mcherry gene  (New England Biolabs)


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    Structured Review

    New England Biolabs mcherry gene
    Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one <t>mCherry-negative</t> chicken in the middle. ( b ) Distribution of <t>mCherry-positive</t> cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.
    Mcherry Gene, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken"

    Article Title: Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14475-w

    Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one mCherry-negative chicken in the middle. ( b ) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.
    Figure Legend Snippet: Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one mCherry-negative chicken in the middle. ( b ) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.

    Techniques Used: FACS, Polymerase Chain Reaction, Amplification

    Analysis of modified PGCs and sperm of recipient rooster. ( a ) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. ( b ) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and ( c ) flow cytometry. ( d ) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. ( e ) mCherry positivity in the spermiogenic epithelium of G 0 recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. ( f ) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.
    Figure Legend Snippet: Analysis of modified PGCs and sperm of recipient rooster. ( a ) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. ( b ) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and ( c ) flow cytometry. ( d ) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. ( e ) mCherry positivity in the spermiogenic epithelium of G 0 recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. ( f ) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.

    Techniques Used: Modification, Transfection, Expressing, Plasmid Preparation, Selection, Fluorescence, Microscopy, Flow Cytometry, Sequencing, Pyrolysis Gas Chromatography, Derivative Assay, Transplantation Assay, Polymerase Chain Reaction

    ( a ) mCherry-positive embryo in the middle of incubation. ( b – e ) mCherry positivity in embryo organs and tissues. From left to right: ( b ) liver, ( c ) spleen, ( d ) skeletal muscle, ( e ) heart.
    Figure Legend Snippet: ( a ) mCherry-positive embryo in the middle of incubation. ( b – e ) mCherry positivity in embryo organs and tissues. From left to right: ( b ) liver, ( c ) spleen, ( d ) skeletal muscle, ( e ) heart.

    Techniques Used: Incubation

    2) Product Images from "Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems in an asexual fungal pathogen"

    Article Title: Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems in an asexual fungal pathogen

    Journal: BMC Biology

    doi: 10.1186/s12915-021-01101-5

    Nuclear interactions in “compatible” and “incompatible” V. dahliae heterokaryons. a Gradual accumulation of sGFP signal in mCherry-labeled nuclei in heterokaryotic cells between strains Ls.17 H1-mCherry × Cf.38 H1-sGFP (“compatible”), and Ls.17 H1-mCherry × PH H1-sGFP (“incompatible”). Arrowheads: recipient nuclei. b Putative nuclear fusion (arrowhead) in strain Ls.17 Δ atg1 . Bars in a , b = 5 μm. c Schematic representation of the possible fates of a non-self cell fusion in V. dahliae . Fused cells can remain viable or be subjected to an incompatibility-triggered reaction. Division of rare hypothetical fused nuclei in viable heterokaryotic cells could generate distinct nuclear lineages in mosaic colonies. d Nuclei of “incompatible” (Ls.17 H1-mCherry × BB sGFP, Ls.17 H1-mCherry × PH sGFP) and self-pairing control (Ls.17 H1-mCherry × Ls.17 sGFP) heterokaryons following growth under non-selective conditions. Bars = 10 μm
    Figure Legend Snippet: Nuclear interactions in “compatible” and “incompatible” V. dahliae heterokaryons. a Gradual accumulation of sGFP signal in mCherry-labeled nuclei in heterokaryotic cells between strains Ls.17 H1-mCherry × Cf.38 H1-sGFP (“compatible”), and Ls.17 H1-mCherry × PH H1-sGFP (“incompatible”). Arrowheads: recipient nuclei. b Putative nuclear fusion (arrowhead) in strain Ls.17 Δ atg1 . Bars in a , b = 5 μm. c Schematic representation of the possible fates of a non-self cell fusion in V. dahliae . Fused cells can remain viable or be subjected to an incompatibility-triggered reaction. Division of rare hypothetical fused nuclei in viable heterokaryotic cells could generate distinct nuclear lineages in mosaic colonies. d Nuclei of “incompatible” (Ls.17 H1-mCherry × BB sGFP, Ls.17 H1-mCherry × PH sGFP) and self-pairing control (Ls.17 H1-mCherry × Ls.17 sGFP) heterokaryons following growth under non-selective conditions. Bars = 10 μm

    Techniques Used: Labeling

    Autophagy is involved in selective nuclear degradation, but not in cell fusion or incompatibility-triggered death. a Live-cell imaging of a self-fusion (Ls.17 H1-mCherry sGFP-Atg8). A ring-like structure with accumulated sGFP-Atg8 (arrows) surrounds the sequestered nucleus (arrowheads). b Live-cell imaging of an “incompatible” fusion (Ls.17 H1-mCherry sGFP-Atg8 × BB H1-sGFP). Localized accumulation of sGFP-Atg8 near the fusion point (arrows) shortly before the incompatibility reaction. Arrowheads: nucleus undergoing degradation. c Frequency of active conidia and their fraction involved in CAT-mediated fusion (left; n = 300 conidia per replicate), non-apical hyphal compartments and fused cells with more than one nuclei (middle; n = 300 hyphal cells or 150 anastomoses per replicate), and inviable fusions determined by staining with methylene blue (right; n = 150 anastomoses per replicate). Wild-type (wt) pairing: Ls.17 H1-mCherry × PH H1-sGFP; pairing of autophagy-deficient mutants (Δ atg1 ): Ls.17 Δ atg1 × PH Δ atg1 . Each strain/pairing was tested in triplicate. Statistical significance of differences was tested with one-way ANOVA followed by Tukey’s post hoc test (left and middle; bars with the same letter do not differ significantly, p value > 0.05) or Student’s t -test (right; * p ≤ 0.05). Error bars: SD. d , e Multinucleate cells (arrowheads) arise frequently in Δ atg1 and Δ atg8 autophagy-deficient mutants from cell fusion ( d ) or sub-apical nuclear division ( e ), in contrast to the wild type that exhibits strictly uninucleate organization. Calcofluor white was used for cell wall staining. Scale bars = 5 μm
    Figure Legend Snippet: Autophagy is involved in selective nuclear degradation, but not in cell fusion or incompatibility-triggered death. a Live-cell imaging of a self-fusion (Ls.17 H1-mCherry sGFP-Atg8). A ring-like structure with accumulated sGFP-Atg8 (arrows) surrounds the sequestered nucleus (arrowheads). b Live-cell imaging of an “incompatible” fusion (Ls.17 H1-mCherry sGFP-Atg8 × BB H1-sGFP). Localized accumulation of sGFP-Atg8 near the fusion point (arrows) shortly before the incompatibility reaction. Arrowheads: nucleus undergoing degradation. c Frequency of active conidia and their fraction involved in CAT-mediated fusion (left; n = 300 conidia per replicate), non-apical hyphal compartments and fused cells with more than one nuclei (middle; n = 300 hyphal cells or 150 anastomoses per replicate), and inviable fusions determined by staining with methylene blue (right; n = 150 anastomoses per replicate). Wild-type (wt) pairing: Ls.17 H1-mCherry × PH H1-sGFP; pairing of autophagy-deficient mutants (Δ atg1 ): Ls.17 Δ atg1 × PH Δ atg1 . Each strain/pairing was tested in triplicate. Statistical significance of differences was tested with one-way ANOVA followed by Tukey’s post hoc test (left and middle; bars with the same letter do not differ significantly, p value > 0.05) or Student’s t -test (right; * p ≤ 0.05). Error bars: SD. d , e Multinucleate cells (arrowheads) arise frequently in Δ atg1 and Δ atg8 autophagy-deficient mutants from cell fusion ( d ) or sub-apical nuclear division ( e ), in contrast to the wild type that exhibits strictly uninucleate organization. Calcofluor white was used for cell wall staining. Scale bars = 5 μm

