Journal: bioRxiv

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1101/2024.05.24.595612

Figure Lengend Snippet: a, Schematic of the fluorescent reporter construct for transcriptional activation; pInducer20-eGFP. An eGFP open reading frame is placed after a Tet Responsive Element (TRE) sequence. Transcription of eGFP can either be facilitated by activation of the co-expressed reverse tet-transactivator rTA3 by addition of doxycycline (1), or by introduction of transcriptional activator dCas9-VPR with a sgRNA targeting the TRE sequence (2). b,c Fluorescence microscopy images (b) and flow cytometry analysis (c) of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 hours after addition of doxycycline (0.5 µg/ml), or transfection with plasmids encoding for dCas9-VPR with non-targeting (NT) sgRNAs, targeting (T) sgRNAs, or targeting MS2-sgRNAs. Doxycyline and dCas9-VPR with targeting sgRNAs increase eGFP expression. MFI: mean fluorescence intensity. Scalebar represents 200 μm. Means + SD, n = 3, One-way ANOVA with post-hoc Dunnett’s multiple comparison test. d, Flow cytometry analysis of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 hours after addition of EVs from HEK293T expressing dCas9-VPR alongside either MCP-PhoCl-CD63 or MCP-PhoCl-CD9, in combination with non-targeting- (NT), or targeting (T) sgRNAs. Both loading constructs facilitate EV-mediated functional dCas9- VPR delivery, resulting in a significant increase in eGFP mean fluorescence intensity (MFI). 4.0x10 11 EVs per well. Means + SD, n = 5, One-way ANOVA with post-hoc Dunnett’s multiple comparison test. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the generation of a lentiviral doxycycline-inducible eGFP expression construct, an eGFP open reading frame was transferred from pDONR221- eGFP (Addgene #25899) into pInducer20 (Addgene #44012) using the Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Construct, Activation Assay, Sequencing, Fluorescence, Microscopy, Flow Cytometry, Expressing, Transfection, Comparison, Functional Assay

Journal:

Article Title: Sequence Elements in both the Intergenic Space and the 3? Untranslated Region of the Crithidia fasciculata KAP3 Gene Are Required for Cell Cycle Regulation of KAP3 mRNA

doi: 10.1128/EC.2.4.671-677.2003

Figure Lengend Snippet: Physical map of the KAP3-GFP expression plasmid pMA1. Full-length EGFP was fused in frame near the 3′ end of the KAP3 coding sequence as a tag to distinguish the chimeric transcript from that of the KAP3 gene. The vector contains the HYG selectable marker flanked by the L. major 5′ and 3′ flanking regions of the DHFR-TS gene for expression of the HYG drug resistance marker. The 5′ and 3′ intergenic sequences flanking the C. fasciculata KAP3 gene are indicated. The locations of the KAP3 splice acceptor site and octameric sequences are also indicated. Mutated sequences are given in parentheses.

Article Snippet: To generate plasmid pMA1, expressing KAP3-GFP transcripts, the full coding region of the enhanced GFP (EGFP) gene was PCR amplified from plasmid pEGFP-1 (BD Clontech, Palo Alto, Calif.) with oligonucleotides G10 (5′-GCA GCCAAGACGGC CATGGTGAGCAAGGGCGAG-3′) and G11 (5′-GAAG GCCGTCTTGGCGATATC CTTGTACAGCTCGTCCATG-3′) (underlining indicates added Bgl I and Eco RV sites), digested with Bgl I, and ligated into Bgl I-digested pJCH2.

Techniques: Expressing, Plasmid Preparation, Sequencing, Marker

Journal: Journal of Virology

Article Title: Neuroattenuation of Vesicular Stomatitis Virus through Picornaviral Internal Ribosome Entry Sites

doi: 10.1128/JVI.02984-12

Figure Lengend Snippet: Construction and recovery of pVSVIRES. (A) The circular map shows the structure of plasmid pVSV-MC11-eGFP with artificially inserted unique restriction sites. (B) A schematic representation of VSV genome constructs with IRES elements. The short hairpin is shown before the IRES at the 5′ end. M* indicates a matrix gene mutation (M51 deletion). (C) VSV genome constructs with FMDV and HRV IRES elements. The gene junction is shown in the middle. The pVSVFMDV and pVSVHRV plasmids were made by insertion of FMDV IRES and HRV IRES elements, respectively, before the start codon of the M gene (boxed). The hairpin is also shown (Hp). Capital bold letters are VSV P and M stop and start codons, respectively. The SmaI restriction site is shown in italics and underlined. The nucleotides (40 nt) between P and M ORF are shown as dotted lines (P/M UTR), and the last five nucleotides of the P/M gene junction are also shown (TGTTA). (D) The circular map shows the structure of plasmid pCI-eGFP, in which the eGFP ORF was inserted between NheI and NotI restriction sites. It was driven by the CMV promoter. A short hairpin (Hp) was inserted before the eGFP start codon. (E) Fluorescence microscopic images of BHK cells transfected with equal amounts of pCI-eGFP plasmid with or without hairpin.

Article Snippet: The enhanced green fluorescent protein (eGFP) open reading frame (ORF) was amplified from plasmid pIRES2-EGFP (Promega Corp., Madison, WI).

Techniques: Plasmid Preparation, Construct, Mutagenesis, Fluorescence, Transfection