Journal: bioRxiv

Article Title: Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

doi: 10.1101/2023.11.08.566127

Figure Lengend Snippet: (A) Schematic illustration and workflow of the Cas9-driven HITI-treatment gene construction. Each donor cassette containing one of the three gRNA sequences (pink) selected from the 5′-UTR sequence of the mouse Rho locus at both ends was prepared. The targeted allele represents the gene structure when the HITI-mediated gene insertion occurs. (B) SSA assay for in vitro evaluation of the cleavage efficiency of the three gRNA sequences targeting Rho. The illustration indicates the SSA-mediated reconstruction of pCAG-EGxxFP fragments by Cas9 cleavage, and the photographs represent HEK293T cells 48 h after transfection with pCAG-EGxxFP, pCAG-mCherry, pCAG-SpCas9, and the three gRNA plasmids. Numbers represent EGFP:mCherry intensity ratios measured by ImageJ. Scale bar: 500 μm. (C) Schematic illustration of HITI-mediated donor insertion by in vivo electroporation into the mouse retina. The construction of each plasmid with a vector backbone is shown. The plasmid cocktail was injected into the subretinal space of postnatal day 0 (P0) mouse retinas. (D) Immunohistochemistry sections of P21 mouse retinas electroporated in vivo at P0 with three plasmid cocktails targeting different mRho-gRNA1 (left), mRho-gRNA2 (middle), and mRho-gRNA3 (right) sequences. Electroporated cells are mCherry positive, and most cells in the outer nuclear layer (ONL) express AcGFP in retinal sections targeting mRho-gRNA1 and mRho-gRNA3 sequences. INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 μm. (E) High-power image of section immunohistochemistry with anti-rhodopsin antibody (white) of the square area shown in . Arrowheads indicate AcGFP-expressing cells. (F) Fraction (Frac.) of AcGFP-positive cells in mCherry-positive ONL cells from three different retinal tissue sections. Numbers, the mean ± SD (n = 6); N.D., not detected. (G) P21 mouse eyecups electroporated in vivo with plasmid cocktails targeting the gRNA sequence, in which the bRho2k promoter was shortened (bRho300bp) to express Cas9 (middle), and the vector backbone for the donor cassette was switched to pAAV (right). The dashed circles represent each dissected eyecup. Scale bar, 1 mm.

Article Snippet: Primary antibodies used in this study were directed against GFP (rat, 1:1000, Nacalai Tesque, Inc., 04404-84), mCherry (rabbit, 1:1000, MBL, PM-005), and rhodopsin (mouse, 1:1000, Merck, MAB5316).

Techniques: Sequencing, SSA Assay, In Vitro, Transfection, In Vivo, Electroporation, Plasmid Preparation, Injection, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

doi: 10.1101/2023.11.08.566127

Figure Lengend Snippet: (A) Schematic illustration of HITI-treatment gene construction for cell sorting. Nuclear localization signals (NLS) were added to the fluorescent markers in the donor and reporter plasmids. (B) Schematic illustration of single-cell sorting and PCR genotyping. The fluorescent region of P56-electroporated retinas was trimmed and dissociated, and 48 cells were sorted using FACS from the GFP- and mCherry-double-positive fractions. WT and KI alleles were amplified from the first PCR, and each allele was amplified using nested PCR. (C) Electrophoresis gel images of the nested PCR of WT and KI alleles of sample numbers 13–17 of the 48 samples tested. (D) Direct sequencing of the reamplified WT (upper) and KI (lower) PCR products. The sequences around the mRho-gRNA1 target (purple shading) are shown. (E) Schematic illustration of the de novo validation of the donor vector insertion. (F) P56-electroporated Rho +/AcGFP retinal sections. AcGFP-positive and -negative cells were observed in HITI-treated mCherry-positive photoreceptor cells. Scale bar: 50 μm. (G) Fraction (Frac.) of mCherry- and/or AcGFP-positive cells counted in retinal sections. Data represents means□±□SD (n = 4).

Article Snippet: Primary antibodies used in this study were directed against GFP (rat, 1:1000, Nacalai Tesque, Inc., 04404-84), mCherry (rabbit, 1:1000, MBL, PM-005), and rhodopsin (mouse, 1:1000, Merck, MAB5316).

Techniques: FACS, Amplification, Nested PCR, Electrophoresis, Sequencing, Plasmid Preparation

Journal: BioTech

Article Title: Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles

doi: 10.3390/biotech11040049

Figure Lengend Snippet: Expression conditions for the photo-switchable proteins.

