m0298  (New England Biolabs)

Journal: Frontiers in Molecular Neuroscience

Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

doi: 10.3389/fnmol.2017.00273

Figure Lengend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

Article Snippet: To differentiate between Fyn expressed in D1R vs. D2R MSNs, we developed a method in which short hairpin RNA (shRNA) that targets a specific gene is expressed only in the presence of Cre-recombinase (Cre).

Techniques: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Scientific Reports

Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

doi: 10.1038/s41598-018-31585-1

Figure Lengend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

Techniques: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

doi: 10.1038/s41598-018-31585-1

Figure Lengend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

Techniques: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

Journal: Scientific Reports

Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

doi: 10.1038/s41598-018-31585-1

Figure Lengend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

doi: 10.1038/s41598-018-31585-1

Figure Lengend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

Techniques: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Scientific Reports

Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

doi: 10.1038/s41598-018-31585-1

Figure Lengend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

Techniques: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Scientific Reports

Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

doi: 10.1038/s41598-018-31585-1

Figure Lengend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

Techniques: Sequencing

Journal: BMC Biotechnology

Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

doi: 10.1186/s12896-018-0462-x

Figure Lengend Snippet: Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation

Article Snippet: Next, we tested whether we can further boost cloning efficiency by increasing the concentrations of cre recombinase.

Techniques: Plasmid Preparation, Incubation, Transformation Assay

Journal: BMC Biotechnology

Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

doi: 10.1186/s12896-018-0462-x

Figure Lengend Snippet: Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion

Article Snippet: Next, we tested whether we can further boost cloning efficiency by increasing the concentrations of cre recombinase.

Techniques: Plasmid Preparation, In Vitro