Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of HEK 293T cells at 48 h post transfection for Ints 13, phiC31, and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays ( n = 3) and circles showing the technical duplicate or triplicate average of each assay on the y axis. The x axis contains the different conditions. For each Int data group, different letters indicate significant differences ( p < 0.05). c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 1021 to 1104 bp for attL and from 1058 to 1084 bp for attR . d Bar graph plots showing the viable cells average (circles corresponding to technical replicates averages) and standard deviation normalized with pGFP of OD measurements obtained after MTT assays ( n = 3). DMSO corresponds to the impairment negative control. Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the EF1 alpha promoter. All the data are representative of two or three technical and three biological replicates.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Transfection, Expressing, Standard Deviation, Amplification, Negative Control, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: The human cell lineage HEK 293T, bovine fibroblasts and A. thaliana protoplasts were the selected model systems. The first step involved the design of two plasmid sets: the integrase expression vectors to express Ints 2, 4, 5, 7, 9, 13, phiC31, or Bxb1 and the switch GFP vectors with the attB and attP recognition sites of the respective Int flanking an egfp coding sequence in a reverse complement (rc) orientation. Acting as a schematic AND gate, combination of the corresponding plasmids of each of the vector sets results in the second step in the process, the test, accomplished by cotransfection or cotransformation assays of mammalian and plant cells, respectively. The third and last step led to the development of analytical methods that include the inputs of an additional schematic AND gate. Microscopy/flow cytometry analyses were used to detect EGFP fluorescence in cells resulting from the flipping action of the integrase. PCR/sequencing was used in the analysis of the egfp coding sequence rotated to the correct forward orientation flanked by the formed attL and attR sites. Both analytical inputs provide evidence of the activated switch vector output. The PCRs used one primer pair to amplify the complete attL site and the entire egfp coding sequence, now in the forward orientation (blue), and a second primer pair to amplify the complete attR site and the egfp sequence (red).

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Plasmid Preparation, Expressing, Sequencing, Cotransfection, Microscopy, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of protoplasts at 24 h post cotransformation for Ints 2, 4, 7, 9, 13, phiC31, and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays (n in Supplementary Table ) and circles showing the technical triplicate average of each assay on the y axis. The x axis contains the different conditions. For each Int data group, different letters indicate significant differences ( p < 0.05). c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 948 to 983 bp for attL and from 1136 to 1132 bp for attR . Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors, plus a mock plasmid. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the CaMV 35S promoter. All the data are representative of three technical and three or more biological replicates. d Bar graph plots showing the viable cells average (circles corresponding to technical replicates averages) and standard deviation normalized with mock plasmid positive cells obtained after FDA assays. DMSO corresponds to the impairment negative control. The cells were transfected with pIE vectors in technical triplicates and three biological replicates (except for Int 7, for which there was three technical and two biological replicates).

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Expressing, Standard Deviation, Amplification, Negative Control, Transfection, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of PBMCs at 48 h post electroporation for GFP switch tests with Int 13 and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays ( n = 3) and circles corresponding to single data points from each donor material. The x axis contains the different conditions. In the PBMCs assays, Ints 2, 13, phiC31, and Bxb1 were evaluated. c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 1021 to 1104 bp for attL and from 1058 to 1084 bp for attR . Negative control cells were electroporated with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the EF1 alpha promoter. All the data were representative of three donors, corresponding to biological triplicates with single measurements.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Electroporation, Expressing, Standard Deviation, Amplification, Negative Control, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: Flow cytometry distribution of NSCs ( a ) and hES cells ( b ) at 48 h post electroporation for GFP switch tests with Ints 2, 9, 13, phiC31, and Bxb1. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. Bar graph plots showing the total average percentage and standard deviation of NSCs ( c ) and hES cell ( d ) populations expressing EGFP in biological repeat assays ( n = 3 for NSCs; n = 2 for hES cells) and circles showing the technical triplicate average of each assay on the y axis. The x axis contains the different conditions. In the stem cell assays, Ints 2, 9, 13, phiC31, and Bxb1 were evaluated. Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors. Positive control cells (pGFP) have an egfp sequence in the forward orientation. All the data are representative of three technical and three (NSCs) or two (hES cells) biological replicates.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Electroporation, Expressing, Standard Deviation, Negative Control, Transfection, Plasmid Preparation, Positive Control, Sequencing

