egfp reporter gene (enhanced green fluorescent protein)  (Addgene inc)

Journal: bioRxiv

Article Title: From Pigments to Precision: Exploring Genetic Transformation and Genome Editing in Wheat and Barley

doi: 10.1101/2024.12.03.626565

Figure Lengend Snippet: Protoplasts were transformed with linearized DNA by PEG-mediated transformation (top images); embryos were transformed with circular DNA by biolistic transformation (bottom images). Promoters activity was evaluated by monitoring GFP fluorescence intensity using a fluorescent microscope. Scale bar in upper and lower images = 100 and 500 µm, respectively.

Article Snippet: We linked each promoter to the wheat codon-optimized enhanced green fluorescent protein ( eGFP ) gene (Figure S2a) and cloned the constructs into the pJET1.2 vector (Thermo Fisher Scientific).

Techniques: Transformation Assay, Activity Assay, Fluorescence, Microscopy

Journal: Frontiers in Plant Science

Article Title: Fine-tuning CRISPR/Cas9 gene editing in common bean ( Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models

doi: 10.3389/fpls.2023.1233418

Figure Lengend Snippet: (A) Position of the sgRNAs targeting the raffinose synthase ( PvRS1 and PvRS2 ) and stachyose synthase ( PvSS ) genes in P. vulgaris . (B) Schematic representation of the T-DNA of the pMR356-GFP and pMR394-GFP vectors. The A. thaliana codon-optimized Cas9 gene ( Atco-Cas9 ) was driven by a Parsley Ubiquitin promoter (PcUbi) in pMR356-GFP whereas Atco-Cas9 was driven by a 2X35S-Ω promoter in pMR394-GFP. In both vectors, the Atco-Cas9 gene was fused to a nuclear localization signal derived from simian virus 40 (SV40 NLS) and transcription was terminated by a A. thaliana heat shock protein 18.2 terminator (AtHSP18.2 ter). The transcription of the sgRNA was driven by a U6 promoter. The enhanced green fluorescent protein ( eGFP ) gene was placed under the regulation of a Cauliflower Mosaic Virus 35S promoter (CaMV 35S) and nopaline synthase terminator (NOS ter).

Article Snippet: For this study, both vectors were further improved by the incorporation of an enhanced green fluorescent protein gene ( eGFP ), under the control of a 35S promoter and terminated by a nopaline synthase terminator (NOS ter) with polyadenylation signal (transcription unit originating from the pGFPGUSPlus vector; RRID: Addgene_64401), using the NEBuilder HiFi DNA Assembly master mix (New England Biolabs, Ipswich, Massachusetts, United States of America).

Techniques: Ubiquitin Proteomics, Derivative Assay, Virus

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: (A) Effect of TmAttacin1a ( TmAtt1a ) knockdown on survival of Pseudomonas entomophila infected Tenebrio molitor females over 35 days. Two independent experiments each performed on 15 females per treatment group. Each dsRNA-treated or dsRNA-untreated group was infected with P. entomophila on day 4 after dsRNA treatment based on the data on knockdown efficiency ( Figure S7 ). Pink dashed line: Full control-PBS; Turquoise dashed line: ds EGFP - PBS; Purple dashed line: ds TmAtt1a - PBS; Pink line: Full control - P . entomophila ; Turquoise line: ds EGFP - P . entomophila ; Purple line: ds TmAtt1a - P . entomophila . (B) Bacterial load in ds TmAtt1a -silenced T. molitor challenged with P. entomophila at 1-, 2-, and 3-day post-infections. The colony-forming units (CFU) recovered from tissues of T. molitor females from ds TmAtt1a (purple) and ds EGFP (control, turquoise). In the box plots, the lower (first) quartile is the closest boundary to zero, the line within the box marks the median (second quartile), and the upper (third) quartile. Each dot represents the CFU count in an individual beetle. The bars represent the 1.5 interquartile. Sample sizes are given in Table S2 .

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Control

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: (A) Effect of TmAttacin1b ( TmAtt1b ) knockdown on survival of Pseudomonas entomophila infected Tenebrio molitor females over 35 days. ds EGFP was injected as a negative control for the nonspecific effects of dsRNA and the third group includes dsRNA untreated control. Two independent experiments each performed on 15 females per treatment group. Each dsRNA-treated or dsRNA-untreated groups were infected with P. entomophila on day 3 after dsRNA treatment based on the data on knockdown efficiency ( Figure S8 ). Pink dashed line: Full control-PBS; Turquoise dashed line: ds EGFP - PBS; Burgundy dashed line: ds TmAtt1b - PBS; Pink line: Full control - P . entomophila ; Turquoise line: ds EGFP - P . entomophila ; Burgundy line: ds TmAtt1b - P . entomophila . (B) Bacterial load in ds TmAtt1b -silenced T. molitor challenged with P. entomophila at 1-, 2-, and 3-day post-infections. The colony-forming units (CFU) recovered from tissues of T. molitor females from ds TmAtt1b (burgundy) and ds EGFP (control, turquoise). In the box plots, the lower (first) quartile is the closest boundary to zero, the line within the box marks the median (second quartile), and the upper (third) quartile. Each dot represents the CFU count in an individual beetle. The bars represent the 1.5 interquartile. Sample sizes are given in Table S2 .

