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  • 94
    Millipore kpn i
    Kpn I, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa kpn i
    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Kpn I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1661 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa kpn i linker
    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Kpn I Linker, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa iii kpn i
    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Iii Kpn I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa kpn i bam hi digested pegfp c1
    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Kpn I Bam Hi Digested Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa nhe i kpn i digested pegfp n1
    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with <t>Kpn</t> I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
    Nhe I Kpn I Digested Pegfp N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Journal: Molecular Biology of the Cell

    Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

    doi: 10.1091/mbc.E19-02-0108

    Figure Lengend Snippet: Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P

    Article Snippet: Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ).

    Techniques: Transfection, Marker, Isolation, Hybridization, Construct, Agarose Gel Electrophoresis, Fluorescence In Situ Hybridization, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Serial Dilution

    Insertion of the LMWP peptide into the PP7 coat protein dimer. The cDNA sequence of LMWP ( in red ) was inserted after the Kpn I site of the vector pETDuet-2PP7 ( a ). The possible structure of the PP7 coat protein dimer carrying LMWP with the AB-loops shown in ribbon was predicted using the program RasMol 2.7.1 ( b )

    Journal: BMC Biotechnology

    Article Title: Intracellular delivery of messenger RNA by recombinant PP7 virus-like particles carrying low molecular weight protamine

    doi: 10.1186/s12896-016-0274-9

    Figure Lengend Snippet: Insertion of the LMWP peptide into the PP7 coat protein dimer. The cDNA sequence of LMWP ( in red ) was inserted after the Kpn I site of the vector pETDuet-2PP7 ( a ). The possible structure of the PP7 coat protein dimer carrying LMWP with the AB-loops shown in ribbon was predicted using the program RasMol 2.7.1 ( b )

    Article Snippet: Briefly, the gene containing LMWP was amplified by PCR using primers 2PP7-Protamine-for and PP7-r (Table ), digested by two restriction enzymes Kpn I and Xho I (Takara, Dalian, China), and ligated with the plasmid pETDuet-2PP7.

    Techniques: Sequencing, Plasmid Preparation

    Schematic illustration of the general idea of this study. The ING4 DNA fragments were first cloned into the pEGFP-C2 expression vector using Xho I and Kpn I restriction enzymes to produce the eukaryotic expression vector pEGFP-ING4. The vector was then transfected into human osteosarcoma cells U-2OS, and stable transfectants of pEGFP-ING4 with successful plasmid transfection were selected. Finally, the effects of overexpressed ING4 on the proliferation, cell cycle, invasion and apoptosis of U-2OS cells were evaluated.

    Journal: Scientific Reports

    Article Title: Delivery of inhibitor of growth 4 (ING4) gene significantly inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells

    doi: 10.1038/srep07380

    Figure Lengend Snippet: Schematic illustration of the general idea of this study. The ING4 DNA fragments were first cloned into the pEGFP-C2 expression vector using Xho I and Kpn I restriction enzymes to produce the eukaryotic expression vector pEGFP-ING4. The vector was then transfected into human osteosarcoma cells U-2OS, and stable transfectants of pEGFP-ING4 with successful plasmid transfection were selected. Finally, the effects of overexpressed ING4 on the proliferation, cell cycle, invasion and apoptosis of U-2OS cells were evaluated.

    Article Snippet: The fragments were cloned into the pEGFP-C2 expression vector (Clontech) using Xho I and Kpn I restriction enzymes (Takara) to produce the eukaryotic expression vector pEGFP-ING4.

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Transfection

    Integration vectors to construct one-hybrid strains with ADE2 and HIS3 reporter genes. ( A ) Integration vector pOHAK for ADE2 reporter gene. We cloned a fragment spanning AFI1-GAL2 UAS -GAL2 TATA -ADE2 from PJ69-2A and replaced the GAL2 UAS with a fragment containing KanMX and an HpaI site to obtain pOHAK. ( B ) Integration vector pOHHZ for HIS3 reporter gene. We cloned a fragment spanning LYS2-GAL1 UAS -GAL1 TATA -HIS3 from PJ69-2A and replaced the GAL1 UAS with a fragment containing Zeo R and a PmeI site to obtain pOHHZ. ( C ) Schematic representation of reporter gene construction in the case of ADE2 as an example. A methylatable bait sequence is cloned at the HpaI site of pOHAK, and the plasmid linearized by SalI-KpnI digestion is used for transformation of PJ69-2A. Homologous recombination occurs within ORFs for AFI1 and ADE2 .