Journal: Nucleic Acids Research

Article Title: CASCADE-Cas3 enables highly efficient genome engineering in Streptomyces species

doi: 10.1093/nar/gkaf214

Figure Lengend Snippet: pCRISPR-Cas3 enables streamlined genome engineering of streptomycetes. ( A ) Distribution of type I and type II CRISPR systems in streptomycetes and the number of PAM sites identified in selected streptomycetes for both systems. Spycas9 and cas3 from S. albidoflavus were used as references. The BLAST search was run on all high quality publicly available Streptomyces genomes ( n = 2401). Type I CRISPR systems appear to be much wider distributed than type II CRISPR systems in streptomycetes. However, only 108 hits with a sequence similarity above 50% were identified. An order of magnitude fewer PAM sites were identified in three selected streptomycetes for the CASCADE-Cas3 PAM compared to the NGG Cas9 PAM, highlighting the much lower number of potential off-target sites. ( B ) Plasmid map of pCRISPR-Cas3. The plasmid is based on the pSG5 replicon and carries the codon optimized type I-C minimal CASCADE under control of the inducible tipA promoter. All elements of the backbone are highlighted in gray. The crRNA is cloned between to repeats in the chromosomal RNA (cRNA) cassette, which is controlled by the constitutive ermE* promoter. Repair templates are cloned into the multiple cloning site (MCS) on the backbone of the plasmid. ( C ) The second repeat has a modified sequence to prevent recombination between the two repeats. The CASCADE complex comprised of Cas5, Cas8, and Cas7 units binds the target sequence and recruits Cas3. Cas3 has a 3′–5′ helicase nuclease activity, resulting in directionally biased deletions. ( D ) cRNAs are cloned between two repeats, posing some challenges due to sequence homologies. Since type IIS restriction enzymes cannot be used in high GC Streptomyces plasmids, a PCR and Gibson Assembly based cloning approach was established, allowing cloning of cRNAs with high efficiencies. ( E ) pCRISPR-Cas3 can be used for targeted deletions of large genomic regions, or for substitutions of such with a specified cargo. It can also be used for random sized deletion experiments.

Article Snippet: Whole genome sequencing using Oxford Nanopore revealed BAC integrations into the native phiC31 attB site, as well as the Δact:: attB and ΔRED:: attB loci in S. coelicolor CW6::act.

Techniques: CRISPR, Sequencing, Plasmid Preparation, Control, Clone Assay, Cloning, Modification, Activity Assay

Journal: Nucleic Acids Research

Article Title: CASCADE-Cas3 enables highly efficient genome engineering in Streptomyces species

doi: 10.1093/nar/gkaf214

Figure Lengend Snippet: pCRISPR-Cas3 introduces genomic deletions with higher efficiencies than pCRISPR-Cas9. ( A ) Plate pictures of S. coelicolor mutants harboring pCRISPR-Cas9 or pCRISPR-Cas3 with spacers targeting the actinorhodin BGC in three different locations, and with or without repair templates. pCRISPR-Cas3 displayed higher toxicity without repair templates but resulted in more exconjugants overall and more with the desired red phenotype once repair templates were provided. ( B ) Representation of sequencing results of selected colonies, both for pCRISPR-Cas3 and pCRISPR-Cas9 with and without repair templates. Both pCRISPR-Cas3 and pCRISPR-Cas9 introduced random sized deletions without repair templates. With repair templates, precise deletions were observed for both pCRISPR-Cas3 and pCRISPR-Cas9. The spacers 1 for Cas9 and CASCADE-Cas3 targeted the left flank of the BGC, spacers 2 the middle, and spacers 3 the right flank. ( C ) Efficiencies for actinorhodin deletions with pCRISPR-Cas9 and pCRISPR-Cas3. For pCRISPR-Cas3, the efficiencies were consistently high (>80%), while with pCRISPR-Cas9 the observed efficiencies were highly sgRNA dependent. Circles correspond to data for spacers 1, triangles for spacers 2, and rectangles for spacers 3. ( D ) Read alignments for the junction site of the two homologous flanks. A HindIII site was integrated, demonstrating that the DSB was repaired using the repair templates cloned into pCRISPR-Cas3 using HindIII. Shown in panel (C) are the means ± standard deviations of three deletion experiments targeting the actinorhodin region with three different spacers.