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Injection, Negative Control, Control

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: (A) Effect of TmAttacin2 ( TmAtt2 ) knockdown on survival of Pseudomonas entomophila infected Tenebrio molitor females over 35 days. ds EGFP was injected as a negative control for the nonspecific effects of dsRNA and the third group includes dsRNA untreated control. Two independent experiments each performed on 15 females per treatment group. Each dsRNA-treated or dsRNA-untreated groups were infected with P. entomophila on day 3 after dsRNA treatment based on the data on knockdown efficiency ( Figure S9 ). Pink dashed line: Full control-PBS; Turquoise dashed line: ds EGFP - PBS; Navy blue dashed line: ds TmAtt2 - PBS; Pink line: Full control - P . entomophila ; Turquoise line: ds EGFP - P . entomophila ; Navy blue line: ds TmAtt2 - P . entomophila . (B) Bacterial load in ds TmAtt2 -silenced T. molitor challenged with P. entomophila at 1-, 2-, and 3-day post-infections. The colony-forming units (CFU) recovered from tissues of T. molitor females from ds TmAtt2 (navy blue) and ds EGFP (control, turquoise). In the box plots, the lower (first) quartile is the closest boundary to zero, the line within the box marks the median (second quartile), and the upper (third) quartile. Each dot represents the CFU count in an individual beetle. The bars represent the 1.5 interquartile. Sample sizes are given in Table S2 .

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Injection, Negative Control, Control

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: (A) Effect of TmAttacin1a-2 ( TmAtt1a-Att2 ) double-knockdown on survival of Pseudomonas entomophila infected Tenebrio molitor females over 35 days. ds EGFP was injected as a negative control for the nonspecific effects of dsRNA and the third group includes dsRNA untreated control. Two independent experiments each performed on 15 females per treatment group. Each dsRNA-treated or dsRNA-untreated groups were infected with P. entomophila on day 4 after dsRNA treatment based on the data on knockdown efficiency ( Figure S10 ). Pink dashed line: Full control-PBS; Turquoise dashed line: ds EGFP - PBS; Orange dashed line: ds TmAtt1a-Att2 - PBS; Pink line: Full control - P . entomophila ; Turquoise line: ds EGFP - P . entomophila ; Orange line: ds TmAtt1a-Att2 - P . entomophila . (B) Bacterial load in ds TmAtt1a-Att2 -silenced T. molitor challenged with P. entomophila at 1-, 2-, and 3-day post-infection. The colony-forming units (CFU) recovered from tissues of T. molitor females from ds TmAtt1a-Att2 (orange) and ds EGFP (control, turquoise). In the box plots, the lower (first) quartile is the closest boundary to zero, the line within the box marks the median (second quartile), and the upper (third) quartile. Each dot represents the CFU count in an individual beetle. The bars represent the 1.5 interquartile. Sample sizes are given in Table S2 .

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Injection, Negative Control, Control

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: (A) Effect of TmAttacin1b-2 ( TmAtt1b-Att2 ) double-knockdown on survival of Pseudomonas entomophila infected Tenebrio molitor females over 35 days. ds EGFP was injected as a negative control for the nonspecific effects of dsRNA and the third group includes dsRNA untreated control. Two independent experiments each performed on 15 females per treatment group. Each dsRNA-treated or dsRNA-untreated groups were infected with P. entomophila on day 3 after dsRNA treatment based on the data on knockdown efficiency ( Figure S11 ). Pink dashed line: Full control-PBS; Turquoise dashed line: ds EGFP - PBS; Olive green dashed line: ds TmAtt1b-Att2 - PBS; Pink line: Full control - P . entomophila ; Turquoise line: ds EGFP - P . entomophila ; Olive green line: ds TmAtt1b-Att2 - P . entomophila . (B) Bacterial load in ds TmAtt1b-Att2 -silenced T. molitor challenged with P. entomophila at 1-, 2-, and 3-day post-infection. The colony-forming units (CFU) recovered from tissues of T. molitor females from ds TmAtt1b-Att2 (olive green) and ds EGFP (control, turquoise). In the box plots, the lower (first) quartile is the closest boundary to zero, the line within the box marks the median (second quartile), and the upper (third) quartile. Each dot represents the CFU count in an individual beetle. The bars represent the 1.5 interquartile. Sample sizes are given in Table S2 .

