S ilencing of SCD and SREBP1 Genes by Short Hairpin RNAs in Chicken Hepatic Cells in Vitro

RNA interference by short hairpin RNAs (shRNAs) is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes involved in denovo lipid biosynthesis were designed upon considering parameters such as secondary structures of shRNAs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target), synthesized and cloned in pENTR/U6 entry vector to knockdown the expression of SCD and SREBP1 genes. After transfection of these shRNA constructs into the chicken embryonic hepatocytes, expressions of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes was observed in hepatocytes. The shRNAs against SCD gene showed the knock down efficiency ranged from 20.4% (shRNA5) to 74.2% (shRNA2). In case of SREBP1 gene, the shRNAs showed knock-down efficiency ranging from 26.8% (shRNA4) to 95.85% (shRNA1). The shRNAs against both the genes introduced in chicken hepatocyte cells did not show any significant impact on expression of immune response genes (IFNA and IFNB) in those cells. These results clearly demonstrated the successful down regulation of the expression of SCD and SREBP1 genes by the shRNA molecules against both the target genes under in vitro condition. It is concluded that the shRNA molecules against SCD and SREBP1 genes showed great potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cells.

The Zymoseptoria tritici ORFeome: A Functional Genomics Community Resource

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

A New Escherichia coli Entry Vector Series (pIIS18) for Seamless Gene Cloning Using Type IIS Restriction Enzymes

A series of new Escherichia coli entry vectors (pIIS18-SapI, pIIS18-BsmBI, pIIS18-BsaI, pIIS18-BfuAI-1, and pIIS18-BfuAI-2) was constructed based on a modified pUC18 backbone, which carried newly designed multiple cloning sites, consisting of two facing type IIS enzyme cleavage sites and one blunt-end enzyme cleavage site. These vectors are useful for seamless gene cloning.

The Zymoseptoria tritici ORFeome: a functional genomics community resource

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.

Gateway Entry Vector Library of Wolbachia pipientis Candidate Effectors from Strain wMel

Wolbachia pipientis is an intracellular symbiont that modifies host biology using a type IV secretion system to inject bacterial effectors into the host cytoplasm. We utilized a bioinformatics approach to predict Wolbachia effectors and cloned the candidates into an entry vector, which can be utilized for subsequent analyses.

Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

Solutions to the modified Korteweg–de Vries equation

This is a continuation of [Notes on solutions in Wronskian form to soliton equations: Korteweg–de Vries-type, arXiv:nlin.SI/0603008]. In the present paper, we review solutions to the modified Korteweg–de Vries equation in terms of Wronskians. The Wronskian entry vector needs to satisfy a matrix differential equation set which contains complex operation. This fact makes the analysis of the modified Korteweg–de Vries to be different from the case of the Korteweg–de Vries equation. To derive complete solution expressions for the matrix differential equation set, we introduce an auxiliary matrix to deal with the complex operation. As a result, the obtained solutions to the modified Korteweg–de Vries equation are categorized into two types: solitons and breathers, together with their limit cases. Besides, we give rational solutions to the modified Korteweg–de Vries equation in Wronskian form. This is derived with the help of a Galilean transformed version of the modified Korteweg–de Vries equation. Finally, typical dynamics of the obtained solutions are analyzed and illustrated. We also list out the obtained solutions and their corresponding basic Wronskian vectors in the conclusion part.