (UWB) Bandpass Filter Design Based on Quintuple/Quadruple/ Triple-Mode Resonator

This paper analyzes the coupling modes when multiple hairpin resonators are coupled. Three kinds of current distribution and direction states exist in the coupled hairpin resonators: the current along the clockwise direction, the current along the counterclockwise direction, and no current density distribution. Different combinations of these current states lead to different coupling modes. In order to distinguish these modes, auxiliary lines are introduced between each two coupled resonators to denote each coupling mode, and this method is theoretically based on the analysis of the eigenvector of the coupling matrix. A simple and effective technique to construct multi-mode resonators (MMRs) is then proposed. An <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">$n$</tex></formula> -mode resonator can be constructed by connecting <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">$n$</tex> </formula> hairpins with small patches. Resonant modes of this class of MMRs were analyzed in detail using the proposed auxiliary lines method. Single stage and four stages of quintuple-mode resonators were used to design high-temperature superconducting ultra-wideband (UWB) filters by properly allocating the first five resonant peaks. Two UWB filters based on quadruple-mode and triple-mode resonators are also given. Parallel-coupled three lines were used to provide tight external couplings, which also generated two transmission poles. Measured results are in good agreement with the simulated ones without any tuning.

Abstract PR09: A genome-wide RNAi screen reveals a protective role of decreased lipogenesis under hypoxia

Abstract As solid tumors form, a number of physiological changes occur within the tumor, including low oxygen levels (hypoxia) and an accumulation of lactic acid with concomitant lowered pH levels (lactic acidosis). In order to adapt to and survive in the strong selective pressures imposed by these tumor microenvironment (TME) “stresses”, cancer cells exhibit significant genetic and metabolic changes. While transcriptional responses and signaling events triggered by stresses are well-studied, a systematic genome-wide, forward-genetics screen to identify which genes are critical or restrictive for cell survival in these TME stresses has not been examined. The purpose of this study is to better understand the function of genes that modulate cell survival under TME stresses in order to improve our knowledge of tumor evolution and allow for the development of treatments to specifically target cancer cells under these stresses. We performed a contextual short-hairpin RNA (shRNA) pooled screen in cancer cells cultured under control, hypoxia or lactic acidosis conditions. Genomic DNA was recovered from each condition to amplify the resulting shRNAs by PCR before quantifying them by microarray. A number of criteria were used to select top candidate hits, including multiple shRNAs targeting a single gene needing to pass stringent statistical criteria. We have successfully validated a number of candidate genes with multiple shRNAs. Importantly, our analysis identified HIF-2α (EPAS1) as critical for cells to survive under hypoxia and this result has been validated. One “multiple hairpin hit” was ACACA. This gene encodes acetyl-CoA carboxylase (ACC1), which is the enzyme that mediates the rate-limiting step of de novo lipogenesis by converting acetyl-CoA to malonyl-CoA. Validation experiments showed that inhibition of ACC1 by four different shRNAs protected cells from hypoxia-induced apoptosis in several cell lines. This protective affect was specific to hypoxia, since cells were not protected under lactic acidosis or glutamine deprivation. Further interrogation of related genes revealed that the depletion of ATP citrate lyase (ACLY) also prevented hypoxia-induced apoptosis. ACLY acts upstream of ACC1, converting citrate to acetyl-CoA. Metabolomic and transcriptomic analysis revealed that the silencing of ACC1 or ACLY significantly diminished the expected hypoxia and stress responses of these cells. We hypothesize that the metabolic shift resulting from the depletion of ACC1 or ACLY switches cancer cells from a “nutrient rich/proliferative” state to a “nutrient poor/survival” state, allowing cells to better cope with hypoxia. Consistent with this concept, when the gene signatures of ACC1 or ACLY depletion were used to stratify patients in breast cancer datasets, the inactivation of these genes correlated with reduced proliferative and hypoxia pathways and more favorable outcomes. These results may have important implications for the use of metformin and lipogenic inhibitors in the clinic. Our forward-genetics screen has identified novel genes and biological processes that impact the survival of cancer cells under TME stresses. These findings inform our understanding of the selection of somatic mutations in cancer cells and help reveal potential new therapeutic strategies to selectively target solid human cancers. This abstract is also presented as Poster B1. Citation Format: Melissa M. Keenan, Beiyu Liu, Jianli Wu, Derek Cyr, Joseph Lucas, Deborah M. Muoio, So Young Kim, Jen-Tsan Chi. A genome-wide RNAi screen reveals a protective role of decreased lipogenesis under hypoxia. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR09. doi:10.1158/1538-7445.CHTME14-PR09

Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model.

