CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (<i>SNAI1</i>) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells

<div><p>Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell–cell adhesion, decreased cell–substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell–substrate adhesion, and increases cell–cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.</p></div

Loss of Snail1 failed to impede TGF-beta induced alterations of morphology and gene expression (A) Representative images of cell morphology of wild-type (WT) RMG (I, II) and Snail1 KO1 (III, IV) cells, after incubation for 72 h in the absence (I, III) or presence (II, IV) of TGF-beta (10 ng/mL).

<p>Scale bar indicates 100 μm. Photos were taken of 5–10 images per sample. Experiments were repeated three times. (B) Quantitative RT-PCR assessment of the cells shown in (A) for the indicated genes. Values are expressed as the mean ± s.e.m. of triplicate samples. Experiments were repeated twice. Statistical significance is indicated by an asterisk (*<i>P</i> < 0.05 and ** <i>P</i> < 0.01 using Students’ <i>t</i>-test). (C) Western blot analysis of the cells shown in (A) for the indicated proteins. Vinculin was used as a loading control. Experiments were repeated twice.</p

Snail1 loss altered cytoskeletal organization and gene expressions in RMG1 cells.

<p>(A) Representative images displaying the cell morphology of wild-type (WT) RMG cells (I), Snail1 KO1 cells (II). Cells are visualized by phase-contrast microscopy. Scale bar indicates 50 μm. WT cells exhibited cobblestone-like morphologies, whereas most of the Snail1 KO1 cells exhibit a more rounded morphology. Representative images of the actin cytoskeletons of WT cells (III) Snail1 KO1 cells (IV) stained with rhodamine X-conjugated phalloidine and visualized using a confocal laser scanning microscope (Zeiss LSM700). Photos of 5 to 10 images per sample were taken. Experiments were repeated 5 times. WT cells are rich in stress fibers, whereas Snail1 KO1 cells have scarce stress fibers, but cortical actin on their cell surfaces. Scale bar indicates 20 μm. (B) Quantitative RT-PCR of indicated genes in wild-type (WT) RMG cells, Snail1 KO1 cells, and Snail1 KO1 cells transiently transfected with a Snail1 expression vector (Snail1 KO1 + Snail1 cells). Values are expressed as the mean ± s.e.m of triplicate samples. Experiments were repeated three times. Statistical significance is indicated by an asterisk (*<i>P</i> < 0.05 and ** <i>P</i> < 0.01 using Students’<i>t</i>-test). Snail1 KO1 cells showed a complete loss of Snail expression and increased expression of <i>E-cadherin</i>, <i>claudin-1</i>, <i>occludin</i>, <i>beta-actin</i>, and <i>alpha-tubulin</i>, as well as decreased expression of <i>integrin alpha 5</i>. Transient expression of Snail1 in Snail1 KO cells reversed these effects, except for the increase in <i>occludin</i> expression. (C) Western blot analysis of indicated proteins in wild-type (WT) RMG cells, Snail1 KO1 cells, and Snail1 KO1 cells transiently transfected with a Snail1 expression vector (Snail1 KO1 + Snail1 cells). Snail1 KO1 cells showed a complete loss of Snail1 expression and an increase in E-cadherin, occludin and claudin-1, beta-actin, alpha- tubulin, as well as a decreased in integrin alpha 5 expression. Transient expression of Snail1 in Snail1 KO cells reversed these effects, except for the decreased in integrin alpha 5 expression. Vinculin was used as a loading control. Experiments were repeated three times.</p

Loss of Snail1 reduced cell–substrate adhesion, cell scattering, cell dissociation, and cell migration in RMG1 cells.

<p>(A) Representative images of substrate adhesion by wild-type (WT) RMG1 cells (I), Snail1 KO1 cells (II) and Snail1 KO cells transiently transfected with a Snail1 expression vector (Snail1 KO1 + Snail1 cells) (III) 4h after seeding in non-coated wells. (B) Determination of the ratios of attached cells represented by the number of attaching cells to non-coated wells (black column) or on wells precoated with DMEM containing FCS (gray column) divided by the number attaching to wells precoated with poly-lysine. Values are expressed as the mean ± s.e.m of triplicate samples. Experiments were repeated three times. (C) Representative images of scattering cells from WT (I), Snail1 KO1 (II) and Snail1 KO1 + Snail1 (III) cells 72 h after seeding. (D) Determination of the relative number of scattering cells represented by the number of isolated cells from the colonies of each type of cells divided by the number of isolated cells from the colonies of WT cells. Values are expressed as the mean ± s.e.m of five images per sample. Experiments were repeated four times. (E) Representative images of cell dissociation among WT cells (I), Snail1 KO1 cells (II) and Snail1 KO1 + Snail1 (III) cells. (F) Determination of the relative number of fragmented particles, presented as the number of fragmented particles of each type of cells divided by the number of fragmented particles of WT cells. Values are expressed as the mean ± s.e.m of 5–10 images per sample. Experiments were repeated twice. (G) Representative images of WT cells (I), Snail1 KO1 cells (II) and Snail1 KO + Snail1 (III) cells that migrated to the lower surfaces of transwell membranes. (H) Determination of the relative number of migrated cells, presented as the number of migrated cells of each type of cells divided by the number of migrated WT cells. Values are expressed as the mean ± s.e.m. of five images per sample. Experiments were repeated three times. Statistical significance is indicated with an asterisk (*<i>P</i> < 0.05 and ** <i>P</i> < 0.01 using Students’ <i>t</i>-test).</p

Primers for qRT-PCR.