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Injection, Negative Control, Control

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: (A) Effect of TmAttacin1a-1b-2 ( TmAtt1a-Att1b-Att2 ) triple-knockdown on survival of Pseudomonas entomophila infected Tenebrio molitor females over 35 days. ds EGFP was injected as a negative control for the nonspecific effects of dsRNA and the third group includes dsRNA untreated control. Two independent experiments each performed on 15 females per treatment group. Each dsRNA-treated or dsRNA-untreated groups were infected with P. entomophila on day 4 after dsRNA treatment based on the data on knockdown efficiency ( Figure S15 ). Pink dashed line: Full control-PBS; Turquoise dashed line: ds EGFP - PBS; Brown dashed line: ds TmAtt1a-Att1b-Att2 - PBS; Pink line: Full control - P . entomophila ; Turquoise line: ds EGFP - P . entomophila ; Brown line: ds TmAtt1a-Att1b-Att2 - P . entomophila . (B) Bacterial load in dsTmAtt1a-Att1b-Att2 -silenced T. molitor challenged with P. entomophila at 1-, 2-, and 3-day post-infection. The colony-forming units (CFU) recovered from tissues of T. molitor females from dsTmAtt1a-Att1b-Att2 (brown) and ds EGFP (control, turquoise). In the box plots, the lower (first) quartile is the closest boundary to zero, the line within the box marks the median (second quartile), and the upper (third) quartile. Each dot represents the CFU count in an individual beetle. The bars represent the 1.5 interquartile. Sample sizes are given in Table S2 .

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Injection, Negative Control, Control

Journal: Frontiers in Immunology

Article Title: The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor

doi: 10.3389/fimmu.2023.1140627

Figure Lengend Snippet: Forest plot representing the effects of the single, double, or triple knockdown of the AMP genes of interest on (A) survival of Tenebrio molitor females infected by Pseudomonas entomophila and (B) the number of P: entomophila CFU retrieved from females at 1-day, (C) 2-days, and (D) 3-days post-infection. In case of the survival, the effect size is calculated as the hazard ratio (HR) between knockdown and ds EGFP control groups (A) . In case of CFU counts (B–D) , it is calculated as the Hedge’s g between knockdown and ds EGFP control groups. Dots represent the HR and Hedge’s g, and the horizontal bars extend from the lower limit to the upper limit of the 95% confidence interval (95CI). An overlap of the 95CI with 1 (vertical line) means the knockdown has no effect on survival compared to control. A HR < 1 indicates a reduction in the hazard, whereas a HR > 1 indicates an increase in hazard In the knockdown compared to control, an overlap of the 95CI with 0 (vertical dotted line) indicates no significant effect of the knockdown treatments compared to control on CFU count, a positive value indicates a positive effect of the knockdown on CFU count, whereas a negative value indicates a negative effect of the knockdown on CFU count compared to control.

Article Snippet: The PCR conditions were as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, and extension at 72 °C for 5 min. A 508 bp PCR product of the Enhanced Green Fluorescent Protein (EGFP) gene derived from the plasmid (pGEM T-easy-GFP, Promega) was similarly amplified and used as a control for dsRNA ( ).

Techniques: Knockdown, Infection, Control

Journal: PLoS ONE

Article Title: Additive Contributions of Two Manganese-Cored Superoxide Dismutases (MnSODs) to Antioxidation, UV Tolerance and Virulence of Beauveria bassiana

doi: 10.1371/journal.pone.0030298

Figure Lengend Snippet: ( a ) The nucleotide and deduced protein sequences of BbSod3 . The uppercase DNA fragment is a 693-bp ORF encoding a protein of 230 amino acids while the lowercase fragments are ORF-flanking regions. Located in 5′ UTR and 3′ UTR are three putative stress-response elements (brown and italisized) and a putative polyadenylation signal (blue), respectively. Note that the first 34 amino acids (highlighted) of the deduced protein were predicted as a mitochondria-targeted signal peptide. The framed residues are the Parker and Blake signatures typical for the Mn-SOD family and the circled residues are metal-binding sites. The sequence double-underlined in blue represents the consensus pattern DXWEHXXY for the Fe/Mn-SOD superfamily. ( b ) Intracellular localization of BbSod3. Mycelia of transgenic strains expressing the fusion BbSod3signal::eGFP (b1–b4) and the signal-free eGFP (b5–b8, control) were stained with the mitochondria-probing stain MitoTracker® Red, emitting the red (stain) and green (eGFP) fluorescences. The differential interference contrast image (b1) and the same image labeled with the stain (b2) and the expressed fusion (b3) overlapped very well, forming the merged image (b4) whose color pattern (entirely brown-yellow in reticulum components) indicates the mitochondrial target of the fused signal peptide, whereas the merged control image (b8) showed more green than yellow. Scale bars: 10 µm.

Article Snippet: The predicted N-terminal mitochondria-targeting signal of 34 amino acids for BbSod3 was amplified from the WT cDNA with SigGFP-F1/R1 primers and fused to the open reading frame (ORF) of the enhanced green fluorescence protein gene eGFP (GenBank ID: U55763) amplified from pEGFP-C1 (BD Biosciences Clontech, CA) with SigGFP-F2/R2.

Techniques: Binding Assay, Sequencing, Transgenic Assay, Expressing, Staining, Labeling