    Techniques Used: Live Cell Imaging, Staining

    “Incompatible” fused cells (via CATs) often escape incompatibility-triggered cell death. a Examples of the four observed types of cellular behavior following CAT-mediated fusion of “compatible” and “incompatible” cells. Strains shown: Ls.17 H1-mCherry paired with Cf.38 H1-sGFP (I), Ls.17 H1-sGFP (II), PH H1-sGFP (III), and BB H1-sGFP (IV). Arrows: nuclei undergoing degradation; arrowheads: migrating nuclei; asterisks: cell shrinkage. Bars = 5 μm. b Frequencies of the four types of cellular behavior in self, “compatible”, and “incompatible” pairings. The numbers of detected events in each case are provided in brackets (detailed results in Additional file 2 : Table S2). c Frequency of nuclear migration through CATs in viable heterokaryons resulting from self, “compatible”, and “incompatible” pairings. Bars = SD. In b , c , statistical significance of differences between the compared groups was tested with one-way ANOVA followed by Tukey’s post hoc test; in b , groups marked by the same superscript character did not differ significantly; in c , ** p value ≤ 0.01; ns: non-significant ( p value > 0.05)
    Figure Legend Snippet: “Incompatible” fused cells (via CATs) often escape incompatibility-triggered cell death. a Examples of the four observed types of cellular behavior following CAT-mediated fusion of “compatible” and “incompatible” cells. Strains shown: Ls.17 H1-mCherry paired with Cf.38 H1-sGFP (I), Ls.17 H1-sGFP (II), PH H1-sGFP (III), and BB H1-sGFP (IV). Arrows: nuclei undergoing degradation; arrowheads: migrating nuclei; asterisks: cell shrinkage. Bars = 5 μm. b Frequencies of the four types of cellular behavior in self, “compatible”, and “incompatible” pairings. The numbers of detected events in each case are provided in brackets (detailed results in Additional file 2 : Table S2). c Frequency of nuclear migration through CATs in viable heterokaryons resulting from self, “compatible”, and “incompatible” pairings. Bars = SD. In b , c , statistical significance of differences between the compared groups was tested with one-way ANOVA followed by Tukey’s post hoc test; in b , groups marked by the same superscript character did not differ significantly; in c , ** p value ≤ 0.01; ns: non-significant ( p value > 0.05)

    Techniques Used: Migration

    Spontaneous non-self fusion via CATs occurs frequently between “incompatible” strains of V. dahliae under starvation. a Wild-type strains used in this experiment and their VCG classification. Single-letter strain codes (in brackets) denote derivative strains expressing sGFP- or mCherry-tagged histone H1 (details in Additional file 1 : Table S1). The structure of the dendrogram reflects the phylogenetic relationships of VCGs as these were previously determined [ 39 ]. b Examples of CAT-mediated fusion between conidia/germlings of strains c-d (originating from the same wild-type strain), c–e (members of VCG 2B) and c–g (“incompatible” members of VCGs 2B and 4A, respectively). Bars = 5 μm. c Frequencies of intra- and inter-strain fusion in pairings of V. dahliae strains a–i. Control combinations indicated with an asterisk involved strains originating from the same wild-type strain. Each pairing was tested in triplicate, and 250-300 fusion events were analyzed per replicate. Bars = standard deviation (SD)
    Figure Legend Snippet: Spontaneous non-self fusion via CATs occurs frequently between “incompatible” strains of V. dahliae under starvation. a Wild-type strains used in this experiment and their VCG classification. Single-letter strain codes (in brackets) denote derivative strains expressing sGFP- or mCherry-tagged histone H1 (details in Additional file 1 : Table S1). The structure of the dendrogram reflects the phylogenetic relationships of VCGs as these were previously determined [ 39 ]. b Examples of CAT-mediated fusion between conidia/germlings of strains c-d (originating from the same wild-type strain), c–e (members of VCG 2B) and c–g (“incompatible” members of VCGs 2B and 4A, respectively). Bars = 5 μm. c Frequencies of intra- and inter-strain fusion in pairings of V. dahliae strains a–i. Control combinations indicated with an asterisk involved strains originating from the same wild-type strain. Each pairing was tested in triplicate, and 250-300 fusion events were analyzed per replicate. Bars = standard deviation (SD)

    Techniques Used: Expressing, Standard Deviation

    3) Product Images from "Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection"

    Article Title: Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

    Journal: Molecular microbiology

    doi: 10.1111/mmi.13946

    Fruitloop gp52 interacts with M. smegmatis Wag31 A. Clarified lysates were prepared from a strain of M. smegmatis expressing either Fruitloop gp52 (−) or Fruitloop gp52 containing a C-terminal HA tag (+). Following immunoprecipitation and washing, proteins were separated on 4–20% SDS-PAGE gel and stained with Colloidal Coomassie Blue. Arrow indicates the expected migration of Fruitloop gp52, and the box shows a prominent band absent in the non-tagged control that was identified by LC/ms-ms as Wag31. B . Wag31 co-immunoprecipitates with Fruitloop gp52. Cleared lysates were prepared of three M. smegmatis strains expressing HA tagged Fruitloop gp52 together with wild type Wag31, a FLAG-tagged derivative of Wag31, or a FLAG-tagged derivative of MSMEG_5831, as indicated, and immunoprecipitated with anti-FLAG antibody. Proteins were resuspended, separated by SDS-PAGE, and probed by Western blotting with anti-FLAG antibody (top panel), or anti-HA antibody (lower panel). C . Polar localization of Fruitloop gp52. M. smegmatis expressing a Fruitloop gp52-GFP fusion was induced for two hours and examined by light amd fluorescence microscopy (left and right panels, respectively). Fluorescent foci are observed at the cell poles (white arrows). D . Overexpression of Wag31 relieves toxicity of Fruitloop gp52. Six strains were constructed containing an inducible Fruitloop gp52 expression plasmid in addition to plasmids overexpressing mCherry (1), CwsA (2), AccA3 (3), AccD5 (4), Wag31 (5), and AccD4 (6). Although CwsA, AccA3, AccD5, and AccD4 are predicted to interact with or are in complex with Wag31, only Wag31 overexpression relieves Fruitloop gp52 toxicity.
    Figure Legend Snippet: Fruitloop gp52 interacts with M. smegmatis Wag31 A. Clarified lysates were prepared from a strain of M. smegmatis expressing either Fruitloop gp52 (−) or Fruitloop gp52 containing a C-terminal HA tag (+). Following immunoprecipitation and washing, proteins were separated on 4–20% SDS-PAGE gel and stained with Colloidal Coomassie Blue. Arrow indicates the expected migration of Fruitloop gp52, and the box shows a prominent band absent in the non-tagged control that was identified by LC/ms-ms as Wag31. B . Wag31 co-immunoprecipitates with Fruitloop gp52. Cleared lysates were prepared of three M. smegmatis strains expressing HA tagged Fruitloop gp52 together with wild type Wag31, a FLAG-tagged derivative of Wag31, or a FLAG-tagged derivative of MSMEG_5831, as indicated, and immunoprecipitated with anti-FLAG antibody. Proteins were resuspended, separated by SDS-PAGE, and probed by Western blotting with anti-FLAG antibody (top panel), or anti-HA antibody (lower panel). C . Polar localization of Fruitloop gp52. M. smegmatis expressing a Fruitloop gp52-GFP fusion was induced for two hours and examined by light amd fluorescence microscopy (left and right panels, respectively). Fluorescent foci are observed at the cell poles (white arrows). D . Overexpression of Wag31 relieves toxicity of Fruitloop gp52. Six strains were constructed containing an inducible Fruitloop gp52 expression plasmid in addition to plasmids overexpressing mCherry (1), CwsA (2), AccA3 (3), AccD5 (4), Wag31 (5), and AccD4 (6). Although CwsA, AccA3, AccD5, and AccD4 are predicted to interact with or are in complex with Wag31, only Wag31 overexpression relieves Fruitloop gp52 toxicity.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Staining, Migration, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Fluorescence, Microscopy, Over Expression, Construct, Plasmid Preparation

