bsiwi  (New England Biolabs)


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    BsiWI
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    BsiWI 1 500 units
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    r0553l
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    Category:
    Restriction Enzymes
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    New England Biolabs bsiwi
    BsiWI
    BsiWI 1 500 units
    https://www.bioz.com/result/bsiwi/product/New England Biolabs
    Average 98 stars, based on 4518 article reviews
    Price from $9.99 to $1999.99
    bsiwi - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    2) Product Images from "Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity"

    Article Title: Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.22.10800-10807.2001

    Schematic diagram of cytochrome c recombinant rabies viruses. The pSPBN vector was derived from SPBN-10 by removing the ψ gene and introducing Bsi WI and Nhe I sites between the G and L genes. Human cytochrome c cDNA was amplified by PCR and, after introduction of Bsi WI and Nhe I sites, ligated into pSPBN, resulting in pSPBN-Cyto c (+). To construct pSPBN-Cyto c (−), a stop codon was introduced into the cytochrome c gene 70 bp after the start codon.
    Figure Legend Snippet: Schematic diagram of cytochrome c recombinant rabies viruses. The pSPBN vector was derived from SPBN-10 by removing the ψ gene and introducing Bsi WI and Nhe I sites between the G and L genes. Human cytochrome c cDNA was amplified by PCR and, after introduction of Bsi WI and Nhe I sites, ligated into pSPBN, resulting in pSPBN-Cyto c (+). To construct pSPBN-Cyto c (−), a stop codon was introduced into the cytochrome c gene 70 bp after the start codon.

    Techniques Used: Recombinant, Plasmid Preparation, Derivative Assay, Amplification, Polymerase Chain Reaction, Construct

    3) Product Images from "A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice"

    Article Title: A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice

    Journal: Virologica Sinica

    doi: 10.1007/s12250-019-00144-x

    Construction and characterization of rRABV expressing IL-15 in vitro and in vivo . A Schematic diagram for construction of LBNSE-Flt3L. The parent vector pLBNSE was constructed based on SAD-B19 strain by deleting the pseudogene between the G and L genes where Bsi wI and Nhe I restriction enzyme sites were introduced. N , P , M , G and L represented the nucleoprotein, phosphoprotein, matrix, glycoprotein, and polymerase genes of RABV, respectively. Multi-step ( B ) and One-step ( C ) virus growth curves were determined on BSR cells. Cells were infected with either LBNSE or LBNSE-Flt3L at a multiplicity of infection (MOI) of 0.01 or 5, respectively, and the culture supernatants were harvested at 1, 2, 3, 4 and 5 dpi for viral titration. The virus growth curves were drawn according to the viral titers measured at each time point. Data are presented as the mean ± SD (n = 3). D Expression of Flt3L was detected in infected BSR cells by ELISA. BSR cells were infected with either the LBNSE or LBNSE-Flt3L at the MOI = 0.001, 0.01, 0.1, or 1, and the culture supernatants were harvested at 24 h post infection for ELISA assay. Data are presented as the mean ± SD (n = 3). E Body weight change curves of mice infected with different rRABVs. Six-week-old female ICR mice (n = 10) were infected by the i.c. route with 1 × 10 7 FFU of LBNSE or LBNSE-Flt3L, or with mock infected with the same volume of DMEM, and the body weights were monitored daily for 2 weeks.
    Figure Legend Snippet: Construction and characterization of rRABV expressing IL-15 in vitro and in vivo . A Schematic diagram for construction of LBNSE-Flt3L. The parent vector pLBNSE was constructed based on SAD-B19 strain by deleting the pseudogene between the G and L genes where Bsi wI and Nhe I restriction enzyme sites were introduced. N , P , M , G and L represented the nucleoprotein, phosphoprotein, matrix, glycoprotein, and polymerase genes of RABV, respectively. Multi-step ( B ) and One-step ( C ) virus growth curves were determined on BSR cells. Cells were infected with either LBNSE or LBNSE-Flt3L at a multiplicity of infection (MOI) of 0.01 or 5, respectively, and the culture supernatants were harvested at 1, 2, 3, 4 and 5 dpi for viral titration. The virus growth curves were drawn according to the viral titers measured at each time point. Data are presented as the mean ± SD (n = 3). D Expression of Flt3L was detected in infected BSR cells by ELISA. BSR cells were infected with either the LBNSE or LBNSE-Flt3L at the MOI = 0.001, 0.01, 0.1, or 1, and the culture supernatants were harvested at 24 h post infection for ELISA assay. Data are presented as the mean ± SD (n = 3). E Body weight change curves of mice infected with different rRABVs. Six-week-old female ICR mice (n = 10) were infected by the i.c. route with 1 × 10 7 FFU of LBNSE or LBNSE-Flt3L, or with mock infected with the same volume of DMEM, and the body weights were monitored daily for 2 weeks.

    Techniques Used: Expressing, In Vitro, In Vivo, Plasmid Preparation, Construct, Infection, Titration, Enzyme-linked Immunosorbent Assay, Mouse Assay

    4) Product Images from "A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase"

    Article Title: A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase

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

    doi: 10.1007/978-1-60761-652-8_6

    Dual-plasmid approach for integrase-mediated attB × attP recombination. ( a ) Map of the attP -carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with Avr II and Afl II can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with Avr II and Bsi WI can place the gene of interest in frame with gfp . The plasmid carries the blasticidin S-deaminase ( bsd ) selectable marker. ( b ) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. ( c ) Schematic representation of the attB × attP recombination allowing integration of a gene of interest ( yfg ) at the cg6 - attB locus. Dd2 attB or 3D7 attB parasites are co-transfected with pINT and an attP -plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2 attB and 3D7 attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP -plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes ( designated as open boxes ), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR : untranslated region. Panel ( c with the permission of the Nature Publishing Group.
    Figure Legend Snippet: Dual-plasmid approach for integrase-mediated attB × attP recombination. ( a ) Map of the attP -carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with Avr II and Afl II can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with Avr II and Bsi WI can place the gene of interest in frame with gfp . The plasmid carries the blasticidin S-deaminase ( bsd ) selectable marker. ( b ) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. ( c ) Schematic representation of the attB × attP recombination allowing integration of a gene of interest ( yfg ) at the cg6 - attB locus. Dd2 attB or 3D7 attB parasites are co-transfected with pINT and an attP -plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2 attB and 3D7 attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP -plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes ( designated as open boxes ), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR : untranslated region. Panel ( c with the permission of the Nature Publishing Group.

