High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

AbstractGene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing make it possible to directly modify the target genes in their native chromosomal location, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the obtained results provided by the direct chromosomal tagging achievable by genome editing strategies. These simple and customizable recombineering toolsets and protocols allow for high-throughput generation of a variety of genetic modifications. In addition, a highly efficient recombinase-mediated cassette exchange system has been developed to facilitate the transfer of the desired sequences from a BAC clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis. The utility of this system is demonstrated by the generation of over 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes, and the characterization of the translational reporter expression patterns for 14 auxin biosynthesis genes.

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High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab369 ◽

2021 ◽

Author(s):

Gerard Terradas ◽

Anita Hermann ◽

Anthony A James ◽

William McGinnis ◽

Ethan Bier

Keyword(s):

Expression Patterns ◽

Resistant Mutant ◽

Gene Drive ◽

Anopheles Mosquitoes ◽

Transgenic Lines ◽

Endogenous Genes ◽

Pathogen Control ◽

Beneficial Traits ◽

Germline Transcript ◽

Female Germline

Abstract Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNos-Cd1 drive (nos-Cas9) in An. gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly co-expressed with endogenous genes in stem and pre-meiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct co-localization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.

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Overexpression of a Banana Aquaporin Gene MaPIP1;1 Enhances Tolerance to Multiple Abiotic Stresses in Transgenic Banana and Analysis of Its Interacting Transcription Factors

Frontiers in Plant Science ◽

10.3389/fpls.2021.699230 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yi Xu ◽

Juhua Liu ◽

Caihong Jia ◽

Wei Hu ◽

Shun Song ◽

...

Keyword(s):

Abiotic Stresses ◽

Promoter Region ◽

Expression Patterns ◽

Soluble Sugar ◽

Ion Leakage ◽

Aquaporin Gene ◽

Gene Response ◽

Transcription Factor Genes ◽

Transgenic Lines ◽

Response To Stress

Aquaporins can improve the ability of plants to resist abiotic stresses, but the mechanism is still not completely clear. In this research, overexpression of MaPIP1;1 in banana improved tolerance to multiple stresses. The transgenic plants resulted in lower ion leakage and malondialdehyde content, while the proline, chlorophyll, soluble sugar, and abscisic acid (ABA) contents were higher. In addition, under high salt and recovery conditions, the content of Na+ and K+ is higher, also under recovery conditions, the ratio of K+/Na+ is higher. Finally, under stress conditions, the expression levels of ABA biosynthesis and response genes in the transgenic lines are higher than those of the wild type. In previous studies, we proved that the MaMADS3 could bind to the promoter region of MaPIP1;1, thereby regulating the expression of MaPIP1;1 and affecting the drought tolerance of banana plants. However, the mechanism of MaPIP1;1 gene response to stress under different adversity conditions might be regulated differently. In this study, we proved that some transcription factor genes, including MaERF14, MaDREB1G, MaMYB1R1, MaERF1/39, MabZIP53, and MaMYB22, showed similar expression patterns with MaPIP1;1 under salt or cold stresses, and their encoded proteins could bind to the promoter region of MaPIP1;1. Here we proposed a novel MaPIP1;1-mediated mechanism that enhanced salt and cold tolerance in bananas. The results of this study have enriched the stress-resistant regulatory network of aquaporins genes and are of great significance for the development of molecular breeding strategies for stress-resistant fruit crops.

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High-Throughput Sequencing is a Crucial Tool to Investigate the Contribution of Human Endogenous Retroviruses (HERVs) to Human Biology and Development

Viruses ◽

10.3390/v12060633 ◽

2020 ◽

Vol 12 (6) ◽

pp. 633 ◽

Cited By ~ 1

Author(s):

Maria Paola Pisano ◽

Nicole Grandi ◽

Enzo Tramontano

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Developmental Stages ◽

Large Fraction ◽

Expression Patterns ◽

Cell Types ◽

Endogenous Retroviruses ◽

Human Endogenous Retroviruses ◽

Retroviral Infections ◽

The Impact

Human Endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that represent a large fraction of our genome. Their transcriptional activity is finely regulated in early developmental stages and their expression is modulated in different cell types and tissues. Such activity has an impact on human physiology and pathology that is only partially understood up to date. Novel high-throughput sequencing tools have recently allowed for a great advancement in elucidating the various HERV expression patterns in different tissues as well as the mechanisms controlling their transcription, and overall, have helped in gaining better insights in an all-inclusive understanding of the impact of HERVs in biology of the host.

