fusx assembly kit  (Addgene inc)

Journal: STAR Protocols

Article Title: An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

doi: 10.1016/j.xpro.2022.101288

Figure Lengend Snippet: Schematic diagram of FusXTBE pairs used in this protocol and their assembly (A) Golden Gate cloning of FusX library enables the rapid synthesis of FusXTBE clones. (B) Each FusXTBE arm consists of a DNA binding domain with either 15 or 16-RVDs targeting 15 or 16-nucleotides DNA binding sequence, which is preceded by a 5′ T nucleotide. Attached to this module is a split half of the DddA tox protein and an UGI molecule. The 14–18 bp protospacer region in between two arms are amenable for deaminase activity. Both the figures were created by using Biorender.com .

Article Snippet: FusX assembly kit , , Addgene kit Cat#1000000063.

Techniques: Cloning, Clone Assay, Binding Assay, Sequencing, Activity Assay

Journal: STAR Protocols

Article Title: An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

doi: 10.1016/j.xpro.2022.101288

Figure Lengend Snippet:

Article Snippet: FusX assembly kit , , Addgene kit Cat#1000000063.

Techniques: Recombinant, Plasmid Preparation, Gel Extraction, Purification, Sequencing, Amplification, Software, Membrane, Pore Size, Spectrophotometry, Microscopy

Journal: eLife

Article Title: Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish

doi: 10.7554/eLife.66393

Figure Lengend Snippet: ( A,B ) Representative images of whole-mount in situ hybridization (ISH) with probes against zebrafish plin2 ( ENSDARG00000042332 ) and zgc: 77,486 ( plin3/4/5 ) ( ENSDARG00000013711 ) at 6 days post fertilization (dpf) either unfed or following feeding with a high-fat meal for 90 min. ISH was performed three times for each gene with n = 10 larvae per probe per experiment; scale = 500 µm ( A ), scale = 100 µm ( B ). Plin2 is expressed in the intestine only following a high-fat meal whereas plin3 is expressed in the intestine in unfed fish and has stronger expression following a high-fat meal. ( C ) Overview of the location and strategy used for TALEN-mediated genome editing. EGFP was fused in-frame at the N-terminus of plin2 . TALEN targets in plin2 are located in exon 1 of the plin2-203 ENSDART00000175378.2 transcript and flank a FokI restriction site, loss of which was used to confirm cutting activity. A donor plasmid with the coding sequence for EGFP and plin2 homology arms was co-injected with TALEN mRNA into one-cell stage embryos to be used as a template for homology directed repair. mTag-RFP-t was fused in-frame at the C-terminus of plin3 . TALEN targets were located in exon 8 of the plin3 ENSDART00000100473.5 transcript and flank the termination codon and an HphI restriction site, loss of which was used to confirm cutting activity. A donor plasmid with the coding sequence for mTagRFP-t and plin3 homology arms was co-injected with TALEN mRNA into one-cell stage embryos to be used as a template for homology directed repair. ( D ) Following identification of fluorescent embryos in the F1 generation, RT-PCR and sequencing of genomic DNA using the primers noted on the knock-in loci depicted in ( C ) were used to confirm successful in-frame integration of the fluorescent tags. The size of the amplicons expected for correct integration were as follows: F1–R1 1033 bp, F2–R2 1340 bp, F3–R3 440 bp for wild-type (WT) and 1224 bp for Fus(EGFP-plin2 ) fusion, F4–R4 1218 bp, F5–R5 1274 bp, F6–R6 401 bp for WT and 1187 for Fus(plin3-RFP ). Arrows indicate the larger amplicon in heterozygous fish carrying the fusion alleles. ( E ) Imaging in live larvae (6 dpf) reveals expression of EGFP-Plin2 only in the intestine of larvae fed a high-fat meal (7 hr post-start of 2 hr meal) and Plin3-RFP is expressed in the intestine of both unfed and fed larvae (4.5 hr post-start of 2 hr meal, larvae are heterozygous for the fusion proteins; the lumen of the intestine has strong autofluorescence in WT and transgenic fish; see for images of whole fish). Scale = 500 µm. ( F ) Examples of larvae expressing both EGFP-Plin2 and Plin3-RFP (7 hr post start of meal). Scale = 500 µm. For ( E and F ), images are representative of at least 15 fish from three independent clutches. ( G ) EGFP-Plin2 (green) and Plin3-RFP (magenta) label the lipid droplet surface in the intestine of 6 dpf larvae fed with a high-fat meal containing either BODIPY 558/568-C12 (magenta) or BODIPY FL-C12 (green) to label the stored lipids. Note the 558/568-C12 is not fully incorporated into stored lipid and is also found diffuse in the cytoplasm. Scale = 10 µm. ( H ) EGFP-Plin2 and Plin3-RFP can decorate the same lipid droplets in the intestine. Arrows denote examples of dual-labeled droplets, scale = 10 µm.

Article Snippet: Recombinant DNA reagent , FusX TALEN assembly system , ; , Addgene kit #1000000063 , .

Techniques: In Situ Hybridization, Expressing, Activity Assay, Plasmid Preparation, Sequencing, Injection, Reverse Transcription Polymerase Chain Reaction, Knock-In, Amplification, Imaging, Transgenic Assay, Labeling

Journal: eLife

Article Title: Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish

doi: 10.7554/eLife.66393

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , FusX TALEN assembly system , ; , Addgene kit #1000000063 , .

Techniques: Sequencing, Recombinant, In Situ, Plasmid Preparation, Modification, TA Cloning, cDNA Synthesis, SYBR Green Assay, Gas Phase Electrophoretic Molecular Mobility Analysis, Software