    Techniques Used: Plasmid Preparation, Marker, Expressing, Selection, Transfection, Polymerase Chain Reaction, DNA Hybridization, Sequencing

    5) Product Images from "Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines"

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines

    Journal: Cells

    doi: 10.3390/cells8010075

    A simplified diagram of the development of a new clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR Cas9) vector in fish cells. ( A ) In the left panel and in colors are represented the names and sizes of the regulatory elements including RNA pol III U6 (U6), short EF1alpha (EFS-NS) promoter, small guide RNA (sgRNA), the antibiotic resistance cassette (puromycin), and the mCherry gene. The novel fish U6 promoter (zebrafish U6 RNA III polymerase (U6ZF) ligated in the new vector is represented in yellow. In orange are highlighted the recognition sequences of the restriction enzymes used in this work ( Bsi WI, Sac II, Kpn I, and BsmB I, respectively). Arrows indicate the downstream activity of U6ZF (yellow) and EFS-NS (violet) promoters. Note that lentiviral elements were omitted in this representation. ( B ) Molecular characterization and isolation of mCherry gene from FU-mCherry-w plasmid (lane 1). In lane 2, a single 0.7 kb fragment (red frame) corresponding to the mCherry sequence was obtained by double digestion (BsiWI and SacII). Lane 3 represents the LentiCRISPR-Cas9 PuroV2 (14 kb) vector, whereas the isolation and removal of puromycin cassette 1.3 kb fragment (red frame) was obtained using the same enzymes mentioned above. ( C ) PCR product of U6 promoter from zebrafish genomic DNA; and ( D ) the new vector LcU6ZF containing the new fish promoter (0.3 kb) highlighted in red, as well as filler fragment (2 kb).
    Figure Legend Snippet: A simplified diagram of the development of a new clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR Cas9) vector in fish cells. ( A ) In the left panel and in colors are represented the names and sizes of the regulatory elements including RNA pol III U6 (U6), short EF1alpha (EFS-NS) promoter, small guide RNA (sgRNA), the antibiotic resistance cassette (puromycin), and the mCherry gene. The novel fish U6 promoter (zebrafish U6 RNA III polymerase (U6ZF) ligated in the new vector is represented in yellow. In orange are highlighted the recognition sequences of the restriction enzymes used in this work ( Bsi WI, Sac II, Kpn I, and BsmB I, respectively). Arrows indicate the downstream activity of U6ZF (yellow) and EFS-NS (violet) promoters. Note that lentiviral elements were omitted in this representation. ( B ) Molecular characterization and isolation of mCherry gene from FU-mCherry-w plasmid (lane 1). In lane 2, a single 0.7 kb fragment (red frame) corresponding to the mCherry sequence was obtained by double digestion (BsiWI and SacII). Lane 3 represents the LentiCRISPR-Cas9 PuroV2 (14 kb) vector, whereas the isolation and removal of puromycin cassette 1.3 kb fragment (red frame) was obtained using the same enzymes mentioned above. ( C ) PCR product of U6 promoter from zebrafish genomic DNA; and ( D ) the new vector LcU6ZF containing the new fish promoter (0.3 kb) highlighted in red, as well as filler fragment (2 kb).

    Techniques Used: CRISPR, Plasmid Preparation, Fluorescence In Situ Hybridization, Activity Assay, Isolation, Sequencing, Polymerase Chain Reaction

    6) Product Images from "Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association"

    Article Title: Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-89

    Comparative genome analysis of K-10 and S397 MAP strains . (A) Comparison of the Bsi WI restriction maps between K-10 (inner circle) and S397 (outer circle). Each box represents a restriction fragment. Green boxes are regions in the same direction and red boxes are regions that are inverted between the two genomes. White boxes are fragments that are not aligned. The red thin line at 12 o'clock is the locus of the gene dnaA . (B) Mauve alignment of all 184 scaffolds of S397 (bottom) with the complete genome of K-10 (top). The colored boxes represent homologous regions present in each genome, which are also connected by lines. Blocks below the centerline of the S397 genome indicate regions with inverse orientation. Regions outside the blocks lack homology between the genomes. Within each block there is a similarity profile of the DNA sequences and the white areas indicate sequences specific to a genome. The scale is in base pairs.
    Figure Legend Snippet: Comparative genome analysis of K-10 and S397 MAP strains . (A) Comparison of the Bsi WI restriction maps between K-10 (inner circle) and S397 (outer circle). Each box represents a restriction fragment. Green boxes are regions in the same direction and red boxes are regions that are inverted between the two genomes. White boxes are fragments that are not aligned. The red thin line at 12 o'clock is the locus of the gene dnaA . (B) Mauve alignment of all 184 scaffolds of S397 (bottom) with the complete genome of K-10 (top). The colored boxes represent homologous regions present in each genome, which are also connected by lines. Blocks below the centerline of the S397 genome indicate regions with inverse orientation. Regions outside the blocks lack homology between the genomes. Within each block there is a similarity profile of the DNA sequences and the white areas indicate sequences specific to a genome. The scale is in base pairs.

