Journal: Frontiers in Cellular Neuroscience

Article Title: Hypoxic culture of umbilical cord mesenchymal stem cell-derived sEVs prompts peripheral nerve injury repair

doi: 10.3389/fncel.2022.897224

Figure Lengend Snippet: Cellular localization and uptake of fluorescently labeled sEVs with SCs. (A) Cellular colocalization: SP8 was used to photograph sEVs prelabeled with PKH67 (green fluorescence) with Hoechst 33,342 (blue fluorescence)-stained SC nuclei. (B) Western blot: Detection of ERK1/2, ZEB2, and c-JUN expression levels in NC Schwann cells and after 48-h treatment with hypoxia sEVs. (C) Real-time PCR: Inflammatory and other restoration-related factors (IL-1β, IL-6, TNF-α, etc.) were detected. (D) ELISA test: GDNF NDF and NT-3 in the supernatant of Schwann cell preparation. Statistical significance, * p < 0.05,** p < 0.01, and **** p < 0.0001. *Significant difference.

Article Snippet: The following ELISA kits were: mouse glial cell line-derived neurotrophic factor (GDNF), ELISA kit (CSB-E07341m, CUSABIO), mouse neurotrophin 3 (NT-3), ELISA kit (CSB-E04687m, CUSABIO), and mouse nerve growth factor (NGF), ELISA kit (CSB-E04684m, CUSABIO).

Techniques: Labeling, Fluorescence, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: Schematic diagram of ultrasound-mediated CRISPR/Cas9 gene editing of Cdh2 to inhibit tumor invasion and metastasis.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: CRISPR

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: ( A ) Fluorescence images of Cas9- and EGFP-stably expressed 4T1 cells transfected with only pU6-sgRNA (EGFP)-mCherry (control), GPD, or GPD + US (GPD: the abbreviation of GVs-PEI-DNA). Scale bar = 200 μm; ( B ) determination of the transfection efficiency by flow cytometry through counting of the red-emitting cells; ( C ) quantitative analysis of the mCherry-expressed cells from ( B ) ( n = 3); ( D ) determination of the EGFP knockout efficiency ( n = 3). ns denotes p > 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: Fluorescence, Stable Transfection, Transfection, Flow Cytometry, Knock-Out

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: ( A ) Fluorescence images of Cas9- and EGFP-stably expressed 4T1 cells transfected with only pU6-sgRNA (Cdh2)-mCherry (control), GPD, or GPD + US (GPD: the abbreviation of GVs–PEI–DNA). Scale bar = 100 μm; ( B ) determination of the transfection efficiency by flow cytometry through the counting of the red-emitting cells in EGFP-expressed cells; ( C ) quantitative analysis of the mCherry-expressed cells from ( B ) ( n = 3) *** p < 0.001. ( D ) Western blotting assay of the expression of N-cadherin in the wild-type (WT) and two Cdh2-KO cell lines. GAPDH was used as an internal marker.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: Fluorescence, Stable Transfection, Transfection, Flow Cytometry, Western Blot, Expressing, Marker