Gene targeting is a critical tool for construction of disease models. However, the application of traditional homologous recombination-mediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods. Although conventional small hairpin RNA (shRNA)-mediated gene silencing offers an alternative for gene targeting, its application is frequently compromised by lower expression efficiency via RNA interference compared to gene knockout. Here we provide an efficient gene targeting strategy involving drug-inducible synergistic silencing with multiple shRNA molecules. On induction, the levels of the target proteins decreased to undetectable levels in all the tested stable transgenic mammalian cell lines, including HEK293 and embryonic stem cell-derived progenies carrying shRNA silencing cassettes. In a transgenic mouse model carrying a silencing cassette targeting the rhodopsin gene, short-time inducer treatment was sufficient to ablate the rhodopsin protein in the retina, resulting in similar retinal phenotypic changes as those observed in rhodopsin mutant mice. Therefore, on a broad basis, this inducible shRNA gene targeting strategy offers a true gene knockout alternative comparable to conventional RNA interference approaches.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-014-9841-9) contains supplementary material, which is available to authorized users.

Evolutionary relationship between 5+5 and 7+7 inverted repeat folds within the amino acid-polyamine-organocation superfamily

Evidence has been presented that 5+5 TMS and 7+7 TMS inverted repeat fold transporters are members of a single superfamily named the Amino acid-Polyamine-organoCation (APC) superfamily. However, the evolutionary relationship between the 5+5 and the 7+7 topological types has not been established. We have identified a common fold, consisting of a spiny membrane helix/sheet, followed by a U-like structure and a V-like structure that is recurrent between domain duplicated units of 5+5 and 7+7 inverted repeat folds. This fold is found in the following protein structures: AdiC, ApcT, LeuT, Mhp1, BetP, CaiT, and SglT (all 5+5 TMS repeats), as well as UraA and SulP (7+7 TMS repeats). AdiC, LeuT and Mhp1 have two extra TMSs after the second duplicated domain, SglT has four extra C-terminal TMSs, and BetP has two extra TMSs before the first duplicated domain. UraA and SulP on the other hand have two extra TMSs at the N-terminus of each duplicated domain unit. These observations imply that multiple hairpin and domain duplication events occurred during the evolution of the APC superfamily. We suggest that the five TMS architecture was primordial and that families gained two TMSs on either side of this basic structure via dissimilar hairpin duplications either before or after intragenic duplication. Evidence for homology between TMSs 1-2 of AdiC and TMSs 1-2 and 3-4 of UraA suggests that the 7+7 topology arose via an internal duplication of the N-terminal hairpin loop within the five TMS repeat unit followed by duplication of the 7 TMS domain.

Turning data on a dime

By enlisting help from a robot to assemble precise structures, researchers have guided telecommunications-wavelength light around multiple hairpin turns in a three-dimensional photonic crystal.

Construction of multiple shRNAs expression vector that inhibits FUT1 gene expression and production of the transgenic SCNT embryos in vitro

Enterotoxigenic Escherichia coli F18 is a major pathogen that causes postweaning diarrhoea and edema disease in piglets. The alpha(1,2)-fucosyltransferase (FUT1) gene has been identified as an ideal candidate gene for controlling the expression of the receptor for ECF18 bacteria. Therefore, the use of RNA interference (RNAi) to study the function of the FUT1 gene and to produce FUT1 knockdown transgenic pig would be highly beneficial. We developed an effective strategy for the expression of multiple small hairpin RNA simultaneously using multiple RNA polymerase III (hU6, hH1, mU6 and h7SK) promoters in a single vector to knockdown the FUT1 gene. Stable FUT1 knockdown transgenic fibroblast lines were generated by transfecting porcine fetal fibroblasts with the constructed vectors. Real-time RT-PCR indicated that the mRNA level of FUT1 in the transgenic fibroblast lines was significantly lower than that in the control, as much as 29 %. Finally, we successfully obtained transgenic SCNT porcine embryos. Overall, the results demonstrated that this vector-based RNAi expression system is an efficient approach to knockdown FUT1 gene expression in porcine fetal fibroblast cells, which could thereby provide donor cells for somatic cell nuclear cloning and the potential production of a marker-free transgenic pig resistant to F18 related diseases. Furthermore, it also provides strong evidence that this approach could be useful both in the production of transgenic livestock resistant to disease, and in the development of effective strategies for the suppression of gene expression in clinical gene therapy.