Article Snippet: The grids were then incubated overnight with the polyclonal antibody against PVX as described above or a monoclonal antibody against mCherry (GeneTex, Irvine, CA, USA, diluted 1:50 in PBS) and subsequently with the gold-labeled secondary antibody for 2 h. The grids were contrasted with 1% ( w / v ) uranyl acetate or 1% ( w / v ) phosphotungstic acid.

Techniques: Expressing

Journal: BioTech

Article Title: Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles

doi: 10.3390/biotech11040049

Figure Lengend Snippet: Buffers for lysis, washing and elution during the purification of each protein component. The LOV2 and Zdk1 proteins were purified as previously described ([ 47 ]). For the Dronpa145N proteins, all cell disruption and purification steps were carried out in the dark or under red light (630 nm) as previously described (Zhou et al., 2012).

Article Snippet: The grids were then incubated overnight with the polyclonal antibody against PVX as described above or a monoclonal antibody against mCherry (GeneTex, Irvine, CA, USA, diluted 1:50 in PBS) and subsequently with the gold-labeled secondary antibody for 2 h. The grids were contrasted with 1% ( w / v ) uranyl acetate or 1% ( w / v ) phosphotungstic acid.

Techniques: Lysis, Purification, Disruption

Journal: BioTech

Article Title: Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles

doi: 10.3390/biotech11040049

Figure Lengend Snippet: Coupling of purified optogenetic proteins to purified PVX-ST particles. ( A ) Conjugation of LOV2/QPAS1 to the surface of PVX particles using the ST-SC system confirmed by SDS-PAGE and Western blot. In addition to the bands for the individual GST-SC-LOV2 (57.1 kDa), GST-SC-QPAS1 (57.4 kDa) and ST-CP (27.4 kDa) proteins, a coupling band of ~85 kDa is present in Western blots probed with anti-GST and anti-PVX antibodies. ( B ) Conjugation of His 6 -SC-Dronpa145N to the surface of PVX particles using the ST-SC system confirmed by SDS-PAGE and Western blot. The anti-His 6 antibody detected the monomeric Dronpa145N protein (42.4 kDa) and a larger protein representing the coupling reaction (70 kDa) which was also detected by the anti-PVX antibody. M = P7719 Color Prestained Protein Standard. Purified GST (28 kDa), PVX-ST (27 kDa) and mCherry-His 6 (27 kDa) were loaded as controls. We mixed 3 µg of PVX-ST with a two-fold molar excess of the SC-fusion protein before loading. The arrows indicate the assembled complexes: PVX-ST/GST-SC-LOV2 = 85.1 kDa, PVX-ST/GST-SC-QPAS1 = 85.4 kDa, PVX-ST/His 6 -SC-G4S-Dronpa145N = 69.2 kDa.

Article Snippet: The grids were then incubated overnight with the polyclonal antibody against PVX as described above or a monoclonal antibody against mCherry (GeneTex, Irvine, CA, USA, diluted 1:50 in PBS) and subsequently with the gold-labeled secondary antibody for 2 h. The grids were contrasted with 1% ( w / v ) uranyl acetate or 1% ( w / v ) phosphotungstic acid.

Techniques: Purification, Conjugation Assay, SDS Page, Western Blot

Journal: BioTech

Article Title: Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles

doi: 10.3390/biotech11040049

Figure Lengend Snippet: Analysis of the modified LOVTRAP system. ( A ) Interaction between the purified LOV2 and Zdk1 proteins analyzed by discontinuous native PAGE. In the dark, the bands representing the individual proteins GST-SC-LOV2 and His 6 -Zdk1-mCherry are joined by a higher-molecular-weight band (indicated by an arrow) representing the complex, which is not present under blue light. M = NativeMark standards. ( B ) Transmission electron micrograph showing the LOVTRAP system attached to PVX particles in the dark. Purified PVX-ST/GST-SC-LOV2 particles were mixed with His 6 -Zdk1-mCherry and incubated for 30 min in the dark. Particles were attached to the grids and His 6 -Zdk1-mCherry was detected with an anti-His 6 /GAM-12 nm gold conjugate. Immunogold decoration was observed only for the mixture of PVX-ST/GST-SC-LOV2 + His 6 -Zdk1-mCherry in the dark. ( C ) Transmission electron micrograph showing purified PVX-ST/GST-SC-LOV2 particles mixed with His 6 -Zdk1-mCherry. The mixture was incubated for 30 min in the dark. Particles were attached to the grids under blue light (455 nm) and His 6 -Zdk1-mCherry was detected with an anti-His 6 /GAM-12 nm gold conjugate. No immunogold decoration was observed on the particles. Scale bar = 100 nm ( B ), 250 nm ( C ). Controls are shown in .