Article Snippet: Whole genome sequencing using Oxford Nanopore revealed BAC integrations into the native phiC31 attB site, as well as the Δact:: attB and ΔRED:: attB loci in S. coelicolor CW6::act.

Techniques: Sequencing, Clone Assay

Journal: Nucleic Acids Research

Article Title: CASCADE-Cas3 enables highly efficient genome engineering in Streptomyces species

doi: 10.1093/nar/gkaf214

Figure Lengend Snippet: Simultaneous deletions and integrations enable streamlined genome engineering. ( A ) The PhiC31 Streptomyces integrase integrates cargo DNA into target attB sites. The consensus attB site from S. coelicolor is 51 bp long and features a central TT sequence where the cargo is integrated. ( B ) Substitution of the entire actinorhodin BGC with an additional attB site. The attB site was cloned between the repair templates. Coverage plots of mappings of ONT data against the wild type and the in silico generated substitution strain reveal precise genome engineering. ( C ) pCRISPR-Cas3 was used for construction of a S. coelicolor expression host using both targeted and semi-targeted deletions and substitutions. ( D ) Oxford Nanopore sequencing results for all deletions based on minimap2 mappings to the reference genome. ( E ) The final strains S. coelicolor CW5 and CW6 both displayed >200% increase in actinorhodin production compared to the base strain S. coelicolor CW1 upon integration of an actinorhodin BGC BAC. ( F ) Phenotypes of S. coelicolor CW6 C and E2 without and with actinorhodin integrations. Shown in panel (E) are the means ± standard deviations of four biological replicates. Significance was tested using unpaired two tailed t -tests, where ** P < .01, *** P < .001, and **** P < .0001.

Article Snippet: Whole genome sequencing using Oxford Nanopore revealed BAC integrations into the native phiC31 attB site, as well as the Δact:: attB and ΔRED:: attB loci in S. coelicolor CW6::act.

Techniques: Sequencing, Clone Assay, In Silico, Generated, Expressing, Nanopore Sequencing, Two Tailed Test

