A Versatile Expression Platform in Insects and Cereals Based on a Cytorhabdovirus

2021 ◽  
pp. 163-170
Author(s):  
Ji-Hui Qiao ◽  
Qiang Gao ◽  
Ying Zang ◽  
Xiao-Dong Fang ◽  
Xian-Bing Wang
Keyword(s):  
Expression Platform

scholarly journals Numaswitch: an efficient high-titer expression platform to produce peptides and small proteins

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bach-Ngan Nguyen ◽  
Florian Tieves ◽  
Thomas Rohr ◽  
Hilke Wobst ◽  
Felix S. Schöpf ◽  
...  
Keyword(s):  
Fermentation Broth ◽  
High Titer ◽  
New Technology ◽  
Active Pharmaceutical Ingredients ◽  
Pharmaceutical Ingredients ◽  
Expression Platform ◽  
Small Proteins ◽  
Production Platform ◽  
Β Amyloid ◽  
Cost Efficient

AbstractThe production of peptides as active pharmaceutical ingredients (APIs) by recombinant technologies is of emerging interest. A reliable production platform, however, is still missing due the inherent characteristics of peptides such as proteolytic sensitivity, aggregation and cytotoxicity. We have developed a new technology named Numaswitch solving present limitations. Numaswitch was successfully employed for the production of diverse peptides and small proteins varying in length, physicochemical and functional characteristics, including Teriparatide, Linaclotide, human β-amyloid and Serum amyloid A3. Additionally, the potential of Numaswitch for a cost-efficient commercial production is demonstrated yielding > 2 g Teriparatide per liter fermentation broth in a quality meeting API standard.

scholarly journals A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 660
Author(s):  
Lu Tan ◽  
Yiwen Zhang ◽  
Xingxing Wang ◽  
Dal Young Kim
Keyword(s):  
Expression Vectors ◽  
Structural Genes ◽  
Host Restriction ◽  
Expression Platform ◽  
Mosquito Cells ◽  
Wide Range ◽  
Restriction Property ◽  
Infected Cells ◽  
Multiple Levels ◽  
Novel Strategy

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.

scholarly journals Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii

Biology ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 46 ◽  
Author(s):  
Lola Esland ◽  
Marco Larrea-Alvarez ◽  
Saul Purton
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Reporter Genes ◽  
Bioactive Metabolites ◽  
Cell Protein ◽  
Cell Factory ◽  
Expression Platform ◽  
Foreign Genes ◽  
Valuable Compounds ◽  
High Level

Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.

scholarly journals Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

2020 ◽  
Author(s):  
Carmen Lopez ◽  
Mingfeng Cao ◽  
Zhanyi Yao ◽  
Zengyi Shao
Keyword(s):  
Yarrowia Lipolytica ◽  
Plasmid Stability ◽  
Critical Role ◽  
Fold Increase ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform ◽  
Regulatory Dna ◽  
Selection Of

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

scholarly journals 75 Epigenetic marks within the swine placenta are differentially expressed due to season

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 33-33
Author(s):  
Robin Cheek ◽  
Lea A Rempel ◽  
Jeremy Miles
Keyword(s):  
Breeding Season ◽  
Random Effect ◽  
Chromatin Modification ◽  
Prenatal Development ◽  
Treatment Groups ◽  
Downstream Targets ◽  
Downstream Target ◽  
Expression Platform ◽  
Semen Storage ◽  
And Control

Abstract The objective of the current project was to validate expression of known chromatin modification genes and assess downstream targets in d45 swine placenta derived from seasonal semen collections and breedings and semen storage. Six treatment groups were developed based upon: season of semen collection (cool or warm), storage of semen (cooled-extended or cryopreserved), and season of breeding (summer or winter) with 8 litters per group as follows: 1) cool/cooled-extended/winter, 2) cool/cryopreserved/winter, 3) warm/cryopreserved/winter, 4) warm/cooled-extended/summer, 5) warm/cooled-extended/summer, and 6) cool/cryopreserved/summer. RNA was extracted from the placenta of a small, medium, and large fetus from each litter, pooled by litter, reverse transcribed, and used for qPCR. Three genes; ATF2, ASH2L, and SMYD3, were validated. Downstream gene targets, ILIB and NANOG, were also tested. Interactions and main effects were tested using the mixed procedure of SAS with boar as a random effect. Placental ASH2L had a tendency for increased expression from summer breedings (P >0.05), similar to previous findings using a different expression platform. Placental expression of ATF2 was greatest (P< 0.05) from placenta derived by summer breedings. A downstream target of ATF2, ILIB, had greater (P< 0.05) placental expression from summer breedings and semen that was cooled-extended as compared to cryopreserved (P< 0.05). Validation of placental expression of SMYD3 was greater from cooled-extended semen in contrast to cryopreserved semen (P< 0.05). These data were unexpected, as previously we determined a difference by breeding season only. However, NANOG, downstream of SMYD3, had greater placental expression from summer breedings (P< 0.05). The current study validated previous results indicating chromatin modification genes were primarily influenced predominantly by breeding season and to a lesser extent, semen storage techniques. Downstream targets were also influenced by breeding season likely influencing prenatal development. These data support that epigenetic modifications persist, even though modern swine systems regulate and control temperature and lighting.

scholarly journals A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

PLoS ONE ◽  
2015 ◽  
Vol 10 (10) ◽  
pp. e0140691 ◽  
Author(s):  
Xiaodong Xiao ◽  
Yan Chen ◽  
Sheila Mugabe ◽  
Changshou Gao ◽  
Christine Tkaczyk ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Screening ◽  
Expression Platform ◽  
Phage Libraries ◽  
Dual Expression

scholarly journals Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 75 ◽  
Author(s):  
Sebastian Escobar-Aguirre ◽  
Duxan Arancibia ◽  
Amanda Escorza ◽  
Cristián Bravo ◽  
María Andrés ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lines ◽  
Gene Function ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Guide Rna ◽  
Bicistronic Vector ◽  
Expression Platform ◽  
Fish Cells ◽  
U6 Rna

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function.

Leveraging Automation toward Development of a High-Throughput Gene Expression Profiling Platform

2020 ◽  
pp. 247255522095659
Author(s):  
Jing Chen ◽  
Alan Futran ◽  
Austin Crithary ◽  
Sha Li ◽  
Alex Wolicki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Discovery ◽  
High Throughput ◽  
Screening Strategy ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Expression Platform ◽  
Program Support ◽  
Drug Discovery Program ◽  
One Step

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

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