Journal: Nature protocols

Article Title: Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

doi: 10.1038/s41596-021-00669-0

Figure Lengend Snippet: Troubleshooting table

Article Snippet: To identify an sgRNA target sequence using the GenScript sgRNA database ( https://www.genscript.com/gRNA-database.html ) or other webtools, input the gene name, symbol or ID and search for human SpCas9 gRNA sequences.

Techniques: Negative Control, Ligation, Transformation Assay, Plasmid Preparation, Selection, Passaging, Clone Assay, Concentration Assay, Sequencing

Journal: Scientific Reports

Article Title: Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

doi: 10.1038/srep27185

Figure Lengend Snippet: ( a ) After 45 days of embryo transfer, pregnancy was confirmed by ultrasonography. ( b ) The calf was delivered without assistant. ( c ) When ultraviolet light was exposed to nose of tg cattle, GFP expression was strongly observed. And the tg cattle grew up to 12 months old without any healthy issue ( d ). To determine GFP or RFP expression in a piece of tissue or primary skin cells via recombination, the tissue and cells were cultured and transfected with Dre recombinase mRNA by nucleofection (( e ) a piece of tissue from tg cattle-brightness, ( e` ) before Dre recombinase transfection (GFP), ( e`` ) after Dre recombinase transfection (RFP)). The primary skin cells from the tg cattle were isolated, cultured and transfected with Dre recombinase mRNA. Before transfection, only GFP expression was observed, RFP expression were observed via GFP gene excision by recombination (( f – f`` ) before transfection brightness, fluorescence, and merged, respectively; ( g – g`` ) after transfection brightness, fluorescence, and merged, respectively). The transgene integration and recombination were confirmed by genomic DNA PCR (( h ) 1: Molecular maker, 2: Wild type cattle, 3: Blood from tg cattle, 4: Positive control (DNAs), 5: Negative control) and RT-PCR (( i ) 1: Wild type cattle, 2: cDNA from tg cattle, 3: Negative control). After Dre recombinase transfection, GFP excision was confirmed by genomic DNA PCR (( j ) 1: Molecular marker, 2: Before transfection, 3: After transfection, 4: Negative control). Gel image was cropped and original image was seen in .

Article Snippet: As briefly, primary cells from a transgenic cattle (SNU-PB-2) were transfected with plasmid DNAs (Cas9 with CMV promoter, single guide RNA for GFP with U6 promoter (Toolgen, Seoul, Republic of Korea), donor DNAs for Knock-In; ) using Nucleofactor technology (Neon ® , Invitrogen; program #16).

Techniques: Expressing, Cell Culture, Transfection, Isolation, Fluorescence, Positive Control, Negative Control, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Scientific Reports

Article Title: Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

doi: 10.1038/srep27185

Figure Lengend Snippet: ( a ) After 45 days of embryo transfer, pregnancy was confirmed by ultrasonography. ( b ) The calf was delivered without any assistance and grew up to 2 months. Analyzing the calf without ultraviolet light, GFP expression was observed in the eyes ( c ) and nose ( d ). The tg cattle have been grown to 5 months old without any health issue ( e ). When ultraviolet light was exposed to the head, GFP expression was strongly observed ( f ). To know GFP in skin cells, the primary skin cells from the tg cattle were isolated and cultured. In over 99% of cells, GFP expression were observed (( g ) brightness; ( g` ) fluorescence). The transgene integration was confirmed by genomic DNA PCR (( h ) 1: Molecular maker, 2: Wild type cattle, 3: Blood from tg cattle, 4: Positive control (DNAs), 5: Negative control) and RT-PCR using primary cells (( i ) 1: cDNA from Wild type cattle, 2: cDNA from tg cattle, 3: Negative control). Gel image was cropped and original image was seen in .

Article Snippet: As briefly, primary cells from a transgenic cattle (SNU-PB-2) were transfected with plasmid DNAs (Cas9 with CMV promoter, single guide RNA for GFP with U6 promoter (Toolgen, Seoul, Republic of Korea), donor DNAs for Knock-In; ) using Nucleofactor technology (Neon ® , Invitrogen; program #16).

Techniques: Expressing, Isolation, Cell Culture, Fluorescence, Positive Control, Negative Control, Reverse Transcription Polymerase Chain Reaction