scholarly journals Rapid and Efficient Colony-PCR for High Throughput Screening of Genetically Transformed Chlamydomonas reinhardtii

Life ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 186
Author(s):  
Serge Basile Nouemssi ◽  
Manel Ghribi ◽  
Rémy Beauchemin ◽  
Fatma Meddeb-Mouelhi ◽  
Hugo Germain ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
High Throughput ◽  
High Throughput Screening ◽  
Culture Media ◽  
Colony Pcr ◽  
Homologous Transformation ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Selection For ◽  
Selection Of

Microalgae biotechnologies are rapidly developing into new commercial settings. Several high value products already exist on the market, and biotechnological development is focused on genetic engineering of microalgae to open up future economic opportunities for food, fuel and pharmacological production. Colony-polymerase chain reaction (colony-PCR or cPCR) is a critical method for screening genetically transformed microalgae cells. However, the ability to rapidly screen thousands of transformants using the current colony-PCR method, becomes a very laborious and time-consuming process. Herein, the non-homologous transformation of Chlamydomonas reinhardtii using the electroporation and glass beads methods generated more than seven thousand transformants. In order to manage this impressive number of clones efficiently, we developed a high-throughput screening (HTS) cPCR method to rapidly maximize the detection and selection of positively transformed clones. For this, we optimized the Chlamydomonas transformed cell layout on the culture media to improve genomic DNA extraction and cPCR in 96-well plate. The application of this optimized HTS cPCR method offers a rapid, less expensive and reliable method for the detection and selection of microalgae transformants. Our method, which saves up to 80% of the experimental time, holds promise for evaluating genetically transformed cells and selection for microalgae-based biotechnological applications such as synthetic biology and metabolic engineering.

A high throughput screening method for the selection of zeolites for binding cations

2005 ◽  
pp. 4167 ◽  
Author(s):  
Edel M. Minogue ◽  
Tammy P. Taylor ◽  
Anthony K. Burrell ◽  
George J. Havrilla ◽  
Benjamin P. Warner ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Selection Of

scholarly journals Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto

2016 ◽  
Vol 106 (11) ◽  
pp. 1366-1375 ◽  
Author(s):  
Xianzhou Nie ◽  
Darcy Sutherland ◽  
Virginia Dickison ◽  
Mathuresh Singh ◽  
Agnes M. Murphy ◽  
...  
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Marker Assisted Selection ◽  
High Resolution Melting ◽  
Chromosome Region ◽  
Sts Markers ◽  
Reverse Primer ◽  
Selection For ◽  
Development And Validation ◽  
Selection Of

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato ‘Barbara’ (Rrrr) and ‘AC Chaleur’ (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

scholarly journals Recent Advances in In-Vitro Assays for Type 2 Diabetes Mellitus: An Overview

2020 ◽  
Vol 16 (1) ◽  
pp. 13-23
Author(s):  
Nazmina Vhora ◽  
Ujjal Naskar ◽  
Aishwarya Hiray ◽  
Abhijeet S. Kate ◽  
Alok Jain
Keyword(s):  
Type 2 Diabetes ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antidiabetic Activity ◽  
Screening Methods ◽  
Selection Of

BACKGROUND: A higher rate of attenuation of molecules in drug discovery has enabled pharmaceutical companies to enhance the efficiency of their hit identification and lead optimization. Selection and development of appropriate in-vitro and in-vivo strategies may improve this process as primary and secondary screening utilize both strategies. In-vivo approaches are too relentless and expensive for assessing hits. Therefore, it has become indispensable to develop and implement suitable in-vitro screening methods to execute the required activities and meet the respective targets. However, the selection of an appropriate in-vitro assay for specific evaluation of cellular activity is no trivial task. It requires thorough investigation of the various parameters involved. AIM: In this review, we aim to discuss in-vitro assays for type 2 diabetes (T2D), which have been utilized extensively by researchers over the last five years, including target-based, non-target based, low-throughput, and high-throughput screening assays. METHODS: The literature search was conducted using databases including Scifinder, PubMed, ScienceDirect, and Google Scholar to find the significant published articles. DISCUSSION and CONCLUSION: The accuracy and relevance of in-vitro assays have a significant impact on the drug discovery process for T2D, especially in assessing the antidiabetic activity of compounds and identifying the site of effect in high-throughput screening. The report reviews the advantages, limitations, quality parameters, and applications of the probed invitro assays, and compares them with one another to enable the selection of the optimal method for any purpose. The information on these assays will accelerate numerous procedures in the drug development process with consistent quality and accuracy.

scholarly journals High-throughput screening of biomolecules using cell-free gene expression systems

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Luis E Contreras-Llano ◽  
Cheemeng Tan
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Screening Methods ◽  
Free Gene ◽  
On Chip ◽  
Translation Systems ◽  
Selection Of

Abstract The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.

scholarly journals Imaging-Based High-Throughput Screening Assay To Identify New Molecules with Transmission-Blocking Potential against Plasmodium falciparum Female Gamete Formation

2015 ◽  
Vol 59 (6) ◽  
pp. 3298-3305 ◽  
Author(s):  
Celia Miguel-Blanco ◽  
Joël Lelièvre ◽  
Michael J. Delves ◽  
Ana I. Bardera ◽  
Jesús L. Presa ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Female Gamete ◽  
Transmission Blocking ◽  
Content Type ◽  
Female Gametocyte ◽  
Gamete Formation ◽  
Selection Of

ABSTRACTIn response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated byPlasmodium falciparumstage V gametocytes. Lately, metabolic activity has been usedin vitroas a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated theP. falciparumfemale gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z′ factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. TheP. falciparumFGAA has been proven to be a robust HTS assay faithful toPlasmodiumtransmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential.

High-throughput screening and selection of mammalian cells for enhanced protein production

2016 ◽  
Vol 11 (7) ◽  
pp. 853-865 ◽  
Author(s):  
Joseph J. Priola ◽  
Nathan Calzadilla ◽  
Martina Baumann ◽  
Nicole Borth ◽  
Christopher G. Tate ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Protein Production ◽  
Selection Of

scholarly journals Data-Driven Derivation of an “Informer Compound Set” for Improved Selection of Active Compounds in High-Throughput Screening

2016 ◽  
Vol 56 (9) ◽  
pp. 1622-1630 ◽  
Author(s):  
Shardul Paricharak ◽  
Adriaan P. IJzerman ◽  
Jeremy L. Jenkins ◽  
Andreas Bender ◽  
Florian Nigsch
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Data Driven ◽  
Active Compounds ◽  
Selection Of
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