Journal: Frontiers in Endocrinology

Article Title: Engineered IRES-mediated promoter-free insulin-producing cells reverse hyperglycemia

doi: 10.3389/fendo.2024.1439351

Figure Lengend Snippet: HDR-mediated modified human insulin gene knock-in in HEK293T cells. (A) Schematics of the donor plasmid and targeting strategy for HDR-mediated knock-in of the modified human insulin at GAPDH 3′-UTR. Dashed lines indicate sections of homology between the GAPDH genomic locus and donor plasmid DNA. Arrows indicate the positions of PCR primers for insulin integration examination. (B) The quantity of insulin in culture media (supernatant) and cell lysis(intracellular) were assessed using ELISA. Fresh culture medium was used as negative control (NC). (C) HDR-mediated integration efficiency of Cas9sg1 and Cas9sg4 using fluorescence images and Flow cytometer analysis. Cas9 plasmid without sgRNA was used as a control. (D) Genome PCR analysis of mcherry + cells produced with Cas9sg1 in sequencing results of the PCR amplicons with expected modifications (human insulin gene) were integrated precisely at both 5′- and 3′-junctions. (E) Insulin secretion from mchP2AIns in response to different types of glucose stimulation. Data are expressed as mean ± SD. n = 5; ***p < 0.001 by student’s t-test.

Article Snippet: Additionally, the total DNA content of each sample was determined using a DNA Quantification Kit (TIANGEN, China) to standardize insulin secretion.

Techniques: Modification, Gene Knock-In, Plasmid Preparation, Knock-In, Lysis, Enzyme-linked Immunosorbent Assay, Negative Control, Fluorescence, Flow Cytometry, Control, Produced, Sequencing

Journal: Nucleic Acids Research

Article Title: Epstein–Barr Virus DNase (BGLF5) induces genomic instability in human epithelial cells

doi: 10.1093/nar/gkp1169

Figure Lengend Snippet: The nuclease activity of EBV DNase is important for the effect of DNA damage. ( a ) TW01 cells were transfected with vector, DNase, Mutant 1, Mutant 2 and Mutant 3, respectively. After 24 h transfection, cells were collected for detection of DNA damage. DNA damage was analyzed by comet assay, which is as described in ‘Materials and Methods’ section. ( b ) The results of comet assay were quantitated by visual scoring. Visual scores were presented as classes 0–4 which increase with the level of genomic DNA damage ( Supplementary Figure S1 ). All data presented represent the means and the standard deviations of at least three independent experiments. Two asterisks denote P < 0.01.

Article Snippet: A single-cell gel electrophoresis (comet assay) kit was employed for evaluating DNA damage (Trevigen, Inc.).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Single Cell Gel Electrophoresis

Journal:

Article Title: Inward rectifier potassium conductance regulates membrane potential of canine colonic smooth muscle

doi: 10.1111/j.1469-7793.1999.0247r.x

Figure Lengend Snippet: A representative gel of Q-PCR for Kir2.1 in the submucosal layer of canine colon is shown in A; 2-fold serial dilutions of mimic DNA were included in the PCR reactions while Kir2.1 cDNA remained constant. The concentration of Kir2.1 cRNA in different regions of the GI tract expressed relative to β-actin cDNA is illustrated in B. The amount of Kir2.1 cDNA in SCM, ICM and MyCM preparations is depicted in C. Results are expressed as means ±s.e.m.; * significant difference in the level of Kir2.1 transcript in ICM and MyCM compared with SCM preparations (P < 0.05).

Article Snippet: Quantitative PCR (Q-PCR) Q-PCR was performed by use of the PCR MIMIC Construction Kit (Clontech, CA, USA), which is based upon a competitive PCR approach: non-homologous engineered DNA standards (referred to as PCR MIMICs) compete with target DNA for the same gene-specific primers.

Techniques: Concentration Assay