Journal: Nucleic Acids Research

Article Title: Vika/ vox , a novel efficient and specific Cre/ loxP -like site-specific recombination system

doi: 10.1093/nar/gks1037

Figure Lengend Snippet: Evaluation of Vika recombination activity on heterologous target sites. ( A ) Scheme of the recombination assay. Site-specific recombination excises a 1.2-kb DNA fragment, flanked by the recombinase target sites (triangles) from the plasmid. Protein coding genes are depicted by arrows and the origin of replication (ori) is shown as a box. CmR, chloramphenicol resistance gene; AraC, gene coding arabinose promoter regulator protein and recombinase, recombinase coding gene. ( B ) Lack of recombination activity of Vika on the loxP , rox and VloxP target sites. Site-specific recombination of native targets by Cre ( loxP ), Dre ( rox ) and VCre ( VloxP ) served as positive control. The − and + indicate presence or absence of L(+)-arabinose (100 μg/ml) in the growth medium. Non-recombined and recombined plasmids are denoted as two triangles and one triangle, respectively. M, marker; 2-log DNA ladder, NEB.

Article Snippet: M, marker; 2-log DNA ladder, NEB. ( B ) Evaluation of DNA damage induction by γ - H2AX assay 72 h after infection with indicated recombinase-expressing virus, n = 3. ( C ) Quantification of the γ - H2AX-positive cells either infected with virus for respective recombinase expression or treated with campthothecin for 2 h. Statistically significant increase of γ - H2AX signals is indicated by asterisks.

Techniques: Activity Assay, Recombination Assay, Plasmid Preparation, Positive Control, Marker

Journal: Nucleic Acids Research

Article Title: Vika/ vox , a novel efficient and specific Cre/ loxP -like site-specific recombination system

doi: 10.1093/nar/gks1037

Figure Lengend Snippet: Evaluation of Vika recombination activity and specificity in E. coli . ( A ) Recombination specificity of indicated recombinases on vox . The − and + indicates absence or presence of L(+)-arabinose (100 μg /ml) in the growth medium. Non-recombined and recombined plasmids are denoted as two triangles and one triangle, respectively. M, marker; 2-log DNA ladder, NEB. ( B ) Matrix of the recombination specificities of indicated recombinase systems. ( C ) Schematic view of LacZ-based reporter assay. The non-recombined reporter plasmid constitutively expresses beta-galactosidase, inferring blue color to cells on X-gal plates. On recombination, the promoter driving LacZ expression is removed, resulting in white colonies on X-gal plates. Triangles depict lox sites. Prom = promoter. ( D ) Recombination activity of indicated recombinases on the loxP , v loxP and vox LacZ-based reporters. Note that white colonies, signifying recombination, only appear when a recombinase is expressed together with its corresponding reporter. Positive controls for the non-recombined (pSVpaX), and recombined form of the reporter (pSVpaXΔ), is shown as Mock. ( E ) Scheme of the integration assay. The host plasmids (pH) carry the coding sequence for the used recombinases, the chloramphenicol resistance gene (CmR), a p15A origin of replication (p15A) and one respective recombinase target site (triangle). The donor plasmids (pD) carry the kanamycin resistance gene (KmR), a R6K origin of replication (R6K) and also one respective recombinase target site (triangle). Only combinations of two identical target sites and their corresponding recombinases will produce co-integrant plasmids that survive kanamycin selection in pir -negative cells. ( F ) Colony formation assay of pir -negative E. coli cells transformed with indicated plasmid combinations. Colonies only form on site-specific integration of pD and pH plasmids. ( G ) Quantification of colonies carrying co-integrant plasmids after transformation with indicated plasmid combinations.

Article Snippet: M, marker; 2-log DNA ladder, NEB. ( B ) Evaluation of DNA damage induction by γ - H2AX assay 72 h after infection with indicated recombinase-expressing virus, n = 3. ( C ) Quantification of the γ - H2AX-positive cells either infected with virus for respective recombinase expression or treated with campthothecin for 2 h. Statistically significant increase of γ - H2AX signals is indicated by asterisks.

Techniques: Activity Assay, Marker, Reporter Assay, Plasmid Preparation, Expressing, Sequencing, Selection, Colony Assay, Transformation Assay

Journal: Nucleic Acids Research

Article Title: Vika/ vox , a novel efficient and specific Cre/ loxP -like site-specific recombination system

doi: 10.1093/nar/gks1037

Figure Lengend Snippet: Evaluation of the recombination activity of indicated recombination systems in E. coli . One and two triangles mark the size of the recombined and non-recombined plasmids, respectively. M = marker, 2-log DNA ladder, NEB, no ind = no addition of L(+)-arabinose to the growth medium, 1, 10 and 100 = growth of the cells in the presence of 1 μg/ml, 10 μg/ml and 100 μg/ml L(+)-arabinose, respectively.

Article Snippet: M, marker; 2-log DNA ladder, NEB. ( B ) Evaluation of DNA damage induction by γ - H2AX assay 72 h after infection with indicated recombinase-expressing virus, n = 3. ( C ) Quantification of the γ - H2AX-positive cells either infected with virus for respective recombinase expression or treated with campthothecin for 2 h. Statistically significant increase of γ - H2AX signals is indicated by asterisks.

Techniques: Activity Assay, Marker

Journal: Nucleic Acids Research

Article Title: Vika/ vox , a novel efficient and specific Cre/ loxP -like site-specific recombination system

doi: 10.1093/nar/gks1037

Figure Lengend Snippet: Evaluation of genotoxicity and cytotoxicity of Vika. ( A ) Recombination specificity of Vika and Cre on the respective cryptic human and mouse chromosomal target sites. The − and + indicates absence or presence of L(+)-arabinose (100 μg/ml) in the growth medium. Non-recombined and recombined plasmids are denoted as two triangles and one triangle, respectively. M, marker; 2-log DNA ladder, NEB. ( B ) Evaluation of DNA damage induction by γ - H2AX assay 72 h after infection with indicated recombinase-expressing virus, n = 3. ( C ) Quantification of the γ - H2AX-positive cells either infected with virus for respective recombinase expression or treated with campthothecin for 2 h. Statistically significant increase of γ - H2AX signals is indicated by asterisks. Error bars indicate standard deviation of the mean value (** indicate P = 0.01). ( D ) Proliferation effects on expression of indicated recombinases in mouse NIH3T3 cells. Cells were infected with bicistronic retroviruses expressing respective recombinase linked to GFP. Every 72 h, cells were analyzed by flow cytometry. Error bars indicate standard deviation of the mean value, n = 2.

Article Snippet: M, marker; 2-log DNA ladder, NEB. ( B ) Evaluation of DNA damage induction by γ - H2AX assay 72 h after infection with indicated recombinase-expressing virus, n = 3. ( C ) Quantification of the γ - H2AX-positive cells either infected with virus for respective recombinase expression or treated with campthothecin for 2 h. Statistically significant increase of γ - H2AX signals is indicated by asterisks.

Techniques: Marker, Infection, Expressing, Standard Deviation, Flow Cytometry