<p>Primers for qRT-PCR.</p

Loss of snail1 impeded TGF-beta induced cell migration and cell dissociation.

<p>(A) Representative images of wild-type (WT) RMG (I, II) and Snail1 KO1 (III, IV) cells that migrated to the lower surface of the membranes of transwell plates. Before seeding into the transwell plates, cells were incubated for 72 h in the absence (I, III) or presence (II, IV) of TGF-beta (10 ng / mL). Photos were taken of 10 images per sample. Experiments were repeated twice. (B) Determination of the relative number of migrated cells, presented as the number of migrated cells shown in (A) divided by the number of migrated WT cells incubated in the absence of TGF-beta. Values are expressed as the mean ± s.e.m. of ten images per sample. Statistical significance is indicated with an asterisk (*<i>P</i> < 0.05 and ** <i>P</i> < 0.01 using Students’ <i>t</i>-test). (C) Representative images of cell dissociation among wild-type (WT) RMG (I, II) and Snail1 KO1 (III, IV) cells. Before seeding, cells were incubated for 72 h in the absence (I, III) or presence (II, IV) of TGF-beta (10 ng/mL). Photos were taken of whole well (I, III) and of 10 images per sample (II, IV). Scale bar indicates 50 μm. Experiments were repeated twice. (D) Determination of the relative number of fragmented particles, presented as the number of fragmented particles of each type of cells shown in (C) divided by the number of fragmented particles of WT cells incubated in the absence of TGF-beta. Values are expressed as the mean ± s.e.m of ten images per sample. Experiments were repeated twice.</p

Generation of Snail1 KO cells using the CRISPR/Cas9n double nicking system.

<p>(A) Schematic illustration of Snail1 gene structure and sequences around the target loci. Yellow boxes indicate exons encoding the Snail1 protein. Blue boxes indicate non-coding exons. The gRNA target sequences and PAM domains are indicated by black and red underlining, respectively. Arrows indicate the locations of PCR primers. (B) The genomic sequences around the target sites of wild type (WT) and Snail1 KO1 cells. (C) Waveform data from a DNA sequencer displaying the sequence obtained from PCR fragments of genomic DNA.</p

Theoretical Prediction of the Reaction Probabilities of H, O, and OH Radicals on the Polypropylene Surface

To determine the H-abstraction reaction probabilities of H/O/OH radicals with a polypropylene (PP) surface, a first-principles calculation was performed based on the DLPNO–CCSD(T)/CBS//M06-2X-D3/def-TZVP theory level. The PP chain model used in this study was 2,4,6-trimethylheptane. The rate constants of the H/O/OH radicals with the isolated PP chain model were calculated based on the conventional transition-state theory. By comparing the experimental values and considering the error factors and their compensation, it was concluded that the orders of magnitude of the predicted rate constants were accurate. The resulting rate constants were converted to reaction probabilities between the H/O/OH radicals and the PP surface. The method used in this study is applicable for obtaining theoretical values of surface reaction probabilities based on first-principles calculations. The calculation at the DLPNO–CCSD(T)/CBS theory level has high accuracy but consumes a large amount of computational resources. The study also demonstrated that the double-hybrid functionals, wB97x-2-D3(BJ) and rev-DSD-PBEP86-D3(BJ), with a 3-ζ or 4-ζ basis set, could reproduce the electronic energy values obtained from DLPNO–CCSD(T)/CBS while using only approximately 1/100 of the computational resources required by the latter under our computer configuration

Computational Study of Excess Electron Mobility in High-Pressure Liquid Benzene

In recent years, excess electron transfer in organic liquids has attracted increasing interest owing to the emerging class of liquid organic semiconductors. In this study, to achieve a comprehensive understanding of electron conduction in liquids, we investigate hopping electron conduction in liquids from an atomistic viewpoint. High-pressure liquid benzene is chosen as a simple model system. Hopping electron mobility is computed using a combination of molecular dynamics simulations, quantum chemical calculations, and kinetic Monte Carlo methods. The computed electron mobility is in good agreement with the experimental values. Because the amplitude of the intermolecular vibration observed in liquids is larger compared to that in solids, the effect of dynamic disorder on electron mobility is investigated. The time scale of the change in electronic couplings due to the rotation of molecules is comparable to that of the electron residence time at each benzene molecule at the absence of change in the arrangement of a benzene dimer. Thus, the effect of dynamic disorder is evaluated by a Monte Carlo-based analysis. It is shown that the effect of dynamic disorder on electron mobility is small even though electronic couplings between molecules fluctuate by more than an order of magnitude

Additional file 3: of Successful production of genome-edited rats by the rGONAD method

Table S1. Tyr-mediated mutations in F1 (DA male/WKY female) rat. Table S2. Tyr-mediated mutations in F1 (WKY male/DA female) rat. Table S3. Tyr-mediated mutations in F1 offspring. Table S4. Coat-color phenotypes recovered from albino in WKY rat. Table S5. CRISPR/Cas9 target sequence and ssODN used. (PDF 46 kb