    Functional requirements for Fruitloop gp52 A . ClustalX alignment of Fruitloop gp52 and non-redundant homologues coded in Cluster F mycobacteriophages. Conserved residues are indicated above, with asterisks noting completely conserved residues. Below is shown a secondary structure prediction (psiPRED) indicating three predicted alpha helices. The positions of single amino acid substitutions in three separate non-toxic mutants of Fruitloop gp52 are shown below. B . Five deletion derivatives (mut1 – mut5) were constructed each of which contains only the segments displayed by the black lines, aligned with the sequences shown in panel A. C . M. smegmatis strains expressing each of the deletion derivatives in panel B were plated onto solid media either with or without inducer; two colonies of each strain were streaked. D . Removal of the C-terminal residues of Fruitloop gp52 enhances its toxicity. M. smegmatis cultures expressing wild-type Fruitloop g52 (uninduced, blue; induced red), or Fruitloop gp52 mut3 (uninduced, green, induced, purple) were induced at time 0 and OD 600 measured up to 8 hrs after induction. E . The C-terminal 19 residues of Fruitloop gp52 confer protein instability. Strains carrying inducible plasmids expressing mCherry, mCherry with C-terminal fusions of 19 residues of Fruitloop gp52,19 residues of Fruitloop gp52 with an A92E substitution at the penultimate residue, C-terminal 19 residues of BPs gp33, or the C-terminal 19 residues of BPs gp33 containing an A135E substitution in its penultimate residue, were induced at fluorescence measured at the following times: 0 (black), 2 hours (blue), 4 hours (green), or 24 hours (yellow) after induction. F . M. smegmatis strains expressing three different non-toxic Fruitloop gp52 mutants were induced, Fruitloop gp52 was immunoprecipitated with anti-HA antibody, and the proteins recovered and separated by SDS-PAGE. Western blotting was used to detect Wag31 with anti-FLAG antibody, followed by HA-tagged Fruitloop gp52 as indicated.
    Figure Legend Snippet: Functional requirements for Fruitloop gp52 A . ClustalX alignment of Fruitloop gp52 and non-redundant homologues coded in Cluster F mycobacteriophages. Conserved residues are indicated above, with asterisks noting completely conserved residues. Below is shown a secondary structure prediction (psiPRED) indicating three predicted alpha helices. The positions of single amino acid substitutions in three separate non-toxic mutants of Fruitloop gp52 are shown below. B . Five deletion derivatives (mut1 – mut5) were constructed each of which contains only the segments displayed by the black lines, aligned with the sequences shown in panel A. C . M. smegmatis strains expressing each of the deletion derivatives in panel B were plated onto solid media either with or without inducer; two colonies of each strain were streaked. D . Removal of the C-terminal residues of Fruitloop gp52 enhances its toxicity. M. smegmatis cultures expressing wild-type Fruitloop g52 (uninduced, blue; induced red), or Fruitloop gp52 mut3 (uninduced, green, induced, purple) were induced at time 0 and OD 600 measured up to 8 hrs after induction. E . The C-terminal 19 residues of Fruitloop gp52 confer protein instability. Strains carrying inducible plasmids expressing mCherry, mCherry with C-terminal fusions of 19 residues of Fruitloop gp52,19 residues of Fruitloop gp52 with an A92E substitution at the penultimate residue, C-terminal 19 residues of BPs gp33, or the C-terminal 19 residues of BPs gp33 containing an A135E substitution in its penultimate residue, were induced at fluorescence measured at the following times: 0 (black), 2 hours (blue), 4 hours (green), or 24 hours (yellow) after induction. F . M. smegmatis strains expressing three different non-toxic Fruitloop gp52 mutants were induced, Fruitloop gp52 was immunoprecipitated with anti-HA antibody, and the proteins recovered and separated by SDS-PAGE. Western blotting was used to detect Wag31 with anti-FLAG antibody, followed by HA-tagged Fruitloop gp52 as indicated.

    Techniques Used: Functional Assay, Construct, Expressing, Fluorescence, Immunoprecipitation, SDS Page, Western Blot

    Expression of Fruitloop gene 52 is toxic in M. smegmatis A . Fruitloop gene 52 was identified in a screen for phage genes that kill M. smegmatis when its expression is induced. Two separate colonies of M. smegmatis carrying Fruitloop 52 regulated by an ATc-inducible promoter (plasmid pCCK1) were streaked either in the presence or absence of inducer. A strain expressing mCherry (plasmid pCCK11) similarly regulated is also shown. B . Fruitloop gp52 expression leads to rapid cessation of M. smegmatis growth. Inducer was added to a liquid culture of M. smegmatis carrying Fruitloop 52 (plasmid pCCK1) at early logarithmic growth (OD 600 0.4) and OD 600 measured at various time points as shown. Uninduced, blue line; induced, red line. C . Fruitloop gp52 expression is bacteriocidal. Samples taken at various times (shown at the right, in hours) from cultures like those shown in panel B were serially diluted 10-fold, and spotted onto solid media lacking inducer. Viability is reduced even after 4 hours of induction. D . Morphological changes to M. smegmatis following induction of Fruitloop 52 expression observed by light microscopy. After 4 hours of induction, an evident bulge is observed at one pole of many cells, followed by cell rounding after 8 hours of induction.
    Figure Legend Snippet: Expression of Fruitloop gene 52 is toxic in M. smegmatis A . Fruitloop gene 52 was identified in a screen for phage genes that kill M. smegmatis when its expression is induced. Two separate colonies of M. smegmatis carrying Fruitloop 52 regulated by an ATc-inducible promoter (plasmid pCCK1) were streaked either in the presence or absence of inducer. A strain expressing mCherry (plasmid pCCK11) similarly regulated is also shown. B . Fruitloop gp52 expression leads to rapid cessation of M. smegmatis growth. Inducer was added to a liquid culture of M. smegmatis carrying Fruitloop 52 (plasmid pCCK1) at early logarithmic growth (OD 600 0.4) and OD 600 measured at various time points as shown. Uninduced, blue line; induced, red line. C . Fruitloop gp52 expression is bacteriocidal. Samples taken at various times (shown at the right, in hours) from cultures like those shown in panel B were serially diluted 10-fold, and spotted onto solid media lacking inducer. Viability is reduced even after 4 hours of induction. D . Morphological changes to M. smegmatis following induction of Fruitloop 52 expression observed by light microscopy. After 4 hours of induction, an evident bulge is observed at one pole of many cells, followed by cell rounding after 8 hours of induction.