    Techniques Used: Blocking Assay

    7) Product Images from "Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice"

    Article Title: Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.24.15405-15416.2005

    Expression of TNF-α by recombinant RV. (A) Schematic of RV-TNF-α recombinant constructs. The pSPBN vector was derived from SN by removing the Ψ gene and introducing BsiWI and NheI sites between the G and L genes. Mouse TNF-α cDNA was amplified by PCR, with the introduction of BsiWI and NheI sites, and was ligated into pSPBN, resulting in pSPBN-TNF-α(+). In pSPBN-TNF-α(MEM), the proteolytic cleavage site of TNF-α was removed by deleting the coding sequence for the first nine amino acids of soluble TNF-α and exchanging the lysine at position 11 with glutamic acid, as described in Materials and Methods. To construct pSPBN-TNF-α(−), the first seven ATG codons of the TNF-α gene were mutated. (B) TNF-α production in BSR cells. BSR cells were infected with SPBN-TNF-α(−), SPBN-TNF-α(MEM), or SPBN-TNF-α(+) at an MOI of 0.1 and incubated at 37°C. TNF-α secreted into the tissue culture supernatants of infected BSR cells was measured at 24 h p.i., using ELISA. OD, optical density. (C to L) Confocal double-immunofluorescence microscopy of TNF-α (green, panels C, F, and I) and rabies ribonucleocapsid (RNP) (red, panels D, G, and K) in NA cells infected with SPBN-TNF-α(+), SPBN-TNF-α(MEM), or SPBN-TNF-α(−). Panels E, H, and L show the composites of TNF-α and RNP double immunofluorescence. Note that TNF-α is absent only in NA cells infected with SPBN-TNF-α(−) (panels I and L).
    Figure Legend Snippet: Expression of TNF-α by recombinant RV. (A) Schematic of RV-TNF-α recombinant constructs. The pSPBN vector was derived from SN by removing the Ψ gene and introducing BsiWI and NheI sites between the G and L genes. Mouse TNF-α cDNA was amplified by PCR, with the introduction of BsiWI and NheI sites, and was ligated into pSPBN, resulting in pSPBN-TNF-α(+). In pSPBN-TNF-α(MEM), the proteolytic cleavage site of TNF-α was removed by deleting the coding sequence for the first nine amino acids of soluble TNF-α and exchanging the lysine at position 11 with glutamic acid, as described in Materials and Methods. To construct pSPBN-TNF-α(−), the first seven ATG codons of the TNF-α gene were mutated. (B) TNF-α production in BSR cells. BSR cells were infected with SPBN-TNF-α(−), SPBN-TNF-α(MEM), or SPBN-TNF-α(+) at an MOI of 0.1 and incubated at 37°C. TNF-α secreted into the tissue culture supernatants of infected BSR cells was measured at 24 h p.i., using ELISA. OD, optical density. (C to L) Confocal double-immunofluorescence microscopy of TNF-α (green, panels C, F, and I) and rabies ribonucleocapsid (RNP) (red, panels D, G, and K) in NA cells infected with SPBN-TNF-α(+), SPBN-TNF-α(MEM), or SPBN-TNF-α(−). Panels E, H, and L show the composites of TNF-α and RNP double immunofluorescence. Note that TNF-α is absent only in NA cells infected with SPBN-TNF-α(−) (panels I and L).

    Techniques Used: Expressing, Recombinant, Construct, Plasmid Preparation, Derivative Assay, Amplification, Polymerase Chain Reaction, Sequencing, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy

    8) Product Images from "Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines"

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines

    Journal: Cells

    doi: 10.3390/cells8010075

    A simplified diagram of the development of a new clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR Cas9) vector in fish cells. ( A ) In the left panel and in colors are represented the names and sizes of the regulatory elements including RNA pol III U6 (U6), short EF1alpha (EFS-NS) promoter, small guide RNA (sgRNA), the antibiotic resistance cassette (puromycin), and the mCherry gene. The novel fish U6 promoter (zebrafish U6 RNA III polymerase (U6ZF) ligated in the new vector is represented in yellow. In orange are highlighted the recognition sequences of the restriction enzymes used in this work ( Bsi WI, Sac II, Kpn I, and BsmB I, respectively). Arrows indicate the downstream activity of U6ZF (yellow) and EFS-NS (violet) promoters. Note that lentiviral elements were omitted in this representation. ( B ) Molecular characterization and isolation of mCherry gene from FU-mCherry-w plasmid (lane 1). In lane 2, a single 0.7 kb fragment (red frame) corresponding to the mCherry sequence was obtained by double digestion (BsiWI and SacII). Lane 3 represents the LentiCRISPR-Cas9 PuroV2 (14 kb) vector, whereas the isolation and removal of puromycin cassette 1.3 kb fragment (red frame) was obtained using the same enzymes mentioned above. ( C ) PCR product of U6 promoter from zebrafish genomic DNA; and ( D ) the new vector LcU6ZF containing the new fish promoter (0.3 kb) highlighted in red, as well as filler fragment (2 kb).
    Figure Legend Snippet: A simplified diagram of the development of a new clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR Cas9) vector in fish cells. ( A ) In the left panel and in colors are represented the names and sizes of the regulatory elements including RNA pol III U6 (U6), short EF1alpha (EFS-NS) promoter, small guide RNA (sgRNA), the antibiotic resistance cassette (puromycin), and the mCherry gene. The novel fish U6 promoter (zebrafish U6 RNA III polymerase (U6ZF) ligated in the new vector is represented in yellow. In orange are highlighted the recognition sequences of the restriction enzymes used in this work ( Bsi WI, Sac II, Kpn I, and BsmB I, respectively). Arrows indicate the downstream activity of U6ZF (yellow) and EFS-NS (violet) promoters. Note that lentiviral elements were omitted in this representation. ( B ) Molecular characterization and isolation of mCherry gene from FU-mCherry-w plasmid (lane 1). In lane 2, a single 0.7 kb fragment (red frame) corresponding to the mCherry sequence was obtained by double digestion (BsiWI and SacII). Lane 3 represents the LentiCRISPR-Cas9 PuroV2 (14 kb) vector, whereas the isolation and removal of puromycin cassette 1.3 kb fragment (red frame) was obtained using the same enzymes mentioned above. ( C ) PCR product of U6 promoter from zebrafish genomic DNA; and ( D ) the new vector LcU6ZF containing the new fish promoter (0.3 kb) highlighted in red, as well as filler fragment (2 kb).