Article Snippet: The grids were then incubated overnight with the polyclonal antibody against PVX as described above or a monoclonal antibody against mCherry (GeneTex, Irvine, CA, USA, diluted 1:50 in PBS) and subsequently with the gold-labeled secondary antibody for 2 h. The grids were contrasted with 1% ( w / v ) uranyl acetate or 1% ( w / v ) phosphotungstic acid.

Techniques: Modification, Purification, Clear Native PAGE, Molecular Weight, Transmission Assay, Incubation

Journal: BioTech

Article Title: Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles

doi: 10.3390/biotech11040049

Figure Lengend Snippet: Analysis of the modified BphP1/QPAS1 system. ( A ) Interaction between the purified BphP1 and QPAS1 proteins analyzed by discontinuous native PAGE. Under far-red light, the bands representing the individual proteins BphP1-mCherry-His 6 and GST-SC-QPAS1 are joined by a higher-molecular-weight band (indicated by an arrow) representing the complex, which is not present under red light. M = NativeMark standards. ( B ) Transmission electron micrograph showing the BphP1/QPAS1 system attached to PVX particles. Purified PVX-ST/GST-SC-QPAS1 particles were mixed with BphP1-mCherry-His 6 and incubated for 30 min under far-red light (780 nm). Particles were attached to the grids and BphP1-mCherry-His 6 was detected with an anti-His 6 /GAM-12 nm gold conjugate. Immunogold decoration was observed only for the mixture of PVX-ST/GST-SC-QPAS1 + BphP1-mCherry-His 6 under far-red light (780 nm). ( C ) Transmission electron micrograph showing purified PVX-ST/GST-SC-QPAS1 particles mixed with BphP1-mCherry-His 6 . The mixture was incubated for 30 min in the dark. Particles were attached to the grids under red light or in the dark and BphP1-mCherry-His 6 was detected with an anti-His 6 /GAM-12 nm gold conjugate. No immunogold decoration was observed for the particles. Scale bar = 250 nm. Controls are shown in .

Article Snippet: The grids were then incubated overnight with the polyclonal antibody against PVX as described above or a monoclonal antibody against mCherry (GeneTex, Irvine, CA, USA, diluted 1:50 in PBS) and subsequently with the gold-labeled secondary antibody for 2 h. The grids were contrasted with 1% ( w / v ) uranyl acetate or 1% ( w / v ) phosphotungstic acid.

Techniques: Modification, Purification, Clear Native PAGE, Molecular Weight, Transmission Assay, Incubation

Journal: BioTech

Article Title: Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles

doi: 10.3390/biotech11040049

Figure Lengend Snippet: Analysis of the Dronpa145N system. ( A ) Analysis of the light-dependent association/dissociation behavior of Dronpa145N by discontinuous native PAGE. The monomeric state is favored under cyan light (505 nm) leading to the loss of signal intensity for the tetrameric form, but the latter is restored by exposure to weak violet light (405 nm) resulting in an increase in signal intensity. M = P7719 Color Prestained Protein Standards. ( B ) Transmission electron micrograph showing Dronpa145N attached to PVX particles. Purified PVX-ST/His 6 -SC-G4S-Dronpa145N particles were irradiated with cyan light (505 nm) before mixing with His 6 -mCh-G4S-Dronpa145N. The mixture was irradiated with violet light (405 nm) and particles were attached to grids. His 6 -mCh-G4S-Dronpa145N was detected with an anti-mCherry/GAM-12 nm gold conjugate. Immunogold decoration was observed for the mixture irradiated successively with cyan and violet light. Scale bar = 500 nm. Controls are shown in .

Article Snippet: The grids were then incubated overnight with the polyclonal antibody against PVX as described above or a monoclonal antibody against mCherry (GeneTex, Irvine, CA, USA, diluted 1:50 in PBS) and subsequently with the gold-labeled secondary antibody for 2 h. The grids were contrasted with 1% ( w / v ) uranyl acetate or 1% ( w / v ) phosphotungstic acid.

Techniques: Clear Native PAGE, Transmission Assay, Purification, Irradiation