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: CRISPR- CLONInG : Replacement of Luciferase (Luc) on FLEx vector. (A) Schematic illustration of FLEx vector with CRISPR cut sites (red scissors) at the two junction sites flanking the undesired Luc fragment. Gray dot dashes: default backbone containing origin of replication and selection for propagation in bacterial host. (B) Luc was cut out with ctRNP (Cas9-ctRNA) complex; FRT-Neo-FRT and tdTomato were PCR-amplified from existing plasmids using primers carrying complementary Gibson overhangs from the adjacent DNA fragment and vector backbone. (C) Two new vector inserts were joined with the CRISPR-digested backbone via Gibson (HiFi) Cloning for final donor assembly. (D) Excised FLEx vector backbone (∼7.5 kb) and Luciferase (∼1.65 kb) (left); PCR amplified FRT-Neo-FRT (∼1.87 kb) and tdTomato (∼1.43 kb) (right). N.S.: non-specific bands. (E) After CRISPR- CLONInG , 14 out of 20 clones verified with PstI RE(s) diagnosis showed correct vector assembly (6 DNA fragments; black arrow); 3 clones validated for sequence integrity. Resolved on 0.9% agarose gel.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Clone Assay, Luciferase, Plasmid Preparation, Selection, Amplification, Sequencing, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: CRISPR- CLONInG : Replacement of partial cargo sequence (Cre-comp) with the desired donor sequence on AAV vector (Addgene #60229). (A) Schematic illustration of AAV vector with CRISPR cut sites (red scissors) at two ends of the Cre-comp segment. Guides (AAV-A and AAV-B) with high on-target scores were selected. (B) Cre-comp was cut out with ctRNP (Cas9-ctRNA) complex; donor for gene replacement (containing 15 bp AA replacement sequence, noted as ‘R’, sandwiched by HA) flanked with complementary Gibson overhangs of the adjacent AAV backbone was PCR-amplified from custom gene synthesized plasmid. (C) Assembled AAV-v1: donor template cloned into the customized AAV backbone via Gibson (HiFi) Assembly. (D) Excised AAV vector backbone (∼3.63 kb) and Cre-comp (∼2.73 kb) (left); PCR amplified donor template (∼0.8 kb) with Gibson overhangs (right). (E) After CRISPR- CLONInG , 15 out of 16 clones showed correct vector assembly, confirmed by BbsI RE(s) diagnosis (two DNA fragments; black arrow); 3 clones further validated by Sanger sequencing. Resolved on 0.9% agarose gel.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Amplification, Synthesized, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: Cloning duo-guides into AAV-v1 vector by exploiting built-in cloning site (originally designed for one sgRNA). Gene editing (AA changes at PSEN1 gene) in N2A cell line. (A) (top) Schematic AAV-v1 construct: blue dotted line zooming out partial sequences of U6 and sgRNA cloning site. (middle) Type IIS RE (SapI) digest creates two unique 5’-overhangs (GGT vs. GTT). (bottom) Showing gene synthesized plasmid carrying duo-sgRNA cassette sequence (Spacer-W+tracrRNA+U6+spacer-X) with complementary overhangs, flanked by uniquely positioned SapI sites, which upon SapI digestion renders two complementary 5’-overhangs (ACC vs. AAC; red dotted line) for cloning. (B) Schematic of the final construct AAV-v2. (C) (top) CRISPR guides (W and X) target sites (purple scissors) on PSEN1 exon 10 region. (bottom) AA replacement donor (R) integrated at the genomic target after rAAV-v2 transduction. (D) PSEN1 exon 10 sequence shown (WT vs. R: 3/5 AA changes - three amino acid codons yellow highlighted). Red arrow: SpCas9 guides (W and X) cutting sites; chromatograms showing WT, mutant (R) and hemizygous (R+indel); green dotted rectangle encompassing the 3/5 AA changes within a 15-nt range.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: Clone Assay, Plasmid Preparation, Construct, Synthesized, Sequencing, CRISPR, Transduction, Mutagenesis