    Journal: Nucleic Acids Research

    Article Title: A yeast one-hybrid system to screen for methylated DNA-binding proteins

    doi: 10.1093/nar/gkq757

    Figure Lengend Snippet: Integration vectors to construct one-hybrid strains with ADE2 and HIS3 reporter genes. ( A ) Integration vector pOHAK for ADE2 reporter gene. We cloned a fragment spanning AFI1-GAL2 UAS -GAL2 TATA -ADE2 from PJ69-2A and replaced the GAL2 UAS with a fragment containing KanMX and an HpaI site to obtain pOHAK. ( B ) Integration vector pOHHZ for HIS3 reporter gene. We cloned a fragment spanning LYS2-GAL1 UAS -GAL1 TATA -HIS3 from PJ69-2A and replaced the GAL1 UAS with a fragment containing Zeo R and a PmeI site to obtain pOHHZ. ( C ) Schematic representation of reporter gene construction in the case of ADE2 as an example. A methylatable bait sequence is cloned at the HpaI site of pOHAK, and the plasmid linearized by SalI-KpnI digestion is used for transformation of PJ69-2A. Homologous recombination occurs within ORFs for AFI1 and ADE2 .

    Article Snippet: We cloned the LexAop8 -Sm4 fragment into the HpaI site of pOHAK, digested the obtained plasmid by SalI and KpnI, used the linearized plasmid DNA to transform PJ69-2A cells (Clonetech) and selected G418-resistant transformants.

    Techniques: Construct, Plasmid Preparation, Clone Assay, Sequencing, Transformation Assay, Homologous Recombination

    Reconstitution of Cre activity among split‐Cre pairs in bacterial and plant cells. (a) Not to scale depiction of DNA constructs used for E. coli and plant cells, only relevant DNA segments shown. N ‐ cre and C‐cre encode N‐ and C‐terminal fragments of Cre, respectively. P T7 = T7 phage promoter; P 35S = CaMV 35S RNA promoter; T T7 = T7 phage transcription terminator; T nos = nos terminator; hpt = hygromycin phosphotransferase gene; gus =beta‐glucuronidase gene; numbers 1–4 refer to PCR primers. Genes transcribed left to right except for hpt in the inversion product where upside‐down lettering indicates transcription from right to left. (b) Depiction of 10 split‐Cre pairs tested along with wild‐type Cre and the 5N/5C positive control. Negative control not shown. % = % of E. coli colonies found to show Cre‐mediated inversion (mean ± SD of three independent experiments; 16 colonies tested per experiments). 0% not shown. (c) Representative PCR analysis of the presence of the 1 + 3 and 2 + 4 PCR products indicative of site‐specific inversion, 3N/1C and 4N/2C are representative pairs that failed to show Cre activity. (d) GUS histochemical staining of bombarded onion epidermis. Blue spots show GUS activity to indicate formation of excision product from site‐specific excision of hpt‐T nos blocking DNA . LZ = leucine zipper of Max and Myc.

    Journal: Plant Biotechnology Journal

    Article Title: A new location to split Cre recombinase for protein fragment complementation

    doi: 10.1111/pbi.12726

    Figure Lengend Snippet: Reconstitution of Cre activity among split‐Cre pairs in bacterial and plant cells. (a) Not to scale depiction of DNA constructs used for E. coli and plant cells, only relevant DNA segments shown. N ‐ cre and C‐cre encode N‐ and C‐terminal fragments of Cre, respectively. P T7 = T7 phage promoter; P 35S = CaMV 35S RNA promoter; T T7 = T7 phage transcription terminator; T nos = nos terminator; hpt = hygromycin phosphotransferase gene; gus =beta‐glucuronidase gene; numbers 1–4 refer to PCR primers. Genes transcribed left to right except for hpt in the inversion product where upside‐down lettering indicates transcription from right to left. (b) Depiction of 10 split‐Cre pairs tested along with wild‐type Cre and the 5N/5C positive control. Negative control not shown. % = % of E. coli colonies found to show Cre‐mediated inversion (mean ± SD of three independent experiments; 16 colonies tested per experiments). 0% not shown. (c) Representative PCR analysis of the presence of the 1 + 3 and 2 + 4 PCR products indicative of site‐specific inversion, 3N/1C and 4N/2C are representative pairs that failed to show Cre activity. (d) GUS histochemical staining of bombarded onion epidermis. Blue spots show GUS activity to indicate formation of excision product from site‐specific excision of hpt‐T nos blocking DNA . LZ = leucine zipper of Max and Myc.

    Article Snippet: Finally, 2NLZ and 3NLZ fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMM23‐CLZ series constructs: 1C‐LZ‐cre and 3C‐LZ‐cre fragments were PCR amplified from pMM23 using primers C‐R2, 1CLZ‐F or 3CLZ‐F for insertion between the Eco RI and Sph I sites of pMD18‐T (Takara).