    Techniques Used: Expressing, Plasmid Preparation, Light Microscopy

    4) Product Images from "Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2"

    Article Title: Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002332

    A dominant negative SmMef2 leads to downregulation of WNT genes in schistosomes. qRT-PCR analysis of (A) Wnt1 transcript levels and (B) Wnt2 transcript levels when transfected with plasmids expressing SmMef2,133 (SmMef2,133 overexpression), wt SmMef2 (wt SmMef2 overexpression) and mCherry (mCherry overexpression). Genes that are overexpressed are regulated by the CMV promoter and compared to samples exposed to plasmid containing the SmMef,133 mutant in the absence of PEI (PEI Negative Control).
    Figure Legend Snippet: A dominant negative SmMef2 leads to downregulation of WNT genes in schistosomes. qRT-PCR analysis of (A) Wnt1 transcript levels and (B) Wnt2 transcript levels when transfected with plasmids expressing SmMef2,133 (SmMef2,133 overexpression), wt SmMef2 (wt SmMef2 overexpression) and mCherry (mCherry overexpression). Genes that are overexpressed are regulated by the CMV promoter and compared to samples exposed to plasmid containing the SmMef,133 mutant in the absence of PEI (PEI Negative Control).

    Techniques Used: Dominant Negative Mutation, Quantitative RT-PCR, Transfection, Expressing, Over Expression, Plasmid Preparation, Mutagenesis, Negative Control

    SmMef2 can autoregulate its transcript levels. Schistosomula were transfected with a plasmid expressing SmMef2, the C-terminal deletion mutant containing the DNA binding domain of SmMef2 (Sm Mef2,133 ), or a negative control mCherry gene in the presence of PEI. Genes that are overexpressed (x-axis) are regulated by the strong CMV promoter. PEI Negative Control samples were exposed to the plasmid containing the SmMef2,133 mutant regulated by control the CMV promoter, but without PEI. qRT-PCR was used to analyze SmMef2 transcript levels or SmMef2,133 transcript levels (y axis) in response to overexpression of (A) the SmMef2,133, (B) SmMef2 (wt SmMef2 overexpression) or a mCherry negative control (mCherry overexpression), (C) SmMef2,133 transcript, and (D) and the wt SmMef2 transcript or a mCherry negative control.
    Figure Legend Snippet: SmMef2 can autoregulate its transcript levels. Schistosomula were transfected with a plasmid expressing SmMef2, the C-terminal deletion mutant containing the DNA binding domain of SmMef2 (Sm Mef2,133 ), or a negative control mCherry gene in the presence of PEI. Genes that are overexpressed (x-axis) are regulated by the strong CMV promoter. PEI Negative Control samples were exposed to the plasmid containing the SmMef2,133 mutant regulated by control the CMV promoter, but without PEI. qRT-PCR was used to analyze SmMef2 transcript levels or SmMef2,133 transcript levels (y axis) in response to overexpression of (A) the SmMef2,133, (B) SmMef2 (wt SmMef2 overexpression) or a mCherry negative control (mCherry overexpression), (C) SmMef2,133 transcript, and (D) and the wt SmMef2 transcript or a mCherry negative control.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay, Negative Control, Quantitative RT-PCR, Over Expression

    Expression constructs used for schistosome transfection. (A) The mCherry reporter gene was cloned into the BamHI site of the pCI-neo vector (Promega) to make plasmid pEJ1175. DNA oligos used for amplification by PCR or RT-PCR are shown as a forward arrow (a) or reverse arrows (b and c) representing forward oligo oEJ1022 (a), and reverse oligos oEJ1023 (b) and oEJ1019 (c). (B) 2000 base pairs of the Sm23 UAS was used to control expression of the mCherry and the Sm23 genes, These genes were cloned into the 7.4 kb pGBKT7 vector to make plasmid pEJ1116. (C) The N-terminal 133 amino acids of SmMef2 are regulated by the CMV promoter and were cloned to make plasmid (pEJ1181). The N-terminus of SmMef2 contains the DNA binding domain, but not its C-terminal transactivation domain. (D) The wild-type SmMef2, regulated by the CMV promoter, was cloned to make plasmid (pLS068). DNA oligos (d) and (e) are used for detection of SmMef2,133 transcript by qRT-PCR, while oligos (f) and (g) are used for specifically measuring wt SmMef2 transcript in qRT-PCR reactions.
    Figure Legend Snippet: Expression constructs used for schistosome transfection. (A) The mCherry reporter gene was cloned into the BamHI site of the pCI-neo vector (Promega) to make plasmid pEJ1175. DNA oligos used for amplification by PCR or RT-PCR are shown as a forward arrow (a) or reverse arrows (b and c) representing forward oligo oEJ1022 (a), and reverse oligos oEJ1023 (b) and oEJ1019 (c). (B) 2000 base pairs of the Sm23 UAS was used to control expression of the mCherry and the Sm23 genes, These genes were cloned into the 7.4 kb pGBKT7 vector to make plasmid pEJ1116. (C) The N-terminal 133 amino acids of SmMef2 are regulated by the CMV promoter and were cloned to make plasmid (pEJ1181). The N-terminus of SmMef2 contains the DNA binding domain, but not its C-terminal transactivation domain. (D) The wild-type SmMef2, regulated by the CMV promoter, was cloned to make plasmid (pLS068). DNA oligos (d) and (e) are used for detection of SmMef2,133 transcript by qRT-PCR, while oligos (f) and (g) are used for specifically measuring wt SmMef2 transcript in qRT-PCR reactions.

    Techniques Used: Expressing, Construct, Transfection, Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Quantitative RT-PCR

    CMV and Sm23 promoters can induce gene expression from a plasmid in transfected schistosomes. mCherry gene expression is regulated by either the CMV promoter, or the schistosome Sm23 promoter. (A) RNA from each sample was extracted to produce cDNA using reverse transcriptase. Sixty ng of cDNA was used for PCR analysis to amplify a 192 bp mCherry gene fragment to test for mCherry transcript expression from either the CMV promoter or the Sm23 promoter. Lanes 1–4 from left to right are: 1] 1 kb Plus DNA Ladder (Invitrogen, Carlsbad, CA). 2] Sample treated with PEI and CMV promoter based vector pEJ1175. 3] Sample treated with PEI and Sm23 promoter based vector, pEJ1116. 4] Sample treated with PEI alone. (B) Total protein was extracted from schistosomes expressing mCherry under control of the CMV promoter (Lane 1) or from untransformed schistosome controls (Lane 2), and assayed by Western blot analysis using an antibody targeting the mCherry protein.
    Figure Legend Snippet: CMV and Sm23 promoters can induce gene expression from a plasmid in transfected schistosomes. mCherry gene expression is regulated by either the CMV promoter, or the schistosome Sm23 promoter. (A) RNA from each sample was extracted to produce cDNA using reverse transcriptase. Sixty ng of cDNA was used for PCR analysis to amplify a 192 bp mCherry gene fragment to test for mCherry transcript expression from either the CMV promoter or the Sm23 promoter. Lanes 1–4 from left to right are: 1] 1 kb Plus DNA Ladder (Invitrogen, Carlsbad, CA). 2] Sample treated with PEI and CMV promoter based vector pEJ1175. 3] Sample treated with PEI and Sm23 promoter based vector, pEJ1116. 4] Sample treated with PEI alone. (B) Total protein was extracted from schistosomes expressing mCherry under control of the CMV promoter (Lane 1) or from untransformed schistosome controls (Lane 2), and assayed by Western blot analysis using an antibody targeting the mCherry protein.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Polymerase Chain Reaction, Western Blot