    Techniques Used: CRISPR, Plasmid Preparation, Fluorescence In Situ Hybridization, Activity Assay, Isolation, Sequencing, Polymerase Chain Reaction

    9) Product Images from "Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice"

    Article Title: Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.24.15405-15416.2005

    Expression of TNF-α by recombinant RV. (A) Schematic of RV-TNF-α recombinant constructs. The pSPBN vector was derived from SN by removing the Ψ gene and introducing BsiWI and NheI sites between the G and L genes. Mouse TNF-α cDNA was amplified by PCR, with the introduction of BsiWI and NheI sites, and was ligated into pSPBN, resulting in pSPBN-TNF-α(+). In pSPBN-TNF-α(MEM), the proteolytic cleavage site of TNF-α was removed by deleting the coding sequence for the first nine amino acids of soluble TNF-α and exchanging the lysine at position 11 with glutamic acid, as described in Materials and Methods. To construct pSPBN-TNF-α(−), the first seven ATG codons of the TNF-α gene were mutated. (B) TNF-α production in BSR cells. BSR cells were infected with SPBN-TNF-α(−), SPBN-TNF-α(MEM), or SPBN-TNF-α(+) at an MOI of 0.1 and incubated at 37°C. TNF-α secreted into the tissue culture supernatants of infected BSR cells was measured at 24 h p.i., using ELISA. OD, optical density. (C to L) Confocal double-immunofluorescence microscopy of TNF-α (green, panels C, F, and I) and rabies ribonucleocapsid (RNP) (red, panels D, G, and K) in NA cells infected with SPBN-TNF-α(+), SPBN-TNF-α(MEM), or SPBN-TNF-α(−). Panels E, H, and L show the composites of TNF-α and RNP double immunofluorescence. Note that TNF-α is absent only in NA cells infected with SPBN-TNF-α(−) (panels I and L).
    Figure Legend Snippet: Expression of TNF-α by recombinant RV. (A) Schematic of RV-TNF-α recombinant constructs. The pSPBN vector was derived from SN by removing the Ψ gene and introducing BsiWI and NheI sites between the G and L genes. Mouse TNF-α cDNA was amplified by PCR, with the introduction of BsiWI and NheI sites, and was ligated into pSPBN, resulting in pSPBN-TNF-α(+). In pSPBN-TNF-α(MEM), the proteolytic cleavage site of TNF-α was removed by deleting the coding sequence for the first nine amino acids of soluble TNF-α and exchanging the lysine at position 11 with glutamic acid, as described in Materials and Methods. To construct pSPBN-TNF-α(−), the first seven ATG codons of the TNF-α gene were mutated. (B) TNF-α production in BSR cells. BSR cells were infected with SPBN-TNF-α(−), SPBN-TNF-α(MEM), or SPBN-TNF-α(+) at an MOI of 0.1 and incubated at 37°C. TNF-α secreted into the tissue culture supernatants of infected BSR cells was measured at 24 h p.i., using ELISA. OD, optical density. (C to L) Confocal double-immunofluorescence microscopy of TNF-α (green, panels C, F, and I) and rabies ribonucleocapsid (RNP) (red, panels D, G, and K) in NA cells infected with SPBN-TNF-α(+), SPBN-TNF-α(MEM), or SPBN-TNF-α(−). Panels E, H, and L show the composites of TNF-α and RNP double immunofluorescence. Note that TNF-α is absent only in NA cells infected with SPBN-TNF-α(−) (panels I and L).

    Techniques Used: Expressing, Recombinant, Construct, Plasmid Preparation, Derivative Assay, Amplification, Polymerase Chain Reaction, Sequencing, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy

    10) Product Images from "The Roles of Chemokines in Rabies Virus Infection: Overexpression May Not Always Be Beneficial ▿"

    Article Title: The Roles of Chemokines in Rabies Virus Infection: Overexpression May Not Always Be Beneficial ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01346-09

    Construction and characterization of recombinant RABVs expressing different chemokins. (A) Construction of full-length recombinant RABVs. Chemokine genes MIP-1α, RANTES, and IP-10 were individually inserted between BsiWI and NheI sites of the
    Figure Legend Snippet: Construction and characterization of recombinant RABVs expressing different chemokins. (A) Construction of full-length recombinant RABVs. Chemokine genes MIP-1α, RANTES, and IP-10 were individually inserted between BsiWI and NheI sites of the

    Techniques Used: Recombinant, Expressing

    11) Product Images from "Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity"

    Article Title: Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.22.10800-10807.2001

    Schematic diagram of cytochrome c recombinant rabies viruses. The pSPBN vector was derived from SPBN-10 by removing the ψ gene and introducing Bsi WI and Nhe I sites between the G and L genes. Human cytochrome c cDNA was amplified by PCR and, after introduction of Bsi WI and Nhe I sites, ligated into pSPBN, resulting in pSPBN-Cyto c (+). To construct pSPBN-Cyto c (−), a stop codon was introduced into the cytochrome c gene 70 bp after the start codon.
    Figure Legend Snippet: Schematic diagram of cytochrome c recombinant rabies viruses. The pSPBN vector was derived from SPBN-10 by removing the ψ gene and introducing Bsi WI and Nhe I sites between the G and L genes. Human cytochrome c cDNA was amplified by PCR and, after introduction of Bsi WI and Nhe I sites, ligated into pSPBN, resulting in pSPBN-Cyto c (+). To construct pSPBN-Cyto c (−), a stop codon was introduced into the cytochrome c gene 70 bp after the start codon.

