Journal: BMC Biotechnology

Article Title: Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research

doi: 10.1186/s12896-018-0491-5

Figure Lengend Snippet: Sequencing analysis of CRISPR/Cas9-mediated TP53 knockout (KO) canine fatal fibroblasts. a Nucleotide sequences of targeted TP53 genomic loci of cells treated with TP53 gRNA #30 (colonies #2, #10, and #11) and b TP53 gRNA #39 (colonies #3, #5, and #6)

Article Snippet: CRISPR/Cas9 vectors targeting TP53 were transfected transiently using Polyexpress TM (Excellgene, Rockville, MD, USA) into K9 fetus 1 cells at passage 1 according to the manufacturer’s instructions.

Techniques: Sequencing, CRISPR, Knock-Out

Journal: BMC Biotechnology

Article Title: Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research

doi: 10.1186/s12896-018-0491-5

Figure Lengend Snippet: Cellular characteristics of canine fibroblasts immortalized by CRISPR/Cas9-mediated TP53 knockout (KO). a No enhanced green fluorescent protein (EGFP) expression was detected by fluorescence activated cell sorting (FACS) analysis in both TP53KO#30 and TP53KO#39 cell lines. b Relative proliferation rates of each cell lines (× 15,000 cells). c Cumulative growth curve data of the control (early passage 2) and two TP53 KO cells were obtained after additional 14 consecutive passages in culture. d Representative images showing the cellular morphologies of the control and two TP53 KO cells at the end time-point (passage 14) of cumulative growth counting analysis. e Representative images of the control and two TP53 KO cells stained with senescence associated β-galactosidase (SA-β-gal) at the end time-point of cumulative growth counting analysis. f Percentage of SA-β-gal-positive cells in each group. g Immunoblot data showing TP53, p21, and SV40LT protein levels in the control, SV40LT-transduced, TP53KO#30, and TP53KO#39 cells grown in the absence or presence of 100 μM TMZ for 48 h. α-Tubulin was used as the loading control. **, p < 0.01

Article Snippet: CRISPR/Cas9 vectors targeting TP53 were transfected transiently using Polyexpress TM (Excellgene, Rockville, MD, USA) into K9 fetus 1 cells at passage 1 according to the manufacturer’s instructions.

Techniques: CRISPR, Knock-Out, Expressing, Fluorescence, FACS, Control, Staining, Western Blot

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: The vector pBSE401 used for CRISPR/Cas9-mediated genome editing. AtU6, Arabidopsis U6 promotor; gRNA, guide RNA; 35S promotor, CaMV 35S promotor; Cas9, codon-optimized Cas9; NLS, nuclear location signal; bar , selective marker gene; KanR, Kanamycin resistance gene; pVS1-RepA, pVS1 replication origin; pVS1-StaA, pVS1 stability function.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: Plasmid Preparation, CRISPR, Marker

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: Identifying transplants in T 0 generation. (A) Detection of the selectable marker gene bar by PAT/Bar test strip. The red arrowhead indicates that bar gene is positive. (B) Gel image of PCR products for T-DNA regions. Cas9, part of the Cas9 coding sequence. sgRNA, region from the U6 promoter to the downstream vector sequence spanning the sgRNA. GmActin was used as a normalization control. V: plasmid of the vector in transformation. WT, wild type soybean plants. Labels 1-16, individual mutant lines.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: Marker, Stripping Membranes, Sequencing, Plasmid Preparation, Transformation Assay, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: CRISPR/Cas9-mediated targeted mutants of E1 in the T 1 generation.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: CRISPR, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: CRISPR/Cas9-induced E1 mutants flowering time under both LD and SD conditions. (A) Phenotypes of wild type (WT, Jack) and homozygous T 2 mutant under LD condition, respectively. Top panel, WT did not have floral buds when T 2 mutant was flowering. Bottom panel, T 2 mutant produced the pods when WT was flowering. Red box, magnified view. (B) Flowering time of WT and homozygous T 2 mutants under LD condition. (C) Phenotypes of wild type (WT, Jack) and homozygous T 2 mutant under SD condition, respectively. (D) Flowering time of WT and homozygous T 2 mutants under SD condition. n, exact numbers of individual plants identified. **, homozygous T 2 mutants exhibit significant early flowering time (P < 0.01). The flowering time is shown as the mean values ± standard deviation.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: CRISPR, Mutagenesis, Produced, Standard Deviation

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: Identifying of trans-clean mutants. (A) Detection of the selectable marker gene bar by PAT/Bar test strip. The red arrowhead indicates that bar gene is positive. (B) Gel image of PCR products for T-DNA elements. Cas9, part of the Cas9 coding sequence. sgRNA, region from the U6 promoter to the downstream vector sequence spanning the sgRNA. GmActin was used as a normalization control. V: plasmid of the vector in transformation. WT, wild type. Labels 1-20, individual mutant lines.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: Marker, Stripping Membranes, Sequencing, Plasmid Preparation, Transformation Assay, Mutagenesis