    5) Product Images from "Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus"

    Article Title: Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus

    Journal: Nature Communications

    doi: 10.1038/ncomms9884

    ΔBd3460 Bdellovibrio self-round upon initiating prey cell entry. Epifluorescence phase contrast microscopy of Bdellovibrio (small, phase dark, comma-shaped cells) preying upon E. coli prey cells which have periplasms constitutively fluorescently labelled by a pMal::mCherry fusion. A cartoon representation is presented above each. ( a ) Control using host independent strain HID22 which is wild-type for Bd3460 (Bb wt) and shows typical attachment to and entry into the prey cell which is rounded up in the process. ( b ) ΔBd3460 host independent strain (Bb Δ3460) attaches to the prey cell in a manner similar to the wild-type control, but then rounds up itself, preventing entry into the prey cell. ( c ) Representative electron micrographs showing the different stages of attachment, Bdellovibrio rounding, and prey rounding. Scale bars, 1 μm ; time is indicated in minutes.
    Figure Legend Snippet: ΔBd3460 Bdellovibrio self-round upon initiating prey cell entry. Epifluorescence phase contrast microscopy of Bdellovibrio (small, phase dark, comma-shaped cells) preying upon E. coli prey cells which have periplasms constitutively fluorescently labelled by a pMal::mCherry fusion. A cartoon representation is presented above each. ( a ) Control using host independent strain HID22 which is wild-type for Bd3460 (Bb wt) and shows typical attachment to and entry into the prey cell which is rounded up in the process. ( b ) ΔBd3460 host independent strain (Bb Δ3460) attaches to the prey cell in a manner similar to the wild-type control, but then rounds up itself, preventing entry into the prey cell. ( c ) Representative electron micrographs showing the different stages of attachment, Bdellovibrio rounding, and prey rounding. Scale bars, 1 μm ; time is indicated in minutes.

    Techniques Used: Microscopy

    Periplasmic localization of Bd3460 protein. Epifluorescence phase contrast microscopy of Bdellovibrio with a Bd3460::mCherry tag. Fluorescence is seen in the small, attack phase cells at times 0 and 240 min, and increases as the Bdellovibrio enter the prey, which rounds up to form a ‘bdelloplast'. As the Bdellovibrio cell grows inside the bdelloplast, the fluorescence becomes dissipated in the larger, cylindrical cell ( T =180 min). Scale bar, 1 μm.
    Figure Legend Snippet: Periplasmic localization of Bd3460 protein. Epifluorescence phase contrast microscopy of Bdellovibrio with a Bd3460::mCherry tag. Fluorescence is seen in the small, attack phase cells at times 0 and 240 min, and increases as the Bdellovibrio enter the prey, which rounds up to form a ‘bdelloplast'. As the Bdellovibrio cell grows inside the bdelloplast, the fluorescence becomes dissipated in the larger, cylindrical cell ( T =180 min). Scale bar, 1 μm.

    Techniques Used: Microscopy, Fluorescence

    6) Product Images from "Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems to enable parasexual interactions in an asexual fungal pathogen"

    Article Title: Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems to enable parasexual interactions in an asexual fungal pathogen

    Journal: bioRxiv

    doi: 10.1101/2021.05.19.444787

    Spontaneous non-self fusion via CATs occurs frequently between “incompatible” strains of V. dahliae under starvation. (a) Wild-type strains used in this experiment and their VCG classification. Single-letter strain codes (in brackets) denote derivative strains expressing sGFP- or mCherry-tagged histone H1 (details in Table S1 ). The structure of the dendrogram reflects the phylogenetic relationships of VCGs as these were previously determined ( Papaioannou et al ., 2013a ). (b) Examples of CAT-mediated fusion between conidia/germlings of strains c-d (originating from the same wild-type strain), c-e (members of VCG 2B) and c-g (“incompatible” members of VCGs 2B and 4A, respectively). Bars = 5 μm. (c) Frequencies of intra- and inter-strain fusion in pairings of V. dahliae strains a-i. Control combinations indicated with an asterisk involved strains originating from the same wild-type strain. Each pairing was tested in triplicate, and 250-300 fusion events were analyzed per replicate. Bars = SD.
    Figure Legend Snippet: Spontaneous non-self fusion via CATs occurs frequently between “incompatible” strains of V. dahliae under starvation. (a) Wild-type strains used in this experiment and their VCG classification. Single-letter strain codes (in brackets) denote derivative strains expressing sGFP- or mCherry-tagged histone H1 (details in Table S1 ). The structure of the dendrogram reflects the phylogenetic relationships of VCGs as these were previously determined ( Papaioannou et al ., 2013a ). (b) Examples of CAT-mediated fusion between conidia/germlings of strains c-d (originating from the same wild-type strain), c-e (members of VCG 2B) and c-g (“incompatible” members of VCGs 2B and 4A, respectively). Bars = 5 μm. (c) Frequencies of intra- and inter-strain fusion in pairings of V. dahliae strains a-i. Control combinations indicated with an asterisk involved strains originating from the same wild-type strain. Each pairing was tested in triplicate, and 250-300 fusion events were analyzed per replicate. Bars = SD.

    Techniques Used: Expressing

    Atg8-mediated nuclear degradation in the strain Ls.17 H1-mCherry sGFP-Atg8. Atg8 is co-localized with the degrading nucleus during this process (arrowheads). Bars = 5 μ m.
    Figure Legend Snippet: Atg8-mediated nuclear degradation in the strain Ls.17 H1-mCherry sGFP-Atg8. Atg8 is co-localized with the degrading nucleus during this process (arrowheads). Bars = 5 μ m.

    Techniques Used:

    Autophagy is involved in selective nuclear degradation in heterokaryons, but it is not required for fusion or cell death due to incompatibility. (a) Live-cell imaging of a self-fusion (Ls.17 H1-mCherry sGFP-Atg8). A ring-like structure with accumulated sGFP-Atg8 (arrows) surrounds the sequestered nucleus (arrowheads). (b) Live-cell imaging of an “incompatible” fusion (Ls.17 H1-mCherry sGFP-Atg8 × BB H1-sGFP). Localized accumulation of sGFP-Atg8 near the fusion point (arrows) shortly before the incompatibility reaction. Arrowheads: nucleus undergoing degradation. (c,d) Multinucleate cells (arrowheads) arise frequently in Δ atg1 and Δ atg8 autophagy-deficient mutants from sub-apical nuclear division (c) or cell fusion (d), in contrast to the wild type that exhibits strictly uninucleate organization. Calcofluor white was used for cell wall staining. Bars in (a-d) = 5 μm. (e) Frequency of active conidia and their fraction involved in CAT-mediated fusion. Each strain was tested in triplicate (n = 300 conidia per replicate). (f) Frequency of non-apical hyphal compartments and fused cells with more than one nuclei. Each strain was tested in triplicate (n = 300 hyphal cells or 150 anastomoses per replicate). In (e,f): statistical significance of differences was tested with one-way ANOVA followed by Tukey’s post-hoc test; bars with the same letter do not differ significantly ( p -value > 0.05). (g) Frequency of inviable fusions, as determined by staining with methylene blue. Wild-type (wt) pairing: Ls.17 H1-mCherry × PH H1-sGFP; pairing of autophagy-deficient mutants (Δ atg1 ): Ls.17 Δ atg1 × PH Δ atg1 . Each pairing was tested in triplicate (n = 150 anastomoses per replicate). Statistical significance of difference between the two groups was assessed using the Student’s t -test (* p ≤ 0.05). Error bars in (e-g): SD.
    Figure Legend Snippet: Autophagy is involved in selective nuclear degradation in heterokaryons, but it is not required for fusion or cell death due to incompatibility. (a) Live-cell imaging of a self-fusion (Ls.17 H1-mCherry sGFP-Atg8). A ring-like structure with accumulated sGFP-Atg8 (arrows) surrounds the sequestered nucleus (arrowheads). (b) Live-cell imaging of an “incompatible” fusion (Ls.17 H1-mCherry sGFP-Atg8 × BB H1-sGFP). Localized accumulation of sGFP-Atg8 near the fusion point (arrows) shortly before the incompatibility reaction. Arrowheads: nucleus undergoing degradation. (c,d) Multinucleate cells (arrowheads) arise frequently in Δ atg1 and Δ atg8 autophagy-deficient mutants from sub-apical nuclear division (c) or cell fusion (d), in contrast to the wild type that exhibits strictly uninucleate organization. Calcofluor white was used for cell wall staining. Bars in (a-d) = 5 μm. (e) Frequency of active conidia and their fraction involved in CAT-mediated fusion. Each strain was tested in triplicate (n = 300 conidia per replicate). (f) Frequency of non-apical hyphal compartments and fused cells with more than one nuclei. Each strain was tested in triplicate (n = 300 hyphal cells or 150 anastomoses per replicate). In (e,f): statistical significance of differences was tested with one-way ANOVA followed by Tukey’s post-hoc test; bars with the same letter do not differ significantly ( p -value > 0.05). (g) Frequency of inviable fusions, as determined by staining with methylene blue. Wild-type (wt) pairing: Ls.17 H1-mCherry × PH H1-sGFP; pairing of autophagy-deficient mutants (Δ atg1 ): Ls.17 Δ atg1 × PH Δ atg1 . Each pairing was tested in triplicate (n = 150 anastomoses per replicate). Statistical significance of difference between the two groups was assessed using the Student’s t -test (* p ≤ 0.05). Error bars in (e-g): SD.

    Techniques Used: Live Cell Imaging, Staining

    Typical features of incompatibility-triggered cell death in V. dahliae . (a) Induction of an incompatibility reaction after cytoplasmic mixing. Strains: PH sGFP and BB H1-sGFP. (b) Induction of an incompatibility reaction without prior cytoplasmic mixing. Strains: PH sGFP and BB. (c) Incompatibility-triggered cell death is characterized by nuclear degradation and cell shrinkage. Strains: PH H1-sGFP and Ls.17 H1-mCherry. In (a-c) arrowheads indicate the contact point of germlings, and arrows indicate cell shrinkage. (d) Staining of CAT-mediated fused cells using methylene blue. Viable fusions remain unstained (top), whereas post-fusion cell death permits the accumulation of the dye in the cytoplasm (bottom). Bars = 5 μ m.
    Figure Legend Snippet: Typical features of incompatibility-triggered cell death in V. dahliae . (a) Induction of an incompatibility reaction after cytoplasmic mixing. Strains: PH sGFP and BB H1-sGFP. (b) Induction of an incompatibility reaction without prior cytoplasmic mixing. Strains: PH sGFP and BB. (c) Incompatibility-triggered cell death is characterized by nuclear degradation and cell shrinkage. Strains: PH H1-sGFP and Ls.17 H1-mCherry. In (a-c) arrowheads indicate the contact point of germlings, and arrows indicate cell shrinkage. (d) Staining of CAT-mediated fused cells using methylene blue. Viable fusions remain unstained (top), whereas post-fusion cell death permits the accumulation of the dye in the cytoplasm (bottom). Bars = 5 μ m.

    Techniques Used: Staining

    Nuclear interactions in “compatible” and “incompatible” V. dahliae heterokaryons. (a) Gradual accumulation of sGFP signal in mCherry-labeled nuclei in heterokaryotic cells between strains Ls.17 H1-mCherry × Cf.38 H1-sGFP (“compatible”), and Ls.17 H1-mCherry × PH H1-sGFP (“incompatible”). Arrowheads: recipient nuclei. (b) Direct nuclear fusion (arrowhead) in strain Ls.17 Δ atg1 . Bars in (a,b) = 5 μm. (c) Schematic representation of the possible fates of a non-self cell fusion in V. dahliae . Fused cells can remain viable or be subjected to an incompatibility-triggered reaction. Division of rare fused nuclei in viable heterokaryotic cells can generate distinct nuclear lineages in mosaic colonies. (d) Nuclei of “incompatible” (Ls.17 H1-mCherry × BB sGFP, Ls.17 H1-mCherry × PH sGFP) and self-pairing control (Ls.17 H1-mCherry × Ls.17 sGFP) heterokaryons following growth under non-selective conditions. Bars = 10 μm.
    Figure Legend Snippet: Nuclear interactions in “compatible” and “incompatible” V. dahliae heterokaryons. (a) Gradual accumulation of sGFP signal in mCherry-labeled nuclei in heterokaryotic cells between strains Ls.17 H1-mCherry × Cf.38 H1-sGFP (“compatible”), and Ls.17 H1-mCherry × PH H1-sGFP (“incompatible”). Arrowheads: recipient nuclei. (b) Direct nuclear fusion (arrowhead) in strain Ls.17 Δ atg1 . Bars in (a,b) = 5 μm. (c) Schematic representation of the possible fates of a non-self cell fusion in V. dahliae . Fused cells can remain viable or be subjected to an incompatibility-triggered reaction. Division of rare fused nuclei in viable heterokaryotic cells can generate distinct nuclear lineages in mosaic colonies. (d) Nuclei of “incompatible” (Ls.17 H1-mCherry × BB sGFP, Ls.17 H1-mCherry × PH sGFP) and self-pairing control (Ls.17 H1-mCherry × Ls.17 sGFP) heterokaryons following growth under non-selective conditions. Bars = 10 μm.

    Techniques Used: Labeling

    “Incompatible” fused cells (via CATs) often escape incompatibility-triggered cell death. (a) Examples of the four observed types of cellular behavior following CAT-mediated fusion of “compatible” and “incompatible” cells. Strains shown: Ls.17 H1-mCherry paired with Cf.38 H1-sGFP (I), Ls.17 H1-sGFP (II), PH H1-sGFP (III) and BB H1-sGFP (IV). Arrows: nuclei undergoing degradation; arrowheads: migrating nuclei; asterisks: cell shrinkage. Bars = 5 μm. (b) Frequencies of the four types of cellular behavior in self, “compatible” and “incompatible” pairings. The numbers of detected events in each case are provided in brackets (detailed results in Table S5 ). (c) Frequency of nuclear migration through CATs in viable heterokaryons resulting from self, “compatible” and “incompatible” pairings. Bars = SD. In (b,c): statistical significance of differences between the compared groups was tested with one-way ANOVA followed by Tukey’s post-hoc test; in (b): groups marked by the same superscript character did not differ significantly; in (c): ** p -value ≤ 0.01; ns: non-significant ( p -value > 0.05).
    Figure Legend Snippet: “Incompatible” fused cells (via CATs) often escape incompatibility-triggered cell death. (a) Examples of the four observed types of cellular behavior following CAT-mediated fusion of “compatible” and “incompatible” cells. Strains shown: Ls.17 H1-mCherry paired with Cf.38 H1-sGFP (I), Ls.17 H1-sGFP (II), PH H1-sGFP (III) and BB H1-sGFP (IV). Arrows: nuclei undergoing degradation; arrowheads: migrating nuclei; asterisks: cell shrinkage. Bars = 5 μm. (b) Frequencies of the four types of cellular behavior in self, “compatible” and “incompatible” pairings. The numbers of detected events in each case are provided in brackets (detailed results in Table S5 ). (c) Frequency of nuclear migration through CATs in viable heterokaryons resulting from self, “compatible” and “incompatible” pairings. Bars = SD. In (b,c): statistical significance of differences between the compared groups was tested with one-way ANOVA followed by Tukey’s post-hoc test; in (b): groups marked by the same superscript character did not differ significantly; in (c): ** p -value ≤ 0.01; ns: non-significant ( p -value > 0.05).

    Techniques Used: Migration

    Fluorescent tagging of V. dahliae Atg8 by a CRISPR/Cas9-based system. (a) Stability of AMA1-containing plasmids under selective and non-selective conditions. Twenty-five randomly selected transformants of V. dahliae Ls.17 were single-spore purified and re-cultured every three days on selective (PDA supplemented with hygromycin B) and non-selective (PDA) medium. After each transfer, the percentage of resistant colonies to hygromycin B was determined. The experiment was performed in triplicate. Bars = SD. (b) Tagging efficiency of Atg8 with mCherry (strain Ls.17), using repair substrates with different lengths of homologous arms. The use of 0.1 kb-long arms yielded mostly chimeric fluorescent colonies consisting of tagged and non-tagged cell populations (13% properly tagged cells on average), in contrast to 1.0 kb-long arms that resulted in more homogeneous colonies (i.e. at least 90% of cells were properly tagged). The experiment was performed in triplicate for each repair construct, and at least 20 independent transformants were checked per replicate (using PCR and microscopy). Bars = SD. (c) Effect of autophagy-inducing conditions on the mCherry-Atg8 localization. Both starvation (i.e. incubation in water) and treatment with rapamycin (i.e. a TOR kinase inhibitor that induces autophagy) lead to the accumulation of the protein in autophagosomes and vacuoles, which indicates the induction of autophagy. (d) Subcellular localization of mCherry-Atg8 during germination in minimal medium. Prior to germination, the protein forms small globular foci (presumably autophagosomes, arrowheads), which seem to accumulate during germination in larger structures (vacuoles, asterisks), which is consistent with the expected participation of autophagy in conidial germination.
    Figure Legend Snippet: Fluorescent tagging of V. dahliae Atg8 by a CRISPR/Cas9-based system. (a) Stability of AMA1-containing plasmids under selective and non-selective conditions. Twenty-five randomly selected transformants of V. dahliae Ls.17 were single-spore purified and re-cultured every three days on selective (PDA supplemented with hygromycin B) and non-selective (PDA) medium. After each transfer, the percentage of resistant colonies to hygromycin B was determined. The experiment was performed in triplicate. Bars = SD. (b) Tagging efficiency of Atg8 with mCherry (strain Ls.17), using repair substrates with different lengths of homologous arms. The use of 0.1 kb-long arms yielded mostly chimeric fluorescent colonies consisting of tagged and non-tagged cell populations (13% properly tagged cells on average), in contrast to 1.0 kb-long arms that resulted in more homogeneous colonies (i.e. at least 90% of cells were properly tagged). The experiment was performed in triplicate for each repair construct, and at least 20 independent transformants were checked per replicate (using PCR and microscopy). Bars = SD. (c) Effect of autophagy-inducing conditions on the mCherry-Atg8 localization. Both starvation (i.e. incubation in water) and treatment with rapamycin (i.e. a TOR kinase inhibitor that induces autophagy) lead to the accumulation of the protein in autophagosomes and vacuoles, which indicates the induction of autophagy. (d) Subcellular localization of mCherry-Atg8 during germination in minimal medium. Prior to germination, the protein forms small globular foci (presumably autophagosomes, arrowheads), which seem to accumulate during germination in larger structures (vacuoles, asterisks), which is consistent with the expected participation of autophagy in conidial germination.

    Techniques Used: CRISPR, Purification, Cell Culture, Construct, Polymerase Chain Reaction, Microscopy, Incubation

    7) Product Images from "Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken"

    Article Title: Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14475-w

    Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one mCherry-negative chicken in the middle. ( b ) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.
    Figure Legend Snippet: Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one mCherry-negative chicken in the middle. ( b ) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.

    Techniques Used: FACS, Polymerase Chain Reaction, Amplification

    Analysis of modified PGCs and sperm of recipient rooster. ( a ) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. ( b ) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and ( c ) flow cytometry. ( d ) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. ( e ) mCherry positivity in the spermiogenic epithelium of G 0 recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. ( f ) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.
    Figure Legend Snippet: Analysis of modified PGCs and sperm of recipient rooster. ( a ) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. ( b ) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and ( c ) flow cytometry. ( d ) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. ( e ) mCherry positivity in the spermiogenic epithelium of G 0 recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. ( f ) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.

    Techniques Used: Modification, Transfection, Expressing, Plasmid Preparation, Selection, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Sequencing, Pyrolysis Gas Chromatography, Derivative Assay, Transplantation Assay, Polymerase Chain Reaction

    ( a ) mCherry-positive embryo in the middle of incubation. ( b – e ) mCherry positivity in embryo organs and tissues. From left to right: ( b ) liver, ( c ) spleen, ( d ) skeletal muscle, ( e ) heart.
    Figure Legend Snippet: ( a ) mCherry-positive embryo in the middle of incubation. ( b – e ) mCherry positivity in embryo organs and tissues. From left to right: ( b ) liver, ( c ) spleen, ( d ) skeletal muscle, ( e ) heart.

    Techniques Used: Incubation

    8) Product Images from "Expression and Targeting of Secreted Proteins from Chlamydia trachomatis"

    Article Title: Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01290-13

    Tetracycline-inducible promoter driven mCherry expression in C. trachomatis . (A) HeLa cells were infected with C. trachomatis L2 transformed with pBOMB4-Tet-mCherry for 8 h, at which time various amounts of anhydrotetracycline hydrochloride (aTC) were
    Figure Legend Snippet: Tetracycline-inducible promoter driven mCherry expression in C. trachomatis . (A) HeLa cells were infected with C. trachomatis L2 transformed with pBOMB4-Tet-mCherry for 8 h, at which time various amounts of anhydrotetracycline hydrochloride (aTC) were

    Techniques Used: Expressing, Infection, Transformation Assay

    9) Product Images from "Genetic requirements for cell division in a genomically minimal cell"

    Article Title: Genetic requirements for cell division in a genomically minimal cell

    Journal: bioRxiv

    doi: 10.1101/2020.10.07.326892

    RGD6 and JCVI-syn3.0 exhibited filamentation, branching, pearling, and other morphological dynamics in the absence of shear flow. (A) RGD6 was capable of filamentous propagation in the shear-free environment of microfluidic chemostats. The left column shows phase contrast images, while the right column shows constitutively expressed mCherry as a marker for cytoplasm. Chemostat walls are indicated as dotted lines. White arrows at 5.5 h indicate the appearance of vesicles at the ends of the filament. Vesicles lacked mCherry from their first appearance. (B) Growth of JCVI-syn3.0 produces pearled and branched filaments, along with other morphologies, shown here in a representative chemostat originally loaded with many cells.
    Figure Legend Snippet: RGD6 and JCVI-syn3.0 exhibited filamentation, branching, pearling, and other morphological dynamics in the absence of shear flow. (A) RGD6 was capable of filamentous propagation in the shear-free environment of microfluidic chemostats. The left column shows phase contrast images, while the right column shows constitutively expressed mCherry as a marker for cytoplasm. Chemostat walls are indicated as dotted lines. White arrows at 5.5 h indicate the appearance of vesicles at the ends of the filament. Vesicles lacked mCherry from their first appearance. (B) Growth of JCVI-syn3.0 produces pearled and branched filaments, along with other morphologies, shown here in a representative chemostat originally loaded with many cells.

    Techniques Used: Marker

    Location of cytoplasmic protein, nucleoids, and membrane in pleomorphic cellular forms. Brightfield and fluorescence optical micrographs of RGD6+mCherry grown in microfluidic chambers show filamentous cells (A), and large cells and vesicles (B,C). (A) Nucleoids appeared separated along the length of filamentous cells, suggesting genome segregation may continue in the absence of complete cell scission, as recently observed in B. subtilis L-forms ( Wu et al., 2020 ). (B) The surface of vesicles, which appeared as lower contrast in phase contrast images, were stained with the membrane dye SP-DiOC18(3). (C) A large vesicle lacked constitutively expressed mCherry but excluded FITC-conjugated dextran in the growth medium, suggesting the vesicle membrane was not permeable to macromolecules. Scale bars: 5 µm (A,B) and 2 µm (C).
    Figure Legend Snippet: Location of cytoplasmic protein, nucleoids, and membrane in pleomorphic cellular forms. Brightfield and fluorescence optical micrographs of RGD6+mCherry grown in microfluidic chambers show filamentous cells (A), and large cells and vesicles (B,C). (A) Nucleoids appeared separated along the length of filamentous cells, suggesting genome segregation may continue in the absence of complete cell scission, as recently observed in B. subtilis L-forms ( Wu et al., 2020 ). (B) The surface of vesicles, which appeared as lower contrast in phase contrast images, were stained with the membrane dye SP-DiOC18(3). (C) A large vesicle lacked constitutively expressed mCherry but excluded FITC-conjugated dextran in the growth medium, suggesting the vesicle membrane was not permeable to macromolecules. Scale bars: 5 µm (A,B) and 2 µm (C).

    Techniques Used: Fluorescence, Staining

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    New England Biolabs mcherry gene
    Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one <t>mCherry-negative</t> chicken in the middle. ( b ) Distribution of <t>mCherry-positive</t> cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.
    Mcherry Gene, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one mCherry-negative chicken in the middle. ( b ) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.

    Journal: Scientific Reports

    Article Title: Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

    doi: 10.1038/s41598-017-14475-w

    Figure Lengend Snippet: Phenotypic and genetic analysis of G 1 offspring. ( a ) Freshly hatched G 1 offspring, one mCherry-negative chicken in the middle. ( b ) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). ( c ) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). ( d ) Photostability of mCherry shown as mCherry signal in adult feather. ( e ) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G 1 offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G 1 roosters Nos 769 and 795 are indicated. ( f ) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.

    Article Snippet: An eGFP plasmid that was used before to generate transgenic chickens was used to swap the eGFP with the mCherry gene by Gibson Assembly (New England Biolabs) using the following oligos: fw: 5′-TATCGCATGCCTGCGATGGTGAGCAAGGGCGAGG-3′, rev: 5′-GCATGGACGAGCTGTACAAGTAGAACTTGTTTATTGCAGCT-3′.

    Techniques: FACS, Polymerase Chain Reaction, Amplification

    Analysis of modified PGCs and sperm of recipient rooster. ( a ) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. ( b ) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and ( c ) flow cytometry. ( d ) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. ( e ) mCherry positivity in the spermiogenic epithelium of G 0 recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. ( f ) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.

    Journal: Scientific Reports

    Article Title: Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

    doi: 10.1038/s41598-017-14475-w

    Figure Lengend Snippet: Analysis of modified PGCs and sperm of recipient rooster. ( a ) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. ( b ) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and ( c ) flow cytometry. ( d ) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. ( e ) mCherry positivity in the spermiogenic epithelium of G 0 recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. ( f ) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.

    Article Snippet: An eGFP plasmid that was used before to generate transgenic chickens was used to swap the eGFP with the mCherry gene by Gibson Assembly (New England Biolabs) using the following oligos: fw: 5′-TATCGCATGCCTGCGATGGTGAGCAAGGGCGAGG-3′, rev: 5′-GCATGGACGAGCTGTACAAGTAGAACTTGTTTATTGCAGCT-3′.

    Techniques: Modification, Transfection, Expressing, Plasmid Preparation, Selection, Fluorescence, Microscopy, Flow Cytometry, Sequencing, Pyrolysis Gas Chromatography, Derivative Assay, Transplantation Assay, Polymerase Chain Reaction

    ( a ) mCherry-positive embryo in the middle of incubation. ( b – e ) mCherry positivity in embryo organs and tissues. From left to right: ( b ) liver, ( c ) spleen, ( d ) skeletal muscle, ( e ) heart.

    Journal: Scientific Reports

    Article Title: Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

    doi: 10.1038/s41598-017-14475-w

    Figure Lengend Snippet: ( a ) mCherry-positive embryo in the middle of incubation. ( b – e ) mCherry positivity in embryo organs and tissues. From left to right: ( b ) liver, ( c ) spleen, ( d ) skeletal muscle, ( e ) heart.

    Article Snippet: An eGFP plasmid that was used before to generate transgenic chickens was used to swap the eGFP with the mCherry gene by Gibson Assembly (New England Biolabs) using the following oligos: fw: 5′-TATCGCATGCCTGCGATGGTGAGCAAGGGCGAGG-3′, rev: 5′-GCATGGACGAGCTGTACAAGTAGAACTTGTTTATTGCAGCT-3′.

    Techniques: Incubation