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of HEK 293T cells at 48 h post transfection for Ints 13, phiC31, and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays ( n = 3) and circles showing the technical duplicate or triplicate average of each assay on the y axis. The x axis contains the different conditions. For each Int data group, different letters indicate significant differences ( p < 0.05). c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 1021 to 1104 bp for attL and from 1058 to 1084 bp for attR . d Bar graph plots showing the viable cells average (circles corresponding to technical replicates averages) and standard deviation normalized with pGFP of OD measurements obtained after MTT assays ( n = 3). DMSO corresponds to the impairment negative control. Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the EF1 alpha promoter. All the data are representative of two or three technical and three biological replicates.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Transfection, Expressing, Standard Deviation, Amplification, Negative Control, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of bovine fibroblasts at 48 h post cotransfection for Ints 9, 13, and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays ( n = 3) and circles showing the technical triplicate average of each assay on the y axis. The x axis contains the different conditions. For each Int data group, different letters indicate significant differences ( p < 0.05). c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 1021 to 1104 bp for attL and from 1058 to 1084 bp for attR . d Bar graph plots showing the viable cells average (circles corresponding to technical replicates averages) and standard deviation normalized with pGFP of OD measurements obtained after MTT assays ( n = 3). DMSO corresponds to the impairment negative control. Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors, plus a mock plasmid. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the EF1 alpha promoter. All the data are representative of three technical and three biological replicates.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Cotransfection, Expressing, Standard Deviation, Amplification, Negative Control, Transfection, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: The human cell lineage HEK 293T, bovine fibroblasts and A. thaliana protoplasts were the selected model systems. The first step involved the design of two plasmid sets: the integrase expression vectors to express Ints 2, 4, 5, 7, 9, 13, phiC31, or Bxb1 and the switch GFP vectors with the attB and attP recognition sites of the respective Int flanking an egfp coding sequence in a reverse complement (rc) orientation. Acting as a schematic AND gate, combination of the corresponding plasmids of each of the vector sets results in the second step in the process, the test, accomplished by cotransfection or cotransformation assays of mammalian and plant cells, respectively. The third and last step led to the development of analytical methods that include the inputs of an additional schematic AND gate. Microscopy/flow cytometry analyses were used to detect EGFP fluorescence in cells resulting from the flipping action of the integrase. PCR/sequencing was used in the analysis of the egfp coding sequence rotated to the correct forward orientation flanked by the formed attL and attR sites. Both analytical inputs provide evidence of the activated switch vector output. The PCRs used one primer pair to amplify the complete attL site and the entire egfp coding sequence, now in the forward orientation (blue), and a second primer pair to amplify the complete attR site and the egfp sequence (red).

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Plasmid Preparation, Expressing, Sequencing, Cotransfection, Microscopy, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of protoplasts at 24 h post cotransformation for Ints 2, 4, 7, 9, 13, phiC31, and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays (n in Supplementary Table ) and circles showing the technical triplicate average of each assay on the y axis. The x axis contains the different conditions. For each Int data group, different letters indicate significant differences ( p < 0.05). c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 948 to 983 bp for attL and from 1136 to 1132 bp for attR . Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors, plus a mock plasmid. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the CaMV 35S promoter. All the data are representative of three technical and three or more biological replicates. d Bar graph plots showing the viable cells average (circles corresponding to technical replicates averages) and standard deviation normalized with mock plasmid positive cells obtained after FDA assays. DMSO corresponds to the impairment negative control. The cells were transfected with pIE vectors in technical triplicates and three biological replicates (except for Int 7, for which there was three technical and two biological replicates).

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Expressing, Standard Deviation, Amplification, Negative Control, Transfection, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Schematic representation of the CaMV 35S promoter as a switchable genetic part. In this case, the switch vector was redesigned to contain the promoter in the reverse complement sequence orientation flanked by the attB and attP sites of three different Ints (2, 4, and 5) in tandem; this construct was named the switch promoter vector (pSP). The integrase expression vectors (pIE) for each Int were cotransformed separately with the pSP, and each resulting activated promoter vector is shown. b Flow cytometry distribution of protoplasts at 24 h post cotransformation. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. c Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays ( n = 3) and circles showing the technical triplicate average of each assay on the y axis. The x axis contains the different conditions. Different letters indicate significant differences ( p < 0.05). d Amplicons obtained through PCR analysis using two specific primer sets. The first primer set (blue) was complementary to the pSP vector backbone sequence (forward) and promoter sequence (reverse). Expected amplicon size: 429 bp. The second primer set (red) was complementary to the promoter sequence (forward) and egfp coding sequence (reverse). Expected amplicon size: 438 bp (primers are colored and marked in letter a). Negative control cells were cotransformed with one of the integrase expression vectors (pIE) plus a mock plasmid or with the switch promoter vector (pSP) plus a mock plasmid. Positive control cells (pGFP) have a plasmid containing the egfp sequence under the control of the CaMV 35S promoter in the forward orientation plus a mock plasmid. Expected amplicon size for pGFP: attL gel: 285 bp; attR gel: 227 bp. These amplicons were smaller than those obtained under the test conditions due to the absence of the Int attachment sites. All the data are representative of three technical and three biological replicates.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Plasmid Preparation, Sequencing, Construct, Expressing, Flow Cytometry, Standard Deviation, Amplification, Negative Control, Positive Control, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: a Flow cytometry distribution of PBMCs at 48 h post electroporation for GFP switch tests with Int 13 and Bxb1, the integrases that led to the highest EGFP-expressing cell frequencies. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. b Bar graph plots showing the total average percentage and standard deviation of a cell population expressing EGFP in biological repeat assays ( n = 3) and circles corresponding to single data points from each donor material. The x axis contains the different conditions. In the PBMCs assays, Ints 2, 13, phiC31, and Bxb1 were evaluated. c Amplicons obtained through PCR analysis using two specific primer sets, the first set to verify attL formation and the second set to verify attR (highlighted in Fig. ). The expected amplicon sizes in the Int test groups varied from 1021 to 1104 bp for attL and from 1058 to 1084 bp for attR . Negative control cells were electroporated with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors. Positive control cells (pGFP) have an egfp sequence in the forward orientation under the control of the EF1 alpha promoter. All the data were representative of three donors, corresponding to biological triplicates with single measurements.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Electroporation, Expressing, Standard Deviation, Amplification, Negative Control, Plasmid Preparation, Positive Control, Sequencing, Control