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Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

PLoS Pathogens ◽

10.1371/journal.ppat.1007903 ◽

2019 ◽

Vol 15 (10) ◽

pp. e1007903

Author(s):

David F. Read ◽

Edmond Atindaana ◽

Kalyani Pyaram ◽

Feng Yang ◽

Sarah Emery ◽

...

Keyword(s):

High Throughput ◽

Expression Patterns ◽

High Throughput Analysis ◽

Throughput Analysis ◽

Hiv 1

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Differential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress

Plants ◽

10.3390/plants9101367 ◽

2020 ◽

Vol 9 (10) ◽

pp. 1367

Author(s):

Edgar Baldemar Sepúlveda-García ◽

José Francisco Pulido-Barajas ◽

Ariana Arlene Huerta-Heredia ◽

Julián Mario Peña-Castro ◽

Renyi Liu ◽

...

Keyword(s):

Oxidative Stress ◽

Zea Mays ◽

High Throughput ◽

Small Rna ◽

Abiotic Stresses ◽

Crop Production ◽

High Throughput Sequencing ◽

Expression Patterns ◽

Evolutionary Divergence ◽

Rna Profiling

Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (Zea mays ssp. parviglumis), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

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Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0663 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1185-1196 ◽

Cited By ~ 34

Author(s):

María-Cruz Marín ◽

José-Rodrigo Rodríguez ◽

Alberto Ferrús

Keyword(s):

Transcription Initiation ◽

Troponin I ◽

Regulatory Element ◽

Expression Patterns ◽

In Silico Analysis ◽

Initiation Site ◽

Transcription Initiation Site ◽

Upstream Regulatory Element ◽

Transgenic Lines

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

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Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation

International Journal of Molecular Sciences ◽

10.3390/ijms23020900 ◽

2022 ◽

Vol 23 (2) ◽

pp. 900

Author(s):

Taja Jeseničnik ◽

Nataša Štajner ◽

Sebastjan Radišek ◽

Ajay Kumar Mishra ◽

Katarina Košmelj ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Expression Patterns ◽

Pathogenic Fungi ◽

Absolute Quantification ◽

Stem Loop ◽

Humulus Lupulus L ◽

Verticillium Nonalfalfae ◽

Highly Virulent ◽

Plant Fungal Pathogen

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

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Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

10.1101/666941 ◽

2019 ◽

Author(s):

David F. Read ◽

Edmond Atindaana ◽

Kalyani Pyaram ◽

Feng Yang ◽

Sarah Emery ◽

...

Keyword(s):

Gene Expression ◽

Infected Cell ◽

High Throughput ◽

Expression Patterns ◽

Clonal Expansion ◽

High Throughput Analysis ◽

Integration Sites ◽

Infected Cells ◽

Hiv 1 ◽

Over Time

AbstractHIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication. Initial findings in immortalized cells were validated using CD4+ cells from donor blood. Tracking clonal behavior by proviral “zip codes” indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in thenefopen reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP-cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in genes’ sense orientation were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP-population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.SummaryVery few HIV-1 infected cells persist in patients for more than a couple days, but those that do pose life-long health risks. Strategies designed to eliminate these cells have been based on assumptions about what viral properties allow infected cell survival. However, such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because much of the research underlying assumptions about virus persistence has been focused on a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views. Here, we developed a high-throughput approach to study hundreds of distinct HIV-1 infected cells and their progeny over time in an unbiased way. This revealed that each virus established its own pattern of gene expression that, upon infected cell division, was stably transmitted to all progeny cells. Expression patterns consisted of alternating waves of activity and inactivity, with the extent of activity differing among infected cell families over a 1000-fold range. The dynamics and variability among infected cells and within complex populations that the work here revealed has not previously been evident, and may help establish more accurate correlates of persistent HIV-1 infection.

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