    Techniques: Activity Assay, Construct, Polymerase Chain Reaction, Positive Control, Negative Control, Staining, Blocking Assay

    Reconstitution of Cre activity for recombination of plasmid and chromosomal DNA. (a) Not to scale depiction of constructs used, only relevant DNA segments shown. Genetic elements as in Figure 3 (a). P 35S = CaMV 35S RNA promoter for plants; T nos = nos terminator; T ubi = ubiquitin gene terminator ; luc = firefly luciferase gene; a to f = primers used. Dashed lines indicate extent of DNA hybridizing to probes p1 and p2 when cleaved with Eco RI (E) plus Bst EII (B) or with Sph I (S). Numbers refer to length of DNA size in kb. (b) Transient expression in Arabidopsis protoplasts. Values are mean ± SD; n = 9. * P

    Journal: Plant Biotechnology Journal

    Article Title: A new location to split Cre recombinase for protein fragment complementation

    doi: 10.1111/pbi.12726

    Figure Lengend Snippet: Reconstitution of Cre activity for recombination of plasmid and chromosomal DNA. (a) Not to scale depiction of constructs used, only relevant DNA segments shown. Genetic elements as in Figure 3 (a). P 35S = CaMV 35S RNA promoter for plants; T nos = nos terminator; T ubi = ubiquitin gene terminator ; luc = firefly luciferase gene; a to f = primers used. Dashed lines indicate extent of DNA hybridizing to probes p1 and p2 when cleaved with Eco RI (E) plus Bst EII (B) or with Sph I (S). Numbers refer to length of DNA size in kb. (b) Transient expression in Arabidopsis protoplasts. Values are mean ± SD; n = 9. * P

    Article Snippet: Finally, 2NLZ and 3NLZ fragments were retrieved by cleavage with Kpn I and Sph I and inserted between P 35S (Kpn I) and T nos (Sph I) of pMM23. pMM23‐CLZ series constructs: 1C‐LZ‐cre and 3C‐LZ‐cre fragments were PCR amplified from pMM23 using primers C‐R2, 1CLZ‐F or 3CLZ‐F for insertion between the Eco RI and Sph I sites of pMD18‐T (Takara).

    Techniques: Activity Assay, Plasmid Preparation, Construct, Luciferase, Expressing

    Restriction fragment length polymorphism (RFLP) of BAC PRV-G and its TK deletion mutants. a RFLP pattern of BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G . Lane 1, 2 and 3 are BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G respectively after digestion with Kpn I. The arrow (lane 2) shows an additional band of 6639 bp and a 5975 bp band missed when compared to lane 3. The arrow(lane 1) showed an additional band of 5628 bp and a missing band of 6639 bp when compared to lane 2. M is 1Kb DNA marker. b Predicted RFLP pattern with PRV ZJ01 strain (GenBank:KM061380.1) as a reference. Lane 1, 2 and 3 are prediction of BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G respectively digested with Kpn I. c Kpn I sites were cited in lane 1, 2 and 3 for BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G respectively. Sites underlined with red lines indicate the changed position of Kpn I restriction site leading to the bands changing accordingly

    Journal: BMC Veterinary Research

    Article Title: Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK gE genes

    doi: 10.1186/s12917-018-1536-7

    Figure Lengend Snippet: Restriction fragment length polymorphism (RFLP) of BAC PRV-G and its TK deletion mutants. a RFLP pattern of BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G . Lane 1, 2 and 3 are BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G respectively after digestion with Kpn I. The arrow (lane 2) shows an additional band of 6639 bp and a 5975 bp band missed when compared to lane 3. The arrow(lane 1) showed an additional band of 5628 bp and a missing band of 6639 bp when compared to lane 2. M is 1Kb DNA marker. b Predicted RFLP pattern with PRV ZJ01 strain (GenBank:KM061380.1) as a reference. Lane 1, 2 and 3 are prediction of BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G respectively digested with Kpn I. c Kpn I sites were cited in lane 1, 2 and 3 for BAC PRV△TK/gE/gI , BAC PRV△TK/gE/gI K+ and BAC PRV-G respectively. Sites underlined with red lines indicate the changed position of Kpn I restriction site leading to the bands changing accordingly

    Article Snippet: Restriction fragment length polymorphism(RFLP) analyses of PRV BAC or BAC mutants was conducted using the restriction endonucleases Kpn I (Takara) as described earlier [ ].

    Techniques: BAC Assay, Marker