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: Validation of lssDNA acquired by CRISPR- CLIP . (A) Cpf1 & WT Cas9 digested the entire plasmid (∼4.9 kb, as shown in ) into ∼2.7 kb and ∼2.2 kb DNA fragments (lane b); acquired lssDNA (∼2.2 kbase) resolved around 1.2-1.3 kb in size $ (lane d, arrow). (B) Control: the uncut plasmid (lane a); control: as described in (A) (lane b); Cpf1 and WT Cas9, along with additional BamHI digested the plasmid into three fragments: BamHI cleaved the ∼2.2 kb dsDNA template into ∼1.2 kb and ∼1 kb, while ∼2.7 kb default plasmid remained intact (lane c); BamHI digestion did not cleave the lssDNA despite bearing a BamHI cut site (lane e, arrow) vs. lssDNA without BamHI digestion (lane d); all resolve in 0.9% agarose gel electrophoresis. (C) Multiple reverse primers (red bent arrow) were used for Sanger sequencing validation of the acquired lssDNA (sense DNA, in this case). $ The lssDNA may not resolve at the exact predicted size (1.1 kb in this case), depending on certain factors, such as buffer condition and potential secondary structure formation due to sequence composition, in which case additional DGLB treatment on acquired lssDNA could aid in size separation with more precise outcomes. The integrity of the procured lssDNA is considered of good quality as long as the majority of the lssDNA resolved close to the predicted size.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: CRISPR- CLIP with add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo-PAM. (A) The dsDNA template carrying lssDNA # cassette is flanked with duo PAM-A (upstream end) and duo-PAM-B (downstream end), anchored in the default plasmid. (B) Each duo-PAM tag contains 23-bp sequence with PAMs for Cpf1 and Cas9 placed at respective 5’ ends of each DNA strand, plus a constant Cpf1 spacer sequence. Duo-PAM-A and -B are assigned with two distinct Cpf1 spacer sequences to enable suitable Cas types to make exclusive incisions on the plasmid at both ends of lssDNA junction sites. Of note, Cas9 spacer sequence is variable and subject to lssDNA donor; Cpf1 and Cas9 incisions on the duo-PAM tags will each remove 4 nts (end sequences of HA) from the lssDNA donor, which merely results in trivial variation in HA length. (C) To procure the sense ssDNA (top strand) as the lssDNA donor, choose Cas9n to cut at duo PAM-A end while choosing Cpf1 to cut at duo PAM-B end. (D) To procure the antisense ssDNA (bottom strand) as the lssDNA donor, choose Cpf1 to cut at the duo PAM-A, while using Cas9n to cut at the duo-PAM-B. (E) Upon DGLB treatment, C and D respectively yielded 3 stand-alone ssDNA units of distinct sizes resolved in 0.9% agarose gel electrophoresis. # Belongs to a locus different from the case shown in and .

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis

Journal: Food Science & Nutrition

Article Title: A mixture of extracts from natural ingredients reduces the neurotoxic polarization of microglia via modulating NF‐κB / NF‐E2 ‐related factor 2 activation

doi: 10.1002/fsn3.4045

Figure Lengend Snippet: The mixture regulated the gene expression and NF‐κB/Nrf2 activation in BV2 microglia during exposure to Aβ and PgLPS. The mRNA expression of TNF‐α (a), IL‐1β (b) at 1 h after exposure to Aβ and PgLPS (AL) with or without pretreatment with propolis (P) or mixture (Mix). (c) Immunofluorescent CLMS images indicating the nuclear translocation of p65 (green) in BV2 cells with Hoechst‐stained nuclei (blue) after exposure to AL for 1 h. Scale bar, 10 μm. The mRNA expression of IL‐10 (d) and BDNF (e) at 1 h after exposure to AL with or without pretreatment with propolis (P) or mixture (Mix). (f) Immunofluorescent CLMS images indicating the nuclear translocation of Nrf2 (green) in BV2 cells with Hoechst‐stained nuclei (blue) after exposure to AL for 1 h. Scale bar, 10 μm. Each column and bar represents the mean ± SD ( n = 3, each). Asterisks indicate a statistically significant difference from the value in the control group (* p < .05, *** p < .001, one‐way ANOVA). Swords indicate a statistically significant difference from the value in the Aβ and PgLPS‐exposed group ( &&& p < .001, one‐way ANOVA). Hash marks indicate a statistically significant difference from the value in the propolis and mixture group ( ### p < .001, one‐way ANOVA).

Article Snippet: The c‐myc‐immortalized mouse microglial cell line MG6 (Cat: RCB2403) was purchased from RIKEN Cell Bank; BV2 cells (the immortalized mouse microglial cell line) were obtained from ACCEGEN (Cat: ABC‐TC212S).

Techniques: Gene Expression, Activation Assay, Expressing, Translocation Assay, Staining, Control