A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants.

Plant expression vectors are essential tools for gene functional analysis and molecular plant breeding. The gene of interest is transferred to the vector by molecular cloning technology. Nimble Cloning is a newly developed molecular cloning method with the advantages of simplicity, efficiency, and standardization. In this study, we developed a "pNC" vector system that contains 55 Nimble Cloning-compatible vectors for functional analysis of genes in plants. These vectors contain the NC frame flanked by unique adapters for one-step and standardized Nimble Cloning. We demonstrate that the pNC vectors are convenient and effective for the functional analysis of plant genes, including the study of gene ectopic expression, protein subcellular localization, protein-protein interaction, gene silencing (RNAi), virus-induced gene silencing, promoter activity, and CRISPR-Cas9-mediated genome editing. The "pNC" vector system represents a high-throughput toolkit that can facilitate the large-scale analysis of plant functional genomics.

ZNF3 regulates proliferation, migration and invasion through MMP1 and TWIST in colorectal cancer.

Colorectal cancer (CRC) is a malignant tumor with a high incidence and mortality worldwide. Currently, the underlying molecular mechanisms of CRC are still unclear. Zinc finger protein 3 (ZNF3) is a zinc-finger transcription factor that has been reported as a candidate for breast cancer prognosis, suggesting its involvement in the regulation of tumorigenesis. However, the association between ZNF3 and CRC remains unknown. To investigate the role of ZNF3 in CRC, we first analyze the correlation between ZNF3 expression and CRC, and the results demonstrate that ZNF3 is highly expressed in CRC tissue and cells, which is associated with the age of CRC patients. In vitro studies show that ZNF3 overexpression promotes CRC cell migration. Compared to control cells, knockdown of ZNF3 markedly suppresses CRC cell proliferation, migration and invasion and promotes G0/G1 phase cell cycle arrest. The expressions of the EMT-related markers TWIST and MMP1 are significantly decreased when ZNF3 is silenced. Additionally, overexpression of MMP1 and TWIST exacerbates CRC cell proliferation, accelerates the S phase cell cycle in ZNF3-knockdown SW480 cells, and increases cell migration and invasion through Transwell chambers. These data suggest that ZNF3 is involved in cellular proliferation, migration and invasion by regulating MMP1 and TWIST in CRC cells.

Meta-Analysis on the Association between Brain-Derived Neurotrophic Factor Polymorphism rs6265 and Ischemic Stroke, Poststroke Depression.

BACKGROUND: Ischemic stroke is a multifactorial neurologic injury that causes mortality and disability worldwide. Poststroke depression is the most important neuropsychiatric consequence of stroke. Brain-derived neurotrophic factor is a neurotrophin family member that plays key role in regulating neuron survival and differentiation. Studies found a polymorphism in brain-derived neurotrophic factor gene (rs6265) may associate with the ischemic stroke and poststroke depression risk. However, the results are inconclusive and inconsistent. METHODS: In the present meta-analysis, the database PubMed, Embase, Cochrane Central Register of Controlled Trials, CNKI, and Chinese Biomedical Literature Database were searched until July 9, 2017. RESULTS: Seven studies with 1287 cases and 1032 controls were included for the meta-analysis of ischemic stroke, and five studies with 272 cases and 503 controls were included for poststroke depression. The results indicated that the GG genotype of brain-derived neurotrophic factor is related to a significantly lower risk of ischemic stroke in the homozygous and dominant models (odds ratio = .57 and .80, respectively). No significant relation was found between rs6265 and poststroke depression. CONCLUSIONS: Thus, brain-derived neurotrophic factor rs6265 might be recommended as a predictor of susceptibility of ischemic stroke. However, the results of this meta-analysis should be interpreted with caution because of the heterogeneity between studies and low sample size. Further studies are needed to evaluate the associations between rs6265 and poststroke depression, especially in Caucasians, with large sample size.

Transient receptor potential melastatin 2 channels (TRPM2) mediate neonatal hypoxic-ischemic brain injury in mice.

Transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, is reported to mediate brain damage following ischemic insults in adult mice. However, the role of TRPM2 channels in neonatal hypoxic-ischemic brain injury remains unknown. We hypothesize that TRPM2+/- and TRPM2-/- neonatal mice have reduced hypoxic-ischemic brain injury. To study the effect of TRPM2 on neonatal brain damage, we used 2,3,5-triphenyltetrazolium chloride (TTC) staining to assess the infarct volume and whole brain imaging to assess morphological changes in the brain. In addition, we also evaluated neurobehavioral outcomes for sensorimotor function 7days following hypoxic-ischemic brain injury. We report that the infarct volumes were significantly smaller and behavioral outcomes were improved in both TRPM2+/- and TRPM2-/- mice compared to that of wildtype mice. Next, we found that TRPM2-null mice showed reduced dephosphorylation of GSK-3ß following hypoxic ischemic injury unlike sham mice. TRPM2+/- and TRPM2-/- mice also had reduced activation of astrocytes and microglia in ipsilateral hemispheres, compared to wildtype mice. These findings suggest that TRPM2 channels play an essential role in mediating hypoxic-ischemic brain injury in neonatal mice. Genetically eliminating TRPM2 channels can provide neuroprotection against hypoxic-ischemic brain injury and this effect is elicited in part through regulation of GSK-3ß.

Modular construction of plasmids by parallel assembly of linear vector components.

Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

Parallel assembly for multiple site-directed mutagenesis of plasmids.

A parallel assembly method for multiple site-directed mutagenesis of plasmids was developed here based on Golden Gate cloning. It takes advantage of type IIs restriction enzymes and T4 DNA ligase to assemble multiple DNA fragments into a plasmid by a defined order. This method can accommodate multiple plasmid mutagenesis at any desired position with all three sequence modification types (substitution, deletion, and insertion) simultaneously. Furthermore, it can be used to create otherwise difficult-to-make mutants-larger deletions and insertions and mutagenesis on larger plasmids. The processes of mutagenesis can be completed quickly by a single restriction-ligation reaction.

High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

Simple construction of chimeric hairpin RNA for virus resistance in plants.

RNA silencing has been adopted to develop virus-resistant plants through expression of virus-derived hairpin RNAs. Due to the high sequence specificity of RNA silencing, this technology has been limited to the targeting of single viruses. Simultaneous targeting of multiple viruses or plant genes can be achieved by using a chimeric cassette. In this study, a simple method was developed to construct chimeric hairpin RNA rapidly and efficiently. This method splices two DNA fragments from viruses or plant genes to be a chimeric sequence using Overlap Extension PCR (OE-PCR); then this chimeric sequence was assembled with an intron sequence to generate an intron-containing hairpin RNA construct in one step mediated by OE-PCR. This method is neither dependent on restriction enzymes nor requires expensive consumables, so a chimeric hairpin RNA can be constructed rapidly and costlessly. Two chimeric hairpin RNA constructs were amplified successfully using this method, with the targeting sequences from both papaya ringspot virus (PRSV) and two plant genes encoding translation initiation factors eIF4E and eIFiso4E. This novel method is a useful strategy to construct chimeric hairpin RNA for RNA silencing in plants.

Rapid one-step construction of hairpin RNA.

Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50bp to 484bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA.