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    Structured Review

    Thermo Fisher hygromycin
    Schematic representation of the reverse engineering approach for the introduction of the mutated alleles. a , b show the direct replacement approach: a two cassettes were amplified, one containing the mutated allele and its terminator region amplified from genomic DNA of the corresponding mutant, flanked downstream by the restriction site for the I-SceI endonuclease and a synthetic homologous recombination sequence (SHR) [ 79 ]. A second cassette was amplified from plasmid pUG6 [ 80 ] for kanMX and from plasmid pUG-natNT2 for natNT2 containing the marker gene flanked upstream by the same SHR sequence as the first cassette and downstream by the restriction site for the I-SceI endonuclease and by 50 bp homologous to the region immediately downstream of the ORF. Upon co-transforming the two cassettes into S. cerevisiae , recombination at the SHR sequence results in integration in the genome and replacement of the wild-type allele. Transformants were selected in YPD containing G418 and/or nourseothricin. b Strains containing the marker gene were transformed with plasmid pUDE206 [ 81 ] expressing the I-SceI endonuclease and selected on YPD agar plates containing <t>hygromycin.</t> I-SceI cuts upstream and downstream of the marker gene, resulting in homologous recombination of the repeated terminator region, and thereby removal of the marker. Removal of marker genes was confirmed by the absence of growth on YPD agar plates containing G418 and/or nourseothricin. For removal of pUDE206, strains containing pUDE206 were grown on YPD and colonies were isolated on YPD agar plates. Plasmid removal was confirmed by the absence of growth on YPD agar plates containing hygromycin. c , d show the deletion/counter-selection approach: c two cassettes were amplified, one containing the GALp-GIN11M86 gene amplified from pGG119 [ 49 ], flanked upstream by 50 bp homologous to the ORF of the gene to be deleted. A second cassette was amplified from plasmid pUG-hphNT1 [ 82 ] containing the hphNT1 marker, flanked upstream by 50 bp homologous to the GALp-GIN11M86 gene and downstream by 50 bp homologous to the ORF to be deleted. Upon co-transforming the two cassettes into S. cerevisiae , they recombine at the SHR sequence insert into the genome, replacing the wild-type allele. d The mutated ORF of the gene was amplified from genomic DNA of the corresponding mutant and was used to transform the strain containing GALp-GIN11M86. Transformants were selected on YPGal agar plates. Replacement of the deleted allele was tested by the ability of transformants to grow in YPGal, inability to grow in the presence of hygromycin, as well as by PCR
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    1) Product Images from "A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations"

    Article Title: A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-016-0583-1

    Schematic representation of the reverse engineering approach for the introduction of the mutated alleles. a , b show the direct replacement approach: a two cassettes were amplified, one containing the mutated allele and its terminator region amplified from genomic DNA of the corresponding mutant, flanked downstream by the restriction site for the I-SceI endonuclease and a synthetic homologous recombination sequence (SHR) [ 79 ]. A second cassette was amplified from plasmid pUG6 [ 80 ] for kanMX and from plasmid pUG-natNT2 for natNT2 containing the marker gene flanked upstream by the same SHR sequence as the first cassette and downstream by the restriction site for the I-SceI endonuclease and by 50 bp homologous to the region immediately downstream of the ORF. Upon co-transforming the two cassettes into S. cerevisiae , recombination at the SHR sequence results in integration in the genome and replacement of the wild-type allele. Transformants were selected in YPD containing G418 and/or nourseothricin. b Strains containing the marker gene were transformed with plasmid pUDE206 [ 81 ] expressing the I-SceI endonuclease and selected on YPD agar plates containing hygromycin. I-SceI cuts upstream and downstream of the marker gene, resulting in homologous recombination of the repeated terminator region, and thereby removal of the marker. Removal of marker genes was confirmed by the absence of growth on YPD agar plates containing G418 and/or nourseothricin. For removal of pUDE206, strains containing pUDE206 were grown on YPD and colonies were isolated on YPD agar plates. Plasmid removal was confirmed by the absence of growth on YPD agar plates containing hygromycin. c , d show the deletion/counter-selection approach: c two cassettes were amplified, one containing the GALp-GIN11M86 gene amplified from pGG119 [ 49 ], flanked upstream by 50 bp homologous to the ORF of the gene to be deleted. A second cassette was amplified from plasmid pUG-hphNT1 [ 82 ] containing the hphNT1 marker, flanked upstream by 50 bp homologous to the GALp-GIN11M86 gene and downstream by 50 bp homologous to the ORF to be deleted. Upon co-transforming the two cassettes into S. cerevisiae , they recombine at the SHR sequence insert into the genome, replacing the wild-type allele. d The mutated ORF of the gene was amplified from genomic DNA of the corresponding mutant and was used to transform the strain containing GALp-GIN11M86. Transformants were selected on YPGal agar plates. Replacement of the deleted allele was tested by the ability of transformants to grow in YPGal, inability to grow in the presence of hygromycin, as well as by PCR
    Figure Legend Snippet: Schematic representation of the reverse engineering approach for the introduction of the mutated alleles. a , b show the direct replacement approach: a two cassettes were amplified, one containing the mutated allele and its terminator region amplified from genomic DNA of the corresponding mutant, flanked downstream by the restriction site for the I-SceI endonuclease and a synthetic homologous recombination sequence (SHR) [ 79 ]. A second cassette was amplified from plasmid pUG6 [ 80 ] for kanMX and from plasmid pUG-natNT2 for natNT2 containing the marker gene flanked upstream by the same SHR sequence as the first cassette and downstream by the restriction site for the I-SceI endonuclease and by 50 bp homologous to the region immediately downstream of the ORF. Upon co-transforming the two cassettes into S. cerevisiae , recombination at the SHR sequence results in integration in the genome and replacement of the wild-type allele. Transformants were selected in YPD containing G418 and/or nourseothricin. b Strains containing the marker gene were transformed with plasmid pUDE206 [ 81 ] expressing the I-SceI endonuclease and selected on YPD agar plates containing hygromycin. I-SceI cuts upstream and downstream of the marker gene, resulting in homologous recombination of the repeated terminator region, and thereby removal of the marker. Removal of marker genes was confirmed by the absence of growth on YPD agar plates containing G418 and/or nourseothricin. For removal of pUDE206, strains containing pUDE206 were grown on YPD and colonies were isolated on YPD agar plates. Plasmid removal was confirmed by the absence of growth on YPD agar plates containing hygromycin. c , d show the deletion/counter-selection approach: c two cassettes were amplified, one containing the GALp-GIN11M86 gene amplified from pGG119 [ 49 ], flanked upstream by 50 bp homologous to the ORF of the gene to be deleted. A second cassette was amplified from plasmid pUG-hphNT1 [ 82 ] containing the hphNT1 marker, flanked upstream by 50 bp homologous to the GALp-GIN11M86 gene and downstream by 50 bp homologous to the ORF to be deleted. Upon co-transforming the two cassettes into S. cerevisiae , they recombine at the SHR sequence insert into the genome, replacing the wild-type allele. d The mutated ORF of the gene was amplified from genomic DNA of the corresponding mutant and was used to transform the strain containing GALp-GIN11M86. Transformants were selected on YPGal agar plates. Replacement of the deleted allele was tested by the ability of transformants to grow in YPGal, inability to grow in the presence of hygromycin, as well as by PCR

    Techniques Used: Amplification, Mutagenesis, Homologous Recombination, Sequencing, Plasmid Preparation, Marker, Transformation Assay, Expressing, Isolation, Selection, Polymerase Chain Reaction

    2) Product Images from "The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass"

    Article Title: The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

    Journal: Genetics and Molecular Biology

    doi: 10.1590/1678-4685-GMB-2014-0300

    Molecular characterization of transgenic switchgrass plants. (A) Polymerase chain reaction (PCR) analysis of DNA samples from regenerated switchgrass plants with Gus and Hyg primers. Marker – 2000-bp DNA molecular markers. Plasmid – pTCK303 vector serving as positive control. Ctrl (Control check) – Wild-type plant serving as a negative control. Lanes 1–13 represent PCR products from individual transgenic plants: 1–4 represent PvPIN1-Ri (RNAi of the PvPIN1 gene), 5–8 represent PvPIN1-OE (overexpression of the PvPIN1 gene), 9 represents asexually reproduced seedlings of PvPIN1-Ri, and 10 represents asexually reproduced seedlings of PvPIN1-OE. (B) Southern blot hybridization analysis of the regenerated plants. The blot was hybridized with a DIG-labeled PCR product of the hygromycin gene. Lane 1 – PvPIN1-OE, 2 – PvPIN1-Ri, 3 and 4 – Asexually propagated plants.
    Figure Legend Snippet: Molecular characterization of transgenic switchgrass plants. (A) Polymerase chain reaction (PCR) analysis of DNA samples from regenerated switchgrass plants with Gus and Hyg primers. Marker – 2000-bp DNA molecular markers. Plasmid – pTCK303 vector serving as positive control. Ctrl (Control check) – Wild-type plant serving as a negative control. Lanes 1–13 represent PCR products from individual transgenic plants: 1–4 represent PvPIN1-Ri (RNAi of the PvPIN1 gene), 5–8 represent PvPIN1-OE (overexpression of the PvPIN1 gene), 9 represents asexually reproduced seedlings of PvPIN1-Ri, and 10 represents asexually reproduced seedlings of PvPIN1-OE. (B) Southern blot hybridization analysis of the regenerated plants. The blot was hybridized with a DIG-labeled PCR product of the hygromycin gene. Lane 1 – PvPIN1-OE, 2 – PvPIN1-Ri, 3 and 4 – Asexually propagated plants.

    Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Marker, Plasmid Preparation, Positive Control, Negative Control, Over Expression, Southern Blot, Hybridization, Labeling

    3) Product Images from "STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus"

    Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007416

    Expression of STAT6 is reduced during KSHV reactivation and early infection. ( A ) Expression of STAT6 but not STAT3 was dramatically reduced during KSHV reactivation. Whole cell lysates from KSHV-infected BCBL1 and BC3 and uninfected BJAB cells individually treated with or without 20 ng/ml of TPA and 1.5mM sodium butyrate (T/NB) for 24 h, were subjected to immunoblotting (IB) with antibodies as indicated in the figure. The relative density (RD) of STAT6 protein band was quantitated. ( B ) Early infection of KSHV reduced STAT6 expression. Whole cell lysate from HUVEC cells with KSHV infection (GFP positive) at different time points, were subjected to immunoblotting (IB) with antibodies as indicated in the figure. ( C ) Doxycycloline (Dox)-induced RTA expression in iSLK-RTA or iSLK-219 cells led to the decreased expression of STAT6 but not of STAT3. Whole cell lysates from the different-induction time points were subjected to immunoblotting (IB) with antibodies as indicated in the figure. ( D ) Quantitative PCR analysis of transcriptional level of STAT6 and STAT3 in iSLK-RTA cells with doxycline induction. ( E ) Quantitative PCR analysis of STAT6 and RTA mRNA transcripts in BJAB, BCBL1 and BC3 cells treated with TPA and sodium butyrate (T/NB) for 0, 12 and 24 h. The relative level of mRNA transcript was present. Beta actin was used as internal control. ( F ) Reporter assays of STAT6 promoter. HEK293 cells co-transfected STAT6 promoter-driven luciferase reporter with different dosage RTA (0, 1, 5, 10μg) were subjected to reporter assay. Relative firefly luciferase unit (RLU) normalization with Renilla activity was analyzed. Data is presented as means±SD of three independent experiments. The expression of exogenous RTA was verified by immunoblotting assays and shown in the middle panel. Schematic of putative RBP-Jκ-binding sites within STAT6 promoter is shown at the bottom panel. ( G ) RTA reduces the protein stability of STAT6. HEK293T cells were co-transfected by FLAG-STAT6 with RTA-myc or vector alone. At 36 h post-transfection, cells were treated with Cycloheximide (CHX) 200μg/ml for the indicated time before harvesting and lysing for immunoblotting. The relative density (RD) of protein level of STAT6 is quantified based on triplicate experiments and shown at the bottom panel.
    Figure Legend Snippet: Expression of STAT6 is reduced during KSHV reactivation and early infection. ( A ) Expression of STAT6 but not STAT3 was dramatically reduced during KSHV reactivation. Whole cell lysates from KSHV-infected BCBL1 and BC3 and uninfected BJAB cells individually treated with or without 20 ng/ml of TPA and 1.5mM sodium butyrate (T/NB) for 24 h, were subjected to immunoblotting (IB) with antibodies as indicated in the figure. The relative density (RD) of STAT6 protein band was quantitated. ( B ) Early infection of KSHV reduced STAT6 expression. Whole cell lysate from HUVEC cells with KSHV infection (GFP positive) at different time points, were subjected to immunoblotting (IB) with antibodies as indicated in the figure. ( C ) Doxycycloline (Dox)-induced RTA expression in iSLK-RTA or iSLK-219 cells led to the decreased expression of STAT6 but not of STAT3. Whole cell lysates from the different-induction time points were subjected to immunoblotting (IB) with antibodies as indicated in the figure. ( D ) Quantitative PCR analysis of transcriptional level of STAT6 and STAT3 in iSLK-RTA cells with doxycline induction. ( E ) Quantitative PCR analysis of STAT6 and RTA mRNA transcripts in BJAB, BCBL1 and BC3 cells treated with TPA and sodium butyrate (T/NB) for 0, 12 and 24 h. The relative level of mRNA transcript was present. Beta actin was used as internal control. ( F ) Reporter assays of STAT6 promoter. HEK293 cells co-transfected STAT6 promoter-driven luciferase reporter with different dosage RTA (0, 1, 5, 10μg) were subjected to reporter assay. Relative firefly luciferase unit (RLU) normalization with Renilla activity was analyzed. Data is presented as means±SD of three independent experiments. The expression of exogenous RTA was verified by immunoblotting assays and shown in the middle panel. Schematic of putative RBP-Jκ-binding sites within STAT6 promoter is shown at the bottom panel. ( G ) RTA reduces the protein stability of STAT6. HEK293T cells were co-transfected by FLAG-STAT6 with RTA-myc or vector alone. At 36 h post-transfection, cells were treated with Cycloheximide (CHX) 200μg/ml for the indicated time before harvesting and lysing for immunoblotting. The relative density (RD) of protein level of STAT6 is quantified based on triplicate experiments and shown at the bottom panel.

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Plasmid Preparation

    RTA interacts with STAT6. ( A ) Ectopic expression of exogenous STAT6 interacted with RTA. Whole cell lysate (WCL) from 293T cells co-transfected with different combination of FLAG-STAT6 and RTA-myc as indicated, were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. ( B ) Endogenous STAT6 associated with RTA in KSHV-positive cells with lytic reactivation. Whole cell lysate (WCL) from BC3 cells treated with 20 ng/ml of TPA and 1.5mM sodium butyrate (T/NB) or iSLK-219 cells treated with 2μg/ml doxycycloline (Dox) for 24h were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. ( C ) RTA associated with the carboxyl terminus of STAT6. 293T cells were transfected with expressing plasmids for 48 h, and the whole cell lysates (WCL) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. Schematic of STAT6 amino acid sequence and its truncation mutants were shown at the bottom panels. The red circle indicates the RTA-associated domains. The nuclear localization sequence (NLS), ubiquitylation (Ub) and phosphorylation (P) sites are shown.
    Figure Legend Snippet: RTA interacts with STAT6. ( A ) Ectopic expression of exogenous STAT6 interacted with RTA. Whole cell lysate (WCL) from 293T cells co-transfected with different combination of FLAG-STAT6 and RTA-myc as indicated, were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. ( B ) Endogenous STAT6 associated with RTA in KSHV-positive cells with lytic reactivation. Whole cell lysate (WCL) from BC3 cells treated with 20 ng/ml of TPA and 1.5mM sodium butyrate (T/NB) or iSLK-219 cells treated with 2μg/ml doxycycloline (Dox) for 24h were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. ( C ) RTA associated with the carboxyl terminus of STAT6. 293T cells were transfected with expressing plasmids for 48 h, and the whole cell lysates (WCL) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. Schematic of STAT6 amino acid sequence and its truncation mutants were shown at the bottom panels. The red circle indicates the RTA-associated domains. The nuclear localization sequence (NLS), ubiquitylation (Ub) and phosphorylation (P) sites are shown.

    Techniques Used: Expressing, Transfection, Immunoprecipitation, Sequencing

    STAT6 knockdown significantly turns over cellular gene expression of PEL cells. ( A ) were individually subjected to RNA deep-sequencing analysis. The cellular genes with significantly change in the presence of STAT6 knockdown are shown. ( B ) Reduction of STAT6 led to the increased gene expression of MHC II-, cytokine- or anti-apoptosis-related proteins in PEL cells. The molecules related to antigen processing and presentation, cell migration, tumor suppressor, and chromosome stability are highlighted. ( C ) and PEL cells with STAT6 knockdown. ( D ) Quantitative PCR analysis of TRIML2, AIM1 and CIITA expression in the iSLK-RTA and iSLK-219 cells treated with Doxycycline, or BCBL1 cells treated with TPA and sodium butyrate (T/NB) for 24 h.
    Figure Legend Snippet: STAT6 knockdown significantly turns over cellular gene expression of PEL cells. ( A ) were individually subjected to RNA deep-sequencing analysis. The cellular genes with significantly change in the presence of STAT6 knockdown are shown. ( B ) Reduction of STAT6 led to the increased gene expression of MHC II-, cytokine- or anti-apoptosis-related proteins in PEL cells. The molecules related to antigen processing and presentation, cell migration, tumor suppressor, and chromosome stability are highlighted. ( C ) and PEL cells with STAT6 knockdown. ( D ) Quantitative PCR analysis of TRIML2, AIM1 and CIITA expression in the iSLK-RTA and iSLK-219 cells treated with Doxycycline, or BCBL1 cells treated with TPA and sodium butyrate (T/NB) for 24 h.

    Techniques Used: Expressing, Sequencing, Migration, Real-time Polymerase Chain Reaction

    4) Product Images from "Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome"

    Article Title: Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

    Journal: Virology Journal

    doi: 10.1186/1743-422X-6-73

    CPE due to DENV infection of C6/36 cells at 5 dpi . Images were taken at the 40× magnification. Cells were those transduced with hRz-encoding retroviruses and selected in hygromycin for stable integration of the transgene. Representative infected cell cultures are shown. These are cells transduced with (A) hRz # 2 , (B) hRz # 5 , (C) hRz # 7 , (D) hRz # 11 , (E) No Rz (transduced with lentivirus vector lacking a hRz) or (F) non-transduced C6/36 cells.
    Figure Legend Snippet: CPE due to DENV infection of C6/36 cells at 5 dpi . Images were taken at the 40× magnification. Cells were those transduced with hRz-encoding retroviruses and selected in hygromycin for stable integration of the transgene. Representative infected cell cultures are shown. These are cells transduced with (A) hRz # 2 , (B) hRz # 5 , (C) hRz # 7 , (D) hRz # 11 , (E) No Rz (transduced with lentivirus vector lacking a hRz) or (F) non-transduced C6/36 cells.

    Techniques Used: Infection, Transduction, Plasmid Preparation

    A: Representative hRz structure and its DENV target sequence . hRz # 1 nucleotide sequence and structure is depicted. Nucleotides flanking the cleavage site (yellow box) in the envelope protein region of the DENV-2 target RNA are enlarged. The ribozyme cleaves the target RNA at the GUC triplet site following antisense recognition and base pairing of the two ribozyme arms. B: Nucleotide alignments of the Human (Hs), D. melanogaster (Dm), and Ae. aegypti (Aa) tRNA val . The position of the concensus internal A and B blocks of the RNA pol III promoter are indicated. C: Plasmid pLAeRzARH was derived from pLXRN as described in Materials and Methods. The RSV promoter was added to drive independent expression of the hygromycin resistance gene. Expression of each hRz is driven by the tRNA val internal RNA pol III promoter to optimize expression and translocation of the hRzs to the cytoplasm, and a stretch of 60As is attached to the 3' end of the hRz sequence for recruitment of RNA helicase.
    Figure Legend Snippet: A: Representative hRz structure and its DENV target sequence . hRz # 1 nucleotide sequence and structure is depicted. Nucleotides flanking the cleavage site (yellow box) in the envelope protein region of the DENV-2 target RNA are enlarged. The ribozyme cleaves the target RNA at the GUC triplet site following antisense recognition and base pairing of the two ribozyme arms. B: Nucleotide alignments of the Human (Hs), D. melanogaster (Dm), and Ae. aegypti (Aa) tRNA val . The position of the concensus internal A and B blocks of the RNA pol III promoter are indicated. C: Plasmid pLAeRzARH was derived from pLXRN as described in Materials and Methods. The RSV promoter was added to drive independent expression of the hygromycin resistance gene. Expression of each hRz is driven by the tRNA val internal RNA pol III promoter to optimize expression and translocation of the hRzs to the cytoplasm, and a stretch of 60As is attached to the 3' end of the hRz sequence for recruitment of RNA helicase.

    Techniques Used: Sequencing, Plasmid Preparation, Derivative Assay, Expressing, Translocation Assay

    5) Product Images from "High-Resolution Mutation Mapping Reveals Parallel Experimental Evolution in YeastMapping Evolution: Linking Adaptive Traits to Genomic Location"

    Article Title: High-Resolution Mutation Mapping Reveals Parallel Experimental Evolution in YeastMapping Evolution: Linking Adaptive Traits to Genomic Location

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0040256

    Successful Mapping of Known Genes Two separate pools of ˜10 7 W303/SK1 segregants, one resistant to canavanine that grew without lysine (can1, LYS5) and one resistant to geneticin, hygromycin, and nourseothricin (KAN R , NAT R , HYG R ), were selected for and mapped. The five genes mapped are: (A) can1, centered on Chromosome 5, position 32.6 kb; (B) LYS5, on Chromosome 7, position 215.7 kb; (C) KAN R , on Chromosome 7, position 413.4 kb; (D) HYG R , on Chromosome 15, position 619.1 kb; and NAT R , on Chromosome 15, position 960.6 kb. The LMS was calculated across the whole genome for each pool (shown in Figure S3 ) and is plotted here along the chromosomes that carry the five target genes, as a function of chromosome position in 100-kb units. The five peaks that correspond to the five target genes all fell above the significant peak thresholds estimated at 99% confidence for each selected pool (horizontal dashed lines, which are so close to the x -axis as to be invisible in [C] and [D]). The arrows mark the actual center of the target genes, the solid lines within the drawn chromosomes mark the predicted center of the genes, and the grey boxes within the chromosomes mark the 95% confidence intervals estimated with simulations (see Materials and Methods ). The peak for LYS5 is low relative to the significant peak cutoff, because of the low local SFP density (see Figure S3 G– S3 I for discussion). Note that the scale of the y -axis is different in the four panels ( Protocol S1 ). (E) The mapping deviations of the genes' predicted centers from their actual centers and their 95% confidence intervals. Their corresponding average genetic distance in cM is written in parentheses. All five genes were found within their 95% confidence intervals.
    Figure Legend Snippet: Successful Mapping of Known Genes Two separate pools of ˜10 7 W303/SK1 segregants, one resistant to canavanine that grew without lysine (can1, LYS5) and one resistant to geneticin, hygromycin, and nourseothricin (KAN R , NAT R , HYG R ), were selected for and mapped. The five genes mapped are: (A) can1, centered on Chromosome 5, position 32.6 kb; (B) LYS5, on Chromosome 7, position 215.7 kb; (C) KAN R , on Chromosome 7, position 413.4 kb; (D) HYG R , on Chromosome 15, position 619.1 kb; and NAT R , on Chromosome 15, position 960.6 kb. The LMS was calculated across the whole genome for each pool (shown in Figure S3 ) and is plotted here along the chromosomes that carry the five target genes, as a function of chromosome position in 100-kb units. The five peaks that correspond to the five target genes all fell above the significant peak thresholds estimated at 99% confidence for each selected pool (horizontal dashed lines, which are so close to the x -axis as to be invisible in [C] and [D]). The arrows mark the actual center of the target genes, the solid lines within the drawn chromosomes mark the predicted center of the genes, and the grey boxes within the chromosomes mark the 95% confidence intervals estimated with simulations (see Materials and Methods ). The peak for LYS5 is low relative to the significant peak cutoff, because of the low local SFP density (see Figure S3 G– S3 I for discussion). Note that the scale of the y -axis is different in the four panels ( Protocol S1 ). (E) The mapping deviations of the genes' predicted centers from their actual centers and their 95% confidence intervals. Their corresponding average genetic distance in cM is written in parentheses. All five genes were found within their 95% confidence intervals.

    Techniques Used:

    6) Product Images from "The synchronous TAG production with the growth by the expression of chloroplast transit peptide-fused ScPDAT in Chlamydomonas reinhardtii"

    Article Title: The synchronous TAG production with the growth by the expression of chloroplast transit peptide-fused ScPDAT in Chlamydomonas reinhardtii

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-018-1160-6

    The construction and effects of exogenous ScPDAT. a The map of pChlamy–ScPDAT plasmid. PUCori: (high-copy replication and growth in E. coli , 673 bp), Hsp70A–RbcS2 (a hybrid constitutive promoter, 495 bp), Int-1 Rbc S2 (maintains the high expression of ScPDAT, 144 bp), β2-tubulin (drives the expression of Aph7 gene, 312 bp), Hygromycin (selection of C. reinhardtii , 1310 bp ), Ampicillin (selection of the plasmid in E. coli , 860 bp), Pbla (expression of the ampicillin resistance gene, 51 bp). b Western blot of ScPDAT in Scpdat , the growth curve of wild type and Scpdat . Cells sampled during a 5-day culture cycle and grown in TAP medium. Each type of algae takes three bottles a day and does not put them back. Shown are mean values and SD; n = 3. Assay conditions are described in “ Methods ”. c The chlorophyll fluorescence of wild type and Scpdat . Shown are mean values and SD; n = 3. Assay conditions are described in “ Methods ”
    Figure Legend Snippet: The construction and effects of exogenous ScPDAT. a The map of pChlamy–ScPDAT plasmid. PUCori: (high-copy replication and growth in E. coli , 673 bp), Hsp70A–RbcS2 (a hybrid constitutive promoter, 495 bp), Int-1 Rbc S2 (maintains the high expression of ScPDAT, 144 bp), β2-tubulin (drives the expression of Aph7 gene, 312 bp), Hygromycin (selection of C. reinhardtii , 1310 bp ), Ampicillin (selection of the plasmid in E. coli , 860 bp), Pbla (expression of the ampicillin resistance gene, 51 bp). b Western blot of ScPDAT in Scpdat , the growth curve of wild type and Scpdat . Cells sampled during a 5-day culture cycle and grown in TAP medium. Each type of algae takes three bottles a day and does not put them back. Shown are mean values and SD; n = 3. Assay conditions are described in “ Methods ”. c The chlorophyll fluorescence of wild type and Scpdat . Shown are mean values and SD; n = 3. Assay conditions are described in “ Methods ”

    Techniques Used: Plasmid Preparation, Expressing, Selection, Western Blot, Fluorescence

    7) Product Images from "Massively parallel assessment of human variants with base editor screens"

    Article Title: Massively parallel assessment of human variants with base editor screens

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100818

    Profiling of 52,034 clinical variants using base editor, related to Figure 7 . (A) Distribution of negative and positive control guides for ClinVar screens in MELJUSO and HT29 (1,461 negative and 496 positive controls) and wtCas9 in HT29 (1,456 negative and 496 positive controls) in the absence of small molecule treatment. Boxes show the quartiles; whiskers show 1.5 times the interquartile range. (B) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO dropout (left) and cisplatin (right) arms. Pearson correlation is indicated. Points are colored by density. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 (left) and BRCA2 (right) between ClinVar and prior tiling screens ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of guides predicted to make a “clean” edit (i.e. in a non-GC motif with no bystanders) in a clinical variant. (E) Distribution of clinical significance for guides predicted to make a clean edit at a range of Z-score cutoffs in HT29 treated with cisplatin. All cutoffs are significant when compared against the unfiltered distribution (*p
    Figure Legend Snippet: Profiling of 52,034 clinical variants using base editor, related to Figure 7 . (A) Distribution of negative and positive control guides for ClinVar screens in MELJUSO and HT29 (1,461 negative and 496 positive controls) and wtCas9 in HT29 (1,456 negative and 496 positive controls) in the absence of small molecule treatment. Boxes show the quartiles; whiskers show 1.5 times the interquartile range. (B) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO dropout (left) and cisplatin (right) arms. Pearson correlation is indicated. Points are colored by density. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 (left) and BRCA2 (right) between ClinVar and prior tiling screens ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of guides predicted to make a “clean” edit (i.e. in a non-GC motif with no bystanders) in a clinical variant. (E) Distribution of clinical significance for guides predicted to make a clean edit at a range of Z-score cutoffs in HT29 treated with cisplatin. All cutoffs are significant when compared against the unfiltered distribution (*p

    Techniques Used: Positive Control, Variant Assay

    GJB2 loss-of-function in HT29 cells treated with hygromycin, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO hygromycin arms. Pearson correlation is indicated. Points are colored by density. (B) Z-scored log-fold change for GJB2 in HT29 treated with hygromycin. The density plot for intergenic controls is plotted. The mean value for controls is indicated as a dashed line. Each bar represents a guide, and the density plot represents the distribution of guides targeting GJB2.
    Figure Legend Snippet: GJB2 loss-of-function in HT29 cells treated with hygromycin, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO hygromycin arms. Pearson correlation is indicated. Points are colored by density. (B) Z-scored log-fold change for GJB2 in HT29 treated with hygromycin. The density plot for intergenic controls is plotted. The mean value for controls is indicated as a dashed line. Each bar represents a guide, and the density plot represents the distribution of guides targeting GJB2.

    Techniques Used:

    Functional profiling of 52,034 clinical variants. (A) Fraction of all single nucleotide clinical variants (n = 388,496) in the ClinVar library that were included in the library (n = 52,034). (B) Timeline of ClinVar screens in HT29 and MELJUSO cells. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 between this ClinVar screen and prior tiling screen ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of sgRNAs targeting genes in the Reactome DNA repair pathway at a range of Z-score cutoffs. All cutoffs are significant when compared against the starting library (*p
    Figure Legend Snippet: Functional profiling of 52,034 clinical variants. (A) Fraction of all single nucleotide clinical variants (n = 388,496) in the ClinVar library that were included in the library (n = 52,034). (B) Timeline of ClinVar screens in HT29 and MELJUSO cells. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 between this ClinVar screen and prior tiling screen ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of sgRNAs targeting genes in the Reactome DNA repair pathway at a range of Z-score cutoffs. All cutoffs are significant when compared against the starting library (*p

    Techniques Used: Functional Assay

    Protein-protein interactions between hits from HT29 and MELJUSO cisplatin screens, related to Figure 7 . Edges represent confidence in STRING. Nodes represent gene hits. Nodes colored in red are in the Reactome DNA repair pathway. Nodes colored in blue are listed in the GO-term for chromatin organization. To define gene hits, we used an independent two sample t-test comparing the log-fold changes of VEP high impact sgRNAs to both intergenic and non-targeting controls in HT29 and MELJUSO treated with cisplatin. We then used the Benjamini-Hochberg procedure to define significant genes, using an FDR cutoff of 0.1. For each cell line, we identified hits which were significant when compared against both sets of controls. We then included all hits from either cell line in the network.
    Figure Legend Snippet: Protein-protein interactions between hits from HT29 and MELJUSO cisplatin screens, related to Figure 7 . Edges represent confidence in STRING. Nodes represent gene hits. Nodes colored in red are in the Reactome DNA repair pathway. Nodes colored in blue are listed in the GO-term for chromatin organization. To define gene hits, we used an independent two sample t-test comparing the log-fold changes of VEP high impact sgRNAs to both intergenic and non-targeting controls in HT29 and MELJUSO treated with cisplatin. We then used the Benjamini-Hochberg procedure to define significant genes, using an FDR cutoff of 0.1. For each cell line, we identified hits which were significant when compared against both sets of controls. We then included all hits from either cell line in the network.

    Techniques Used:

    Counter-screen with wtCas9 in HT29 cells confirms loss-of-function alleles and uncovers potential gain-of-function mutants, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between wildtype (WT) and base editor (BE) Cas9 in the HT29 untreated arm. Bins for defining hits are outlined. Pearson correlation coefficient is indicated. Points are colored by density (B). Same as (A) but for HT29 treated with cisplatin. (C) Distribution of predicted impact for sgRNAs in the HT29 untreated arm. Bins correspond to those indicated in panel (A). Positive controls include sgRNAs targeting essential splice sites. Negative controls include sgRNAs targeting non-essential splice sites, intergenic and non-targeting controls. “No edit” sgRNAs only have C’s in a GC-motif in the edit window. (D) Same as (C) but for HT29 treated with cisplatin. (E) Same as (C) but for predicted consequence. All consequences with a maximum fraction of 0.05 across bins are grouped as “other”. (F) Same as (E) but for HT29 treated with cisplatin.
    Figure Legend Snippet: Counter-screen with wtCas9 in HT29 cells confirms loss-of-function alleles and uncovers potential gain-of-function mutants, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between wildtype (WT) and base editor (BE) Cas9 in the HT29 untreated arm. Bins for defining hits are outlined. Pearson correlation coefficient is indicated. Points are colored by density (B). Same as (A) but for HT29 treated with cisplatin. (C) Distribution of predicted impact for sgRNAs in the HT29 untreated arm. Bins correspond to those indicated in panel (A). Positive controls include sgRNAs targeting essential splice sites. Negative controls include sgRNAs targeting non-essential splice sites, intergenic and non-targeting controls. “No edit” sgRNAs only have C’s in a GC-motif in the edit window. (D) Same as (C) but for HT29 treated with cisplatin. (E) Same as (C) but for predicted consequence. All consequences with a maximum fraction of 0.05 across bins are grouped as “other”. (F) Same as (E) but for HT29 treated with cisplatin.

    Techniques Used:

    8) Product Images from "Selective monitoring of insulin secretion after CRISPR interference in intact pancreatic islets despite submaximal infection"

    Article Title: Selective monitoring of insulin secretion after CRISPR interference in intact pancreatic islets despite submaximal infection

    Journal: Islets

    doi: 10.1080/19382014.2020.1752072

    CRISPRi-coupled insulin nanoluciferase reporter. (A) Schematic of the reporter coupled to a sgRNA expression cassette and a dCas9-KRAB cDNA. (B) Validation of a potent Gck sgRNA. MIN6 expressing dCas9KRAB were infected with a virus expressing a Gck sgRNA or a control sgRNA selected with hygromycin and RT-qPCR was performed for Gck . n = 3. As determined by Student’s t-test, **p
    Figure Legend Snippet: CRISPRi-coupled insulin nanoluciferase reporter. (A) Schematic of the reporter coupled to a sgRNA expression cassette and a dCas9-KRAB cDNA. (B) Validation of a potent Gck sgRNA. MIN6 expressing dCas9KRAB were infected with a virus expressing a Gck sgRNA or a control sgRNA selected with hygromycin and RT-qPCR was performed for Gck . n = 3. As determined by Student’s t-test, **p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    9) Product Images from "Homologous recombination is required for AAV-mediated gene targeting"

    Article Title: Homologous recombination is required for AAV-mediated gene targeting

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl455

    Schematic representation of the vectors used for gene targeting. ( A ) Plasmid pEGFPΔ32 containing the mutant target (thick white arrow) expressed under human CMV promoter was integrated in the genome of MO59K cells. Thirty-two base pair deletion at position 198 (5′-ctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaa-3′) was replaced by an in-frame stop-codon supplied within a unique SpeI restriction site (5′-CAC TAG TTGAAGCa-3′). Resistance to hygromycin was provided by a hygromycin phosphotransferase (Hygro) expressed from an SV40 promoter. ( B ) Schematic of rAAV used as repair substrates in gene targeting experiments. All viral vectors consisted of single-stranded DNA enclosed by native AAV subtype 2 inverted repeats. Both substrates (white arrows) lacked 14 bp from the 5′ end of the EGFP gene including the ATG codon (Δ14EGFP). A short and a long fragment from the P.pastoris alcohol oxidase termination sequence (Pp AOX TT and TT) replaced the CMV and the CAAT+TATA signals of the CBA/rabbit β-globin promoter (CBA/β-globin). The rAAV-NeoX contained additional homology to the genomic target (thick black line). The recombinant viruses also contained a neomycin phosphotransferase gene (Neo) expressed under a HSV-TK promoter. ( C ) Diagram of the vectors used to test cell infectivity. rAAV-RFPX and rAAV-RFP were identical to rAAV-NeoX and rAAV-Neo, respectively, except for the replacement of the neomycin phosphotransferase expression cassete with a RFP expression.
    Figure Legend Snippet: Schematic representation of the vectors used for gene targeting. ( A ) Plasmid pEGFPΔ32 containing the mutant target (thick white arrow) expressed under human CMV promoter was integrated in the genome of MO59K cells. Thirty-two base pair deletion at position 198 (5′-ctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaa-3′) was replaced by an in-frame stop-codon supplied within a unique SpeI restriction site (5′-CAC TAG TTGAAGCa-3′). Resistance to hygromycin was provided by a hygromycin phosphotransferase (Hygro) expressed from an SV40 promoter. ( B ) Schematic of rAAV used as repair substrates in gene targeting experiments. All viral vectors consisted of single-stranded DNA enclosed by native AAV subtype 2 inverted repeats. Both substrates (white arrows) lacked 14 bp from the 5′ end of the EGFP gene including the ATG codon (Δ14EGFP). A short and a long fragment from the P.pastoris alcohol oxidase termination sequence (Pp AOX TT and TT) replaced the CMV and the CAAT+TATA signals of the CBA/rabbit β-globin promoter (CBA/β-globin). The rAAV-NeoX contained additional homology to the genomic target (thick black line). The recombinant viruses also contained a neomycin phosphotransferase gene (Neo) expressed under a HSV-TK promoter. ( C ) Diagram of the vectors used to test cell infectivity. rAAV-RFPX and rAAV-RFP were identical to rAAV-NeoX and rAAV-Neo, respectively, except for the replacement of the neomycin phosphotransferase expression cassete with a RFP expression.

    Techniques Used: Plasmid Preparation, Mutagenesis, Sequencing, Crocin Bleaching Assay, Recombinant, Infection, Expressing

    10) Product Images from "Massively parallel assessment of human variants with base editor screens"

    Article Title: Massively parallel assessment of human variants with base editor screens

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100818

    Profiling of 52,034 clinical variants using base editor, related to Figure 7 . (A) Distribution of negative and positive control guides for ClinVar screens in MELJUSO and HT29 (1,461 negative and 496 positive controls) and wtCas9 in HT29 (1,456 negative and 496 positive controls) in the absence of small molecule treatment. Boxes show the quartiles; whiskers show 1.5 times the interquartile range. (B) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO dropout (left) and cisplatin (right) arms. Pearson correlation is indicated. Points are colored by density. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 (left) and BRCA2 (right) between ClinVar and prior tiling screens ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of guides predicted to make a “clean” edit (i.e. in a non-GC motif with no bystanders) in a clinical variant. (E) Distribution of clinical significance for guides predicted to make a clean edit at a range of Z-score cutoffs in HT29 treated with cisplatin. All cutoffs are significant when compared against the unfiltered distribution (*p
    Figure Legend Snippet: Profiling of 52,034 clinical variants using base editor, related to Figure 7 . (A) Distribution of negative and positive control guides for ClinVar screens in MELJUSO and HT29 (1,461 negative and 496 positive controls) and wtCas9 in HT29 (1,456 negative and 496 positive controls) in the absence of small molecule treatment. Boxes show the quartiles; whiskers show 1.5 times the interquartile range. (B) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO dropout (left) and cisplatin (right) arms. Pearson correlation is indicated. Points are colored by density. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 (left) and BRCA2 (right) between ClinVar and prior tiling screens ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of guides predicted to make a “clean” edit (i.e. in a non-GC motif with no bystanders) in a clinical variant. (E) Distribution of clinical significance for guides predicted to make a clean edit at a range of Z-score cutoffs in HT29 treated with cisplatin. All cutoffs are significant when compared against the unfiltered distribution (*p

    Techniques Used: Positive Control, Variant Assay

    GJB2 loss-of-function in HT29 cells treated with hygromycin, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO hygromycin arms. Pearson correlation is indicated. Points are colored by density. (B) Z-scored log-fold change for GJB2 in HT29 treated with hygromycin. The density plot for intergenic controls is plotted. The mean value for controls is indicated as a dashed line. Each bar represents a guide, and the density plot represents the distribution of guides targeting GJB2.
    Figure Legend Snippet: GJB2 loss-of-function in HT29 cells treated with hygromycin, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between HT29 and MELJUSO hygromycin arms. Pearson correlation is indicated. Points are colored by density. (B) Z-scored log-fold change for GJB2 in HT29 treated with hygromycin. The density plot for intergenic controls is plotted. The mean value for controls is indicated as a dashed line. Each bar represents a guide, and the density plot represents the distribution of guides targeting GJB2.

    Techniques Used:

    Functional profiling of 52,034 clinical variants. (A) Fraction of all single nucleotide clinical variants (n = 388,496) in the ClinVar library that were included in the library (n = 52,034). (B) Timeline of ClinVar screens in HT29 and MELJUSO cells. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 between this ClinVar screen and prior tiling screen ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of sgRNAs targeting genes in the Reactome DNA repair pathway at a range of Z-score cutoffs. All cutoffs are significant when compared against the starting library (*p
    Figure Legend Snippet: Functional profiling of 52,034 clinical variants. (A) Fraction of all single nucleotide clinical variants (n = 388,496) in the ClinVar library that were included in the library (n = 52,034). (B) Timeline of ClinVar screens in HT29 and MELJUSO cells. (C) Correlation of log-fold changes of sgRNAs targeting BRCA1 between this ClinVar screen and prior tiling screen ( Figure 2 ). Pearson correlation is indicated. (D) Fraction of sgRNAs targeting genes in the Reactome DNA repair pathway at a range of Z-score cutoffs. All cutoffs are significant when compared against the starting library (*p

    Techniques Used: Functional Assay

    Protein-protein interactions between hits from HT29 and MELJUSO cisplatin screens, related to Figure 7 . Edges represent confidence in STRING. Nodes represent gene hits. Nodes colored in red are in the Reactome DNA repair pathway. Nodes colored in blue are listed in the GO-term for chromatin organization. To define gene hits, we used an independent two sample t-test comparing the log-fold changes of VEP high impact sgRNAs to both intergenic and non-targeting controls in HT29 and MELJUSO treated with cisplatin. We then used the Benjamini-Hochberg procedure to define significant genes, using an FDR cutoff of 0.1. For each cell line, we identified hits which were significant when compared against both sets of controls. We then included all hits from either cell line in the network.
    Figure Legend Snippet: Protein-protein interactions between hits from HT29 and MELJUSO cisplatin screens, related to Figure 7 . Edges represent confidence in STRING. Nodes represent gene hits. Nodes colored in red are in the Reactome DNA repair pathway. Nodes colored in blue are listed in the GO-term for chromatin organization. To define gene hits, we used an independent two sample t-test comparing the log-fold changes of VEP high impact sgRNAs to both intergenic and non-targeting controls in HT29 and MELJUSO treated with cisplatin. We then used the Benjamini-Hochberg procedure to define significant genes, using an FDR cutoff of 0.1. For each cell line, we identified hits which were significant when compared against both sets of controls. We then included all hits from either cell line in the network.

    Techniques Used:

    Counter-screen with wtCas9 in HT29 cells confirms loss-of-function alleles and uncovers potential gain-of-function mutants, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between wildtype (WT) and base editor (BE) Cas9 in the HT29 untreated arm. Bins for defining hits are outlined. Pearson correlation coefficient is indicated. Points are colored by density (B). Same as (A) but for HT29 treated with cisplatin. (C) Distribution of predicted impact for sgRNAs in the HT29 untreated arm. Bins correspond to those indicated in panel (A). Positive controls include sgRNAs targeting essential splice sites. Negative controls include sgRNAs targeting non-essential splice sites, intergenic and non-targeting controls. “No edit” sgRNAs only have C’s in a GC-motif in the edit window. (D) Same as (C) but for HT29 treated with cisplatin. (E) Same as (C) but for predicted consequence. All consequences with a maximum fraction of 0.05 across bins are grouped as “other”. (F) Same as (E) but for HT29 treated with cisplatin.
    Figure Legend Snippet: Counter-screen with wtCas9 in HT29 cells confirms loss-of-function alleles and uncovers potential gain-of-function mutants, related to Figure 7 . (A) Correlation of z-scored log-fold changes (relative to intergenic controls) between wildtype (WT) and base editor (BE) Cas9 in the HT29 untreated arm. Bins for defining hits are outlined. Pearson correlation coefficient is indicated. Points are colored by density (B). Same as (A) but for HT29 treated with cisplatin. (C) Distribution of predicted impact for sgRNAs in the HT29 untreated arm. Bins correspond to those indicated in panel (A). Positive controls include sgRNAs targeting essential splice sites. Negative controls include sgRNAs targeting non-essential splice sites, intergenic and non-targeting controls. “No edit” sgRNAs only have C’s in a GC-motif in the edit window. (D) Same as (C) but for HT29 treated with cisplatin. (E) Same as (C) but for predicted consequence. All consequences with a maximum fraction of 0.05 across bins are grouped as “other”. (F) Same as (E) but for HT29 treated with cisplatin.

    Techniques Used:

    11) Product Images from "Expression Ratios of the Anti-Apoptotic BCL2 Family Members Dictate the Selective Addiction of KSHV-Transformed Primary Effusion Lymphoma Cell Lines to MCL1"

    Article Title: Expression Ratios of the Anti-Apoptotic BCL2 Family Members Dictate the Selective Addiction of KSHV-Transformed Primary Effusion Lymphoma Cell Lines to MCL1

    Journal: bioRxiv

    doi: 10.1101/2022.09.07.507059

    A . Western blots for BAX, BAK1 and tubulin proteins of WT or BAX/BAK1 double knockout (DKO) clone 28 (BC-1) and clone 12 (BC-3). B . Summary of CRISPR edits in BC-1 DKO clone #28 and BC-3 DKO clone #12. C-G . Dose response curves of BC-1 or BC-3 cells with BAX/BAK1 wildtype or DKO background upon exposure to different inducers of cell death: C . staurosporine, D . puromycin, E . blasticidin, F . hygromycin, or G . zeocin. Statistical differences were calculated using two-way ANOVA with Tukey’s multiple comparison post-hoc test (n=3). Adjusted p values reflect post-hoc comparisons between the responses of the wt cell line compared to the DKO cell line at the specific time point. Error bars, standard error of mean. n.s., not significant.
    Figure Legend Snippet: A . Western blots for BAX, BAK1 and tubulin proteins of WT or BAX/BAK1 double knockout (DKO) clone 28 (BC-1) and clone 12 (BC-3). B . Summary of CRISPR edits in BC-1 DKO clone #28 and BC-3 DKO clone #12. C-G . Dose response curves of BC-1 or BC-3 cells with BAX/BAK1 wildtype or DKO background upon exposure to different inducers of cell death: C . staurosporine, D . puromycin, E . blasticidin, F . hygromycin, or G . zeocin. Statistical differences were calculated using two-way ANOVA with Tukey’s multiple comparison post-hoc test (n=3). Adjusted p values reflect post-hoc comparisons between the responses of the wt cell line compared to the DKO cell line at the specific time point. Error bars, standard error of mean. n.s., not significant.

    Techniques Used: Western Blot, Double Knockout, CRISPR

    12) Product Images from "A mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species"

    Article Title: A mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07995-y

    Chr RC and the two smaller accessory chromosomes of Forc016 are conditionally dispensable. Electrophoretic karyotyping shows complete absence of chr RC (marked with a red asterisk in lane 6) in all five hygromycin sensitive strains (arrowhead 1, lanes 1–5). Additionally, chromosome loss strain #2 displays absence of the two smallest chromosomes (arrowheads 2). The left and right lanes show the karyotypes of S . pombe and S . cerevisiae , respectively, applied as markers. This image is cropped, the original gel photograph can be found in Supplementary Fig. S13 .
    Figure Legend Snippet: Chr RC and the two smaller accessory chromosomes of Forc016 are conditionally dispensable. Electrophoretic karyotyping shows complete absence of chr RC (marked with a red asterisk in lane 6) in all five hygromycin sensitive strains (arrowhead 1, lanes 1–5). Additionally, chromosome loss strain #2 displays absence of the two smallest chromosomes (arrowheads 2). The left and right lanes show the karyotypes of S . pombe and S . cerevisiae , respectively, applied as markers. This image is cropped, the original gel photograph can be found in Supplementary Fig. S13 .

    Techniques Used:

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    Thermo Fisher hygromycin b
    The CORE plasmids used in the  delitto perfetto  technique. Each of the five plasmids used in the non-break system ( a ) contains a counterselectable marker, either  KlURA3  from  Kluyveromyces lactis  or a mutant form (V122A) of the human p53 cDNA, and a reporter marker, either  kanMX4  conveying resistance to Geneticin (G418) or  hyg  for resistance to the antibiotic hygromycin B. In addition to these markers, the two plasmids used in the break system ( b ) contain the inducible  GAL1  promoter and I- Sce I gene used to express the I- Sce I endonuclease and generate a DSB at the I- Sce I site. The origin of replication ( ori ) for all CORE plasmids is indicated as well as the  bla  marker gene, which provides resistance to the β-lactam antibiotic ampicillin and is used for selection.
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    The CORE plasmids used in the  delitto perfetto  technique. Each of the five plasmids used in the non-break system ( a ) contains a counterselectable marker, either  KlURA3  from  Kluyveromyces lactis  or a mutant form (V122A) of the human p53 cDNA, and a reporter marker, either  kanMX4  conveying resistance to Geneticin (G418) or  hyg  for resistance to the antibiotic hygromycin B. In addition to these markers, the two plasmids used in the break system ( b ) contain the inducible  GAL1  promoter and I- Sce I gene used to express the I- Sce I endonuclease and generate a DSB at the I- Sce I site. The origin of replication ( ori ) for all CORE plasmids is indicated as well as the  bla  marker gene, which provides resistance to the β-lactam antibiotic ampicillin and is used for selection.

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: In Vivo Site-Specific Mutagenesis and Gene Collage Using the Delitto Perfetto System in Yeast Saccharomyces cerevisiae

    doi: 10.1007/978-1-61779-129-1_11

    Figure Lengend Snippet: The CORE plasmids used in the delitto perfetto technique. Each of the five plasmids used in the non-break system ( a ) contains a counterselectable marker, either KlURA3 from Kluyveromyces lactis or a mutant form (V122A) of the human p53 cDNA, and a reporter marker, either kanMX4 conveying resistance to Geneticin (G418) or hyg for resistance to the antibiotic hygromycin B. In addition to these markers, the two plasmids used in the break system ( b ) contain the inducible GAL1 promoter and I- Sce I gene used to express the I- Sce I endonuclease and generate a DSB at the I- Sce I site. The origin of replication ( ori ) for all CORE plasmids is indicated as well as the bla marker gene, which provides resistance to the β-lactam antibiotic ampicillin and is used for selection.

    Article Snippet: Hygromycin B (Hygro; per 1 l): YPD agar media is autoclaved, then cooled to 55–60°C, and Hygro solution (300 μg/ml; Invitrogen) is then mixed with media prior to pouring.

    Techniques: Marker, Mutagenesis, Selection

    The construction of ST-EGFP-APPV-N pro cell lines. The construction of lentiviral expression plasmid was shown in (a). (b) ST cells were infected by lentiviruses expressing EGFP-APPV-N pro , and then subjected to selection with Hygromycin B to generate ST-EGFP-APPV-N pro stable cell line. (c) The ST-EGFP-APPV-N pro cells and ST cells were observed by fluorescence microscope. (d) The expression of fusion protein in the ST-EGFP-APPV-N pro cells were analyzed by western blot using mouse anti-GFP and rabbit anti-myc antibodies

    Journal: Virulence

    Article Title: Disruption of interferon-β production by the Npro of atypical porcine pestivirus

    doi: 10.1080/21505594.2021.1880773

    Figure Lengend Snippet: The construction of ST-EGFP-APPV-N pro cell lines. The construction of lentiviral expression plasmid was shown in (a). (b) ST cells were infected by lentiviruses expressing EGFP-APPV-N pro , and then subjected to selection with Hygromycin B to generate ST-EGFP-APPV-N pro stable cell line. (c) The ST-EGFP-APPV-N pro cells and ST cells were observed by fluorescence microscope. (d) The expression of fusion protein in the ST-EGFP-APPV-N pro cells were analyzed by western blot using mouse anti-GFP and rabbit anti-myc antibodies

    Article Snippet: The ST-EGFP-APPV-Npro cell line was cultured in the medium supplemented with 300 μg/ml Hygromycin B (Thermo Fisher Scientific, China).

    Techniques: Expressing, Plasmid Preparation, Infection, Selection, Stable Transfection, Fluorescence, Microscopy, Western Blot

    Treatment with hygromycin B of transgenic cells lowers levels of EphA4. ( A ) Levels of LRP-1 on TIMP-3/HEK or control cells was measured by flow cytometry and plotted as the average of mean fluorescent intensities (MFI) ± standard deviation ( n = 3). ( B ) Expression levels of EphA4 mRNA in TIMP-3/HEK and control HEK 293 cells plotted as relative 2 −ΔΔCT ( n = 4) ( C ) Immunoblots and their respective quantification ( n = 4) showing levels of EphA4 in TIMP-3/HEK or control HEK 293 cell lysates, in the absence or presence of 500 nM RAP. Actin was used as a loading control. ( D ) Immunoblots and their respective quantification ( n = 9) showed levels of EphA4 in lysates of HEK 293 cells supplemented with TIMP-3-containing or control conditioned media. GAPDH was used as a lading control. ( E ) Immunoblots and their respective quantification ( n = 3) showed levels of EphA4 TIMP-1 overexpressing (TIMP-1/HEK) or control HEK 293 cells. Calnexin was used as a loading control. ( F ) Immunoblots and their respective quantification ( n = 3) showed levels of TIMP-3 in the conditioned media, and EphA4 in the lysate of TIMP-3/HEK or control HEK 293 cells treated with different concentrations of hygromycin B (0–800 μg/mL). Actin was used as a loading control. ( G ) Immunoblots showed levels of EphA4 in the cell lysate of HEK 293 cells, transfected with pCEP4 and treated with or without 400 μg/mL hygromycin B. Actin was used a loading control. All densitometric quantifications shown as mean values ± standard deviation; (** p

    Journal: International Journal of Molecular Sciences

    Article Title: Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates

    doi: 10.3390/ijms22052392

    Figure Lengend Snippet: Treatment with hygromycin B of transgenic cells lowers levels of EphA4. ( A ) Levels of LRP-1 on TIMP-3/HEK or control cells was measured by flow cytometry and plotted as the average of mean fluorescent intensities (MFI) ± standard deviation ( n = 3). ( B ) Expression levels of EphA4 mRNA in TIMP-3/HEK and control HEK 293 cells plotted as relative 2 −ΔΔCT ( n = 4) ( C ) Immunoblots and their respective quantification ( n = 4) showing levels of EphA4 in TIMP-3/HEK or control HEK 293 cell lysates, in the absence or presence of 500 nM RAP. Actin was used as a loading control. ( D ) Immunoblots and their respective quantification ( n = 9) showed levels of EphA4 in lysates of HEK 293 cells supplemented with TIMP-3-containing or control conditioned media. GAPDH was used as a lading control. ( E ) Immunoblots and their respective quantification ( n = 3) showed levels of EphA4 TIMP-1 overexpressing (TIMP-1/HEK) or control HEK 293 cells. Calnexin was used as a loading control. ( F ) Immunoblots and their respective quantification ( n = 3) showed levels of TIMP-3 in the conditioned media, and EphA4 in the lysate of TIMP-3/HEK or control HEK 293 cells treated with different concentrations of hygromycin B (0–800 μg/mL). Actin was used as a loading control. ( G ) Immunoblots showed levels of EphA4 in the cell lysate of HEK 293 cells, transfected with pCEP4 and treated with or without 400 μg/mL hygromycin B. Actin was used a loading control. All densitometric quantifications shown as mean values ± standard deviation; (** p

    Article Snippet: Methods to Validate Surfaceomics TIMP-3/HEK cells were maintained in complete media supplemented with 800 μg/mL hygromycin B.

    Techniques: Transgenic Assay, Flow Cytometry, Standard Deviation, Expressing, Western Blot, Transfection

    Efficiency of stable cell line generation. (A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of pcDNA5/FRT/TO and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 pg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).

    Journal: bioRxiv

    Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

    doi: 10.1101/160101

    Figure Lengend Snippet: Efficiency of stable cell line generation. (A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of pcDNA5/FRT/TO and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 pg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).

    Article Snippet: Twenty four hours after transfection, cells were replated to 60 mm dishes and subjected to selection with hygromycin B (50 and 175 μg/ml for 293 and HeLa cells, respectively) (Thermo Fisher Scientific) and blasticidin (10 μg/ml) (Invivogen) for up to a month.

    Techniques: Stable Transfection, Plasmid Preparation, Selection, Transfection, Concentration Assay, Staining