    Techniques Used: Recombinant, Plasmid Preparation, Derivative Assay, Amplification, Polymerase Chain Reaction, Construct

    12) Product Images from "Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair"

    Article Title: Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16308-2

    Correction of the mutation in TGFBI R124H mutant keratocytes using CRISPR-mediated HDR. (a) Result of an RFLP analysis of edited R124H cells. TGFBI exon 4 was amplified by PCR, and the products were treated with the BsiWI restriction enzyme. The lane with three bands was edited heterozygously and the lane with two bands was edited homozygously. (b) DNA sequences of PCR products amplified from the TGFBI gene of wild-type cells, a heterogeneous R124H mutant, and a repaired allele by HDR after transfection of Cas9 guide RNA and ssDNA. Two peaks were observed in the sequence of the R124H heterogeneous mutant, while the base of HDR-repaired cells was corrected to T. (c) Editing efficiency of CRISPR/Cas9-mediated HDR of an R124H mutation. RFLP, restriction fragment length polymorphism TGFBI, transforming growth factor β-induced; HDR, homology-directed repair.
    Figure Legend Snippet: Correction of the mutation in TGFBI R124H mutant keratocytes using CRISPR-mediated HDR. (a) Result of an RFLP analysis of edited R124H cells. TGFBI exon 4 was amplified by PCR, and the products were treated with the BsiWI restriction enzyme. The lane with three bands was edited heterozygously and the lane with two bands was edited homozygously. (b) DNA sequences of PCR products amplified from the TGFBI gene of wild-type cells, a heterogeneous R124H mutant, and a repaired allele by HDR after transfection of Cas9 guide RNA and ssDNA. Two peaks were observed in the sequence of the R124H heterogeneous mutant, while the base of HDR-repaired cells was corrected to T. (c) Editing efficiency of CRISPR/Cas9-mediated HDR of an R124H mutation. RFLP, restriction fragment length polymorphism TGFBI, transforming growth factor β-induced; HDR, homology-directed repair.

    Techniques Used: Mutagenesis, CRISPR, Amplification, Polymerase Chain Reaction, Transfection, Sequencing

    13) Product Images from "High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases"

    Article Title: High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004090

    Herpes simplex viral genome editing targeted by the CRISPR-Cas system. (A) Schematic of the HSV1 genome and the gRNA-206 target site in the UL23 gene. PAM sequences are highlighted in orange. The BsiWI restriction site used for RFLP is highlighted in aqua green. (B) The fragment amplified from an HSV1 genome using oligo 8 and oligo 9 was used in the RFLP assay. Co-expression of Cas9 and gRNA-206 in HSV1-infected cells leads to indel mutations in the TK gene of the HSV1 genome, which destroys the BsiWI restriction site. Arrows indicate fragments generated by BsiWI digestion. (C) Titration of HSV1 using an endpoint dilution assay in the presence or absence of 100 µg/ml ACV. The titers of total HSV1 progeny are represented by a blue bar. The titers of ACV-resistant HSV1 progeny virus are represented by an orange bar. N = 3; error bars show the means ± SEM. ND, not detected. (D) Sequences of indel mutations identified from eight ACV-resistant HSV1 strains. Red dashes, deletions; red bases, insertions. The incidence of each genotype is listed in the right-most column. (E) Strategy of Cas9:gRNA-induced HDR used to insert the EGFP gene into the HSV1 genome using donor DNA. (F) Titration of recombinant HSV1 using a plaque assay. HDR was induced by the Cas9 protein or Cas9:gRNA206. Blue bars represent the total number of macroscopically visible plaques; and green bars represent the number of green fluorescent plaques that were counted using a fluorescence microscope. N > 5; error bars show the means ± SEM. ND, not detected. (G) An isolated green HSV1 plaque that was observed using a fluorescence microscope and merged with an image of DAPI staining. Scale bar, 50 µm. (H) PCR amplification of HSV1 genomes extracted from a green fluorescent plaque using oligo 8 and oligo 9 to verify the lack of wild-type HSV1 contamination.
    Figure Legend Snippet: Herpes simplex viral genome editing targeted by the CRISPR-Cas system. (A) Schematic of the HSV1 genome and the gRNA-206 target site in the UL23 gene. PAM sequences are highlighted in orange. The BsiWI restriction site used for RFLP is highlighted in aqua green. (B) The fragment amplified from an HSV1 genome using oligo 8 and oligo 9 was used in the RFLP assay. Co-expression of Cas9 and gRNA-206 in HSV1-infected cells leads to indel mutations in the TK gene of the HSV1 genome, which destroys the BsiWI restriction site. Arrows indicate fragments generated by BsiWI digestion. (C) Titration of HSV1 using an endpoint dilution assay in the presence or absence of 100 µg/ml ACV. The titers of total HSV1 progeny are represented by a blue bar. The titers of ACV-resistant HSV1 progeny virus are represented by an orange bar. N = 3; error bars show the means ± SEM. ND, not detected. (D) Sequences of indel mutations identified from eight ACV-resistant HSV1 strains. Red dashes, deletions; red bases, insertions. The incidence of each genotype is listed in the right-most column. (E) Strategy of Cas9:gRNA-induced HDR used to insert the EGFP gene into the HSV1 genome using donor DNA. (F) Titration of recombinant HSV1 using a plaque assay. HDR was induced by the Cas9 protein or Cas9:gRNA206. Blue bars represent the total number of macroscopically visible plaques; and green bars represent the number of green fluorescent plaques that were counted using a fluorescence microscope. N > 5; error bars show the means ± SEM. ND, not detected. (G) An isolated green HSV1 plaque that was observed using a fluorescence microscope and merged with an image of DAPI staining. Scale bar, 50 µm. (H) PCR amplification of HSV1 genomes extracted from a green fluorescent plaque using oligo 8 and oligo 9 to verify the lack of wild-type HSV1 contamination.