Journal: Communications Biology

Article Title: Genetic switches designed for eukaryotic cells and controlled by serine integrases

doi: 10.1038/s42003-020-0971-8

Figure Lengend Snippet: Flow cytometry distribution of NSCs ( a ) and hES cells ( b ) at 48 h post electroporation for GFP switch tests with Ints 2, 9, 13, phiC31, and Bxb1. The heat map indicates the scattering of high cell concentrations (warm colors) to low cell concentrations (cool colors). The gate encompasses the EGFP-expressing cell population. Bar graph plots showing the total average percentage and standard deviation of NSCs ( c ) and hES cell ( d ) populations expressing EGFP in biological repeat assays ( n = 3 for NSCs; n = 2 for hES cells) and circles showing the technical triplicate average of each assay on the y axis. The x axis contains the different conditions. In the stem cell assays, Ints 2, 9, 13, phiC31, and Bxb1 were evaluated. Negative control cells were transfected with only one of the two vector sets, that is, integrase expression (pIE) or switch GFP (pSG) vectors. Positive control cells (pGFP) have an egfp sequence in the forward orientation. All the data are representative of three technical and three (NSCs) or two (hES cells) biological replicates.

Article Snippet: For the switch GFP vectors, the egfp coding sequence (Addgene, 11154) was cloned in reverse complement orientation, flanked by the attB site and the reverse complement sequence of the attP site of the individual Ints 2, 4, 5, 7, 9, 13, phiC31, and Bxb1.

Techniques: Flow Cytometry, Electroporation, Expressing, Standard Deviation, Negative Control, Transfection, Plasmid Preparation, Positive Control, Sequencing

Journal: eLife

Article Title: Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity

doi: 10.7554/eLife.41769

Figure Lengend Snippet:

Article Snippet: For integration of the tetO and cuO array, 150,000 cells were electroporated with 300 ng each of (1) a tetOx224 repeat plasmid bearing a PhiC31 attB sequence and a FRT-flanked neomycin resistance cassette, (2) a cuOx144 repeat plasmid bearing a Bxb1 attB sequence and a floxed puromycin or blasticidin resistance cassette, (3) an expression plasmid for the PhiC31 integrase (a gift from Philippe Soriano, Addgene Plasmid #13795), and (4) an expression plasmid for the Bxb1 integrase using the Neon Transfection System.

Techniques: Stable Transfection, Control, Derivative Assay, Recombinant, Plasmid Preparation, Selection, Expressing, Sequencing, CRISPR, Software, Library Quantification