Journal: Nucleic Acids Research

Article Title: Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation

doi: 10.1093/nar/gkm496

Figure Lengend Snippet: Time course assay for continuous expression of both Nat and Mod PV L1 genes in cultured mouse KCs. Mouse KCs grown in medium for 1 day were transiently transfected with the four L1 gene expression constructs for 16 h and then collected for analysis of the L1 gene expression for a 12-day period post-transfection. (A) Quantitative RT-PCR analysis of L1 mRNA in cultured KC cells. RNA samples were prepared at D3, D6, D9 and D12 post-transfection. 0.5 μg of each sample of DNase I-digested total RNA was used for RT-PCR. Upper panel : The RT-PCR products from L1 and actin mRNAs were electrophoresed on a 1% agarose gel. Lower panel : Relative L1 mRNAs are shown with the means (± S.E.M) of four separate assays from two independent experiments. Statistical analysis of the results was conducted. P < 0.01 and P < 0.05 represent the significant degree of the differences between Nat and Mod L1 mRNAs using t Test, respectively. (B) Western blotting analysis of L1 protein and β-tubulin in cultured mouse KCs. Monoclonal antibody against L1 protein was used to probe the blot. Upper panel shows the results of the L1 immunoblotting assay; lower panel shows the signals of β-tubulin showing comparable loading of the protein samples.

Article Snippet: Following transcription, 2 μl RNase-free DNase (2000 U/ml, Promega, Australia) was added into each reaction to digest the DNA template at 37°C for 1 h. The transcribed PV L1 mRNAs were then purified by phenol–chloroform extraction, followed by precipitation with 2 volumes of ethanol and washed twice with 70% ethanol.

Techniques: Expressing, Cell Culture, Transfection, Gene Expression, Construct, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

Journal: Nucleic Acids Research

Article Title: Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation

doi: 10.1093/nar/gkm496

Figure Lengend Snippet: Time course assay for continuous expression of both Nat and Mod PV L1 genes in cultured human KCs. KCs at a density of 6.5 × 10 4 cells/cm 2 in medium grown for 1 day were transiently transfected with the four L1 gene expression constructs for 16 h and then collected for analysis of the L1 gene expression for a 9-day period post-transfection. (A) Quantitative RT-PCR analysis of L1 mRNA in cultured KC cells. RNA samples were prepared at D3, D6 and D9 post-transfection. 0.5 μg of each sample of DNase I-digested total RNA was used for RT-PCR. Upper panel : The RT-PCR products from L1 and actin mRNAs were electrophoresed on a 1% agarose gel. Lower panel : Relative L1 mRNAs are shown with the means (± S.E.M) of four separate assays from two separate transfections. Statistical analysis of the results was conducted. P < 0.01 and P < 0.05 represent the significant degree of the differences between Nat and Mod L1 mRNAs using t Test, respectively. (B) Western blotting analysis of L1 protein and β-tubulin in cultured human KCs. Monoclonal antibody against L1 protein was used to probe the blot. Upper panel shows the results of the L1 immunoblotting assay; lower panel shows the signals of β-tubulin showing comparable loading of the protein samples.

Article Snippet: Following transcription, 2 μl RNase-free DNase (2000 U/ml, Promega, Australia) was added into each reaction to digest the DNA template at 37°C for 1 h. The transcribed PV L1 mRNAs were then purified by phenol–chloroform extraction, followed by precipitation with 2 volumes of ethanol and washed twice with 70% ethanol.

Techniques: Expressing, Cell Culture, Transfection, Gene Expression, Construct, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

Journal: Chemical Science

Article Title: Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification † †Electronic supplementary information (ESI) available: Sequence information, reaction conditions optimization and Cas9 cleavage analysis in a time dependent manner. See DOI: 10.1039/c6sc01355d

doi: 10.1039/c6sc01355d

Figure Lengend Snippet: Schematic diagram of the nicking triggered exponential amplification reaction (NTEXPAR) for the detection of Cas9 cleaved dsDNA. (a) The NTEXPAR probe designed for Cas9 cleavage assay contains a nicking site, target sequence and protospacer adjacent motif (PAM) which can be recognized and cleaved by a Cas9–sgRNA complex. (b) After Cas9 cleavage, the exposed blunt-ended cleavage site can be recognized by a nicking enzyme and DNA polymerase to generate a short single-stranded DNA trigger by a nicking and strand displacement mechanism. The generated ssDNA trigger could be further exponentially amplified with the same enzymes in the presence of a designed amplification template. The amount of cleaved dsDNA could be quantified by measuring the product related fluorescence signal.

Article Snippet: The dNTPs mix, Vent (exo-) DNA polymerase (2000 U mL –1 ), Nt.BstNBI nicking endonuclease (10 000 U mL –1 ), Cas9 nuclease S. pyogenes (1000 nM), HiScribe™ T7 Quick High Yield RNA Synthesis Kit, 10× NEBuffer 3.1 and 10× ThermoPol buffer were purchased from NEB (New England Biolabs, Ipswich, MA, USA).

Techniques: Amplification, Cleavage Assay, Sequencing, Generated, Fluorescence