    Techniques Used: CRISPR, Amplification, RFLP Assay, Expressing, Infection, Generated, Titration, Endpoint Dilution Assay, Recombinant, Plaque Assay, Fluorescence, Microscopy, Isolation, Staining, Polymerase Chain Reaction

    14) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    15) Product Images from "The Roles of Chemokines in Rabies Virus Infection: Overexpression May Not Always Be Beneficial ▿"

    Article Title: The Roles of Chemokines in Rabies Virus Infection: Overexpression May Not Always Be Beneficial ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01346-09

    Construction and characterization of recombinant RABVs expressing different chemokins. (A) Construction of full-length recombinant RABVs. Chemokine genes MIP-1α, RANTES, and IP-10 were individually inserted between BsiWI and NheI sites of the
    Figure Legend Snippet: Construction and characterization of recombinant RABVs expressing different chemokins. (A) Construction of full-length recombinant RABVs. Chemokine genes MIP-1α, RANTES, and IP-10 were individually inserted between BsiWI and NheI sites of the

    Techniques Used: Recombinant, Expressing

    16) Product Images from "Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?"

    Article Title: Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?

    Journal: Nanotheranostics

    doi: 10.7150/ntno.23826

    SSTR2, 3 and 5 expression in cell lines employed in the study: indirect immunolabelling in a flow cytometry analysis. SSTR2, 3 and 5 immunolabelling results in paraformaldehyde (PFA)-fixed and saponin-permeabilized HEK293 and BON1 cells, along with matched control stains in viable non-permeabilized HEK293 cells are presented on panels A and B, respectively. As all the anti-SSTR antibodies (Abs) employed in the series on panel A target native epitopes within C -tails of the receptors (confined to cytoplasmic compartment), the cells were fixed with PFA and permeabilized with saponin before immunolabelling. Conversely, the immunolabelling of the cells on panel B involved primary Ab against distinct tags within extracellular N -termini of SSTRs, hence no permeabilization was required and the staining was done on viable non-permeabilized cells. Noteworthy, the pattern of signal from matched samples stained for the same target with Abs against its different epitopes (Abs to intracellular C -tails of receptors on panel A vs Abs to tags within extracellular domains of the same receptors on panel B) is almost identical, which signifies specificity of the data. Bimodal appearance of the populations on histograms, especially obvious in case of SSTR3- and 5-overexpressing cells, is explained by the oligoclonal nature of the cultures, with the resulting distribution being formed by progeny of two (or more) dominant clones. Staining for β-tubulin, a component of a cytoskeleton, on panels A and B was implemented as a positive or negative control of permeabilization, respectively. The cells were analyzed on LSRII cytometer; at least 15 000 of the gated events were captured. Every image represents an overlay histogram of two samples: black transparent charts stand for either non-stained controls or fully stained samples; shaded green charts reflect the corresponding secondary antibody - only stained controls. x-axis denotes sample emission [(505 nm longpass)/(530/30 nm bandpass)] upon stimulation with 488 nm laser; y-axis indicates the number of events registered. The data from a single representative experiment (performed in duplicate) is shown; the complete series has been independently performed at least three times.
    Figure Legend Snippet: SSTR2, 3 and 5 expression in cell lines employed in the study: indirect immunolabelling in a flow cytometry analysis. SSTR2, 3 and 5 immunolabelling results in paraformaldehyde (PFA)-fixed and saponin-permeabilized HEK293 and BON1 cells, along with matched control stains in viable non-permeabilized HEK293 cells are presented on panels A and B, respectively. As all the anti-SSTR antibodies (Abs) employed in the series on panel A target native epitopes within C -tails of the receptors (confined to cytoplasmic compartment), the cells were fixed with PFA and permeabilized with saponin before immunolabelling. Conversely, the immunolabelling of the cells on panel B involved primary Ab against distinct tags within extracellular N -termini of SSTRs, hence no permeabilization was required and the staining was done on viable non-permeabilized cells. Noteworthy, the pattern of signal from matched samples stained for the same target with Abs against its different epitopes (Abs to intracellular C -tails of receptors on panel A vs Abs to tags within extracellular domains of the same receptors on panel B) is almost identical, which signifies specificity of the data. Bimodal appearance of the populations on histograms, especially obvious in case of SSTR3- and 5-overexpressing cells, is explained by the oligoclonal nature of the cultures, with the resulting distribution being formed by progeny of two (or more) dominant clones. Staining for β-tubulin, a component of a cytoskeleton, on panels A and B was implemented as a positive or negative control of permeabilization, respectively. The cells were analyzed on LSRII cytometer; at least 15 000 of the gated events were captured. Every image represents an overlay histogram of two samples: black transparent charts stand for either non-stained controls or fully stained samples; shaded green charts reflect the corresponding secondary antibody - only stained controls. x-axis denotes sample emission [(505 nm longpass)/(530/30 nm bandpass)] upon stimulation with 488 nm laser; y-axis indicates the number of events registered. The data from a single representative experiment (performed in duplicate) is shown; the complete series has been independently performed at least three times.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Clone Assay, Negative Control

    17) Product Images from "Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome"

    Article Title: Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-10-25

    A high-resolution optical map of M. ap ATCC 19698 . Genomic DNA of M. ap ATCC 19698 was mounted on derivatized glass using a microfluidic device and digested with Bsi WI. Fragment images were collected and processed by an automatic data acquisition system. A total of 970 optical contigs were assembled into one circular consensus map, giving an approximately 82-fold genome coverage. Optical contigs are represented by arcs of various lengths. Each arc is intersected by radiating lines that represent Bsi WI cutting sites, and arbitrary colors represent homologous overlapping fragments.
    Figure Legend Snippet: A high-resolution optical map of M. ap ATCC 19698 . Genomic DNA of M. ap ATCC 19698 was mounted on derivatized glass using a microfluidic device and digested with Bsi WI. Fragment images were collected and processed by an automatic data acquisition system. A total of 970 optical contigs were assembled into one circular consensus map, giving an approximately 82-fold genome coverage. Optical contigs are represented by arcs of various lengths. Each arc is intersected by radiating lines that represent Bsi WI cutting sites, and arbitrary colors represent homologous overlapping fragments.

    Techniques Used:

    Southern blotting analysis of the M. ap genomes . Genomic DNA from the two strains were digested with either Bsi WI or Kpn I, and subsequently probed with labeled fragments indicated in blue boxes, in supplementary Figure Two in Additional file 1 . Note the equal size of restriction fragments detected in both genomes with either probes.
    Figure Legend Snippet: Southern blotting analysis of the M. ap genomes . Genomic DNA from the two strains were digested with either Bsi WI or Kpn I, and subsequently probed with labeled fragments indicated in blue boxes, in supplementary Figure Two in Additional file 1 . Note the equal size of restriction fragments detected in both genomes with either probes.

    Techniques Used: Southern Blot, Labeling

    Related Articles

    Amplification:

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine
    Article Snippet: .. The amplified PCR product was digested with BsiWI and AsiSI (New England Biolabs, Ipswich, MA, USA) and then ligated to pSPBNGAS-GAS-GAS previously digested with BsiWI and AsiSI . .. The resulting plasmid was designated pSPBNGAS-LBVG-GAS ( ).

    Ligation:

    Article Title: A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice
    Article Snippet: .. Briefly, PCR production ( Flt3L gene) and LBNSE infectious clone were both digested with Bsi WI and Nhe I (New England BioLabs, Ipswich, MA, USA), and after recycling, the Flt3L gene was inserted between G and L genes of LBNSE by ligation to finish the construction of LBNSE-Flt3L infectious clone. .. For the rescue of LBNSE-Flt3L, BSR cells were transfected with 2.0 µg of LBNSE-Flt3L infectious clone, 0.5 µg of pH-N, 0.25 µg of pH-P, 0.15 µg of pH-G, and 0.1 µg of pH-L using the SuperFect transfection reagent (Qiagen) according to the manufacturer’s protocol.

    Polymerase Chain Reaction:

    Article Title: A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice
    Article Snippet: .. Briefly, PCR production ( Flt3L gene) and LBNSE infectious clone were both digested with Bsi WI and Nhe I (New England BioLabs, Ipswich, MA, USA), and after recycling, the Flt3L gene was inserted between G and L genes of LBNSE by ligation to finish the construction of LBNSE-Flt3L infectious clone. .. For the rescue of LBNSE-Flt3L, BSR cells were transfected with 2.0 µg of LBNSE-Flt3L infectious clone, 0.5 µg of pH-N, 0.25 µg of pH-P, 0.15 µg of pH-G, and 0.1 µg of pH-L using the SuperFect transfection reagent (Qiagen) according to the manufacturer’s protocol.

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine
    Article Snippet: .. The amplified PCR product was digested with BsiWI and AsiSI (New England Biolabs, Ipswich, MA, USA) and then ligated to pSPBNGAS-GAS-GAS previously digested with BsiWI and AsiSI . .. The resulting plasmid was designated pSPBNGAS-LBVG-GAS ( ).

    Article Title: Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity
    Article Snippet: .. The PCR product was digested with Bsi WI and Nhe I (New England Biolabs) and ligated into rabies virus vector pSPBN, which had been digested with Bsi WI and Nhe I ( ). .. The resulting plasmid was designated pSPBN-Cyto c (+) (Fig. ).

    Article Title: Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice
    Article Snippet: .. The PCR product was digested with BsiWI and NheI (New England Biolabs, Beverly, MA) and ligated into RV vector pSPBN that had been previously digested with BsiWI and NheI. .. The resulting plasmid was designated pSPBN-TNF-α(+) (Fig. ).

    Plasmid Preparation:

    Article Title: Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity
    Article Snippet: .. The PCR product was digested with Bsi WI and Nhe I (New England Biolabs) and ligated into rabies virus vector pSPBN, which had been digested with Bsi WI and Nhe I ( ). .. The resulting plasmid was designated pSPBN-Cyto c (+) (Fig. ).

    Article Title: Overexpression of Tumor Necrosis Factor Alpha by a Recombinant Rabies Virus Attenuates Replication in Neurons and Prevents Lethal Infection in Mice
    Article Snippet: .. The PCR product was digested with BsiWI and NheI (New England Biolabs, Beverly, MA) and ligated into RV vector pSPBN that had been previously digested with BsiWI and NheI. .. The resulting plasmid was designated pSPBN-TNF-α(+) (Fig. ).

    Sequencing:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: .. To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: .. To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Derivative Assay:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: .. To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: .. To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

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    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . <t>BsiWI</t> , <t>AsiSI</t> , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
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    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Journal: Tropical Medicine and Infectious Disease

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    doi: 10.3390/tropicalmed2030037

    Figure Lengend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Article Snippet: The amplified PCR product was digested with BsiWI and AsiSI (New England Biolabs, Ipswich, MA, USA) and then ligated to pSPBNGAS-GAS-GAS previously digested with BsiWI and AsiSI .

    Techniques: Recombinant

    Schematic diagram of cytochrome c recombinant rabies viruses. The pSPBN vector was derived from SPBN-10 by removing the ψ gene and introducing Bsi WI and Nhe I sites between the G and L genes. Human cytochrome c cDNA was amplified by PCR and, after introduction of Bsi WI and Nhe I sites, ligated into pSPBN, resulting in pSPBN-Cyto c (+). To construct pSPBN-Cyto c (−), a stop codon was introduced into the cytochrome c gene 70 bp after the start codon.

    Journal: Journal of Virology

    Article Title: Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity

    doi: 10.1128/JVI.75.22.10800-10807.2001

    Figure Lengend Snippet: Schematic diagram of cytochrome c recombinant rabies viruses. The pSPBN vector was derived from SPBN-10 by removing the ψ gene and introducing Bsi WI and Nhe I sites between the G and L genes. Human cytochrome c cDNA was amplified by PCR and, after introduction of Bsi WI and Nhe I sites, ligated into pSPBN, resulting in pSPBN-Cyto c (+). To construct pSPBN-Cyto c (−), a stop codon was introduced into the cytochrome c gene 70 bp after the start codon.

    Article Snippet: The PCR product was digested with Bsi WI and Nhe I (New England Biolabs) and ligated into rabies virus vector pSPBN, which had been digested with Bsi WI and Nhe I ( ).

    Techniques: Recombinant, Plasmid Preparation, Derivative Assay, Amplification, Polymerase Chain Reaction, Construct

    Construction and characterization of rRABV expressing IL-15 in vitro and in vivo . A Schematic diagram for construction of LBNSE-Flt3L. The parent vector pLBNSE was constructed based on SAD-B19 strain by deleting the pseudogene between the G and L genes where Bsi wI and Nhe I restriction enzyme sites were introduced. N , P , M , G and L represented the nucleoprotein, phosphoprotein, matrix, glycoprotein, and polymerase genes of RABV, respectively. Multi-step ( B ) and One-step ( C ) virus growth curves were determined on BSR cells. Cells were infected with either LBNSE or LBNSE-Flt3L at a multiplicity of infection (MOI) of 0.01 or 5, respectively, and the culture supernatants were harvested at 1, 2, 3, 4 and 5 dpi for viral titration. The virus growth curves were drawn according to the viral titers measured at each time point. Data are presented as the mean ± SD (n = 3). D Expression of Flt3L was detected in infected BSR cells by ELISA. BSR cells were infected with either the LBNSE or LBNSE-Flt3L at the MOI = 0.001, 0.01, 0.1, or 1, and the culture supernatants were harvested at 24 h post infection for ELISA assay. Data are presented as the mean ± SD (n = 3). E Body weight change curves of mice infected with different rRABVs. Six-week-old female ICR mice (n = 10) were infected by the i.c. route with 1 × 10 7 FFU of LBNSE or LBNSE-Flt3L, or with mock infected with the same volume of DMEM, and the body weights were monitored daily for 2 weeks.

    Journal: Virologica Sinica

    Article Title: A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice

    doi: 10.1007/s12250-019-00144-x

    Figure Lengend Snippet: Construction and characterization of rRABV expressing IL-15 in vitro and in vivo . A Schematic diagram for construction of LBNSE-Flt3L. The parent vector pLBNSE was constructed based on SAD-B19 strain by deleting the pseudogene between the G and L genes where Bsi wI and Nhe I restriction enzyme sites were introduced. N , P , M , G and L represented the nucleoprotein, phosphoprotein, matrix, glycoprotein, and polymerase genes of RABV, respectively. Multi-step ( B ) and One-step ( C ) virus growth curves were determined on BSR cells. Cells were infected with either LBNSE or LBNSE-Flt3L at a multiplicity of infection (MOI) of 0.01 or 5, respectively, and the culture supernatants were harvested at 1, 2, 3, 4 and 5 dpi for viral titration. The virus growth curves were drawn according to the viral titers measured at each time point. Data are presented as the mean ± SD (n = 3). D Expression of Flt3L was detected in infected BSR cells by ELISA. BSR cells were infected with either the LBNSE or LBNSE-Flt3L at the MOI = 0.001, 0.01, 0.1, or 1, and the culture supernatants were harvested at 24 h post infection for ELISA assay. Data are presented as the mean ± SD (n = 3). E Body weight change curves of mice infected with different rRABVs. Six-week-old female ICR mice (n = 10) were infected by the i.c. route with 1 × 10 7 FFU of LBNSE or LBNSE-Flt3L, or with mock infected with the same volume of DMEM, and the body weights were monitored daily for 2 weeks.

    Article Snippet: Briefly, PCR production ( Flt3L gene) and LBNSE infectious clone were both digested with Bsi WI and Nhe I (New England BioLabs, Ipswich, MA, USA), and after recycling, the Flt3L gene was inserted between G and L genes of LBNSE by ligation to finish the construction of LBNSE-Flt3L infectious clone.

    Techniques: Expressing, In Vitro, In Vivo, Plasmid Preparation, Construct, Infection, Titration, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Dual-plasmid approach for integrase-mediated attB × attP recombination. ( a ) Map of the attP -carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with Avr II and Afl II can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with Avr II and Bsi WI can place the gene of interest in frame with gfp . The plasmid carries the blasticidin S-deaminase ( bsd ) selectable marker. ( b ) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. ( c ) Schematic representation of the attB × attP recombination allowing integration of a gene of interest ( yfg ) at the cg6 - attB locus. Dd2 attB or 3D7 attB parasites are co-transfected with pINT and an attP -plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2 attB and 3D7 attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP -plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes ( designated as open boxes ), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR : untranslated region. Panel ( c with the permission of the Nature Publishing Group.

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

    Article Title: A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase

    doi: 10.1007/978-1-60761-652-8_6

    Figure Lengend Snippet: Dual-plasmid approach for integrase-mediated attB × attP recombination. ( a ) Map of the attP -carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with Avr II and Afl II can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with Avr II and Bsi WI can place the gene of interest in frame with gfp . The plasmid carries the blasticidin S-deaminase ( bsd ) selectable marker. ( b ) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. ( c ) Schematic representation of the attB × attP recombination allowing integration of a gene of interest ( yfg ) at the cg6 - attB locus. Dd2 attB or 3D7 attB parasites are co-transfected with pINT and an attP -plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2 attB and 3D7 attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP -plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes ( designated as open boxes ), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR : untranslated region. Panel ( c with the permission of the Nature Publishing Group.

    Article Snippet: Digestion with Avr II and Bsi WI (New England Biolabs) can also be performed to place the gene of interest in frame with gfp ) under the identifiers and for pLN-ENR-GFP and pINT, respectively.

    Techniques: Plasmid Preparation, Marker, Expressing, Selection, Transfection, Polymerase Chain Reaction, DNA Hybridization, Sequencing