Journal: Frontiers in Genome Editing

Article Title: T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

doi: 10.3389/fgeed.2023.1130736

Figure Lengend Snippet: Obligate-heterodimerization mitigates off-target activity of CCR5 -TALEN (A) Schematic of possible TALEN pairing combinations. Combinations are displayed for TALENs with wild-type (WT) and obligate-heterodimeric (OH) FokI domains. (B) Genotyping. Results of T7E1 assay of WT-TALEN and OH-TALEN (KKR-ELD and KVR-EAD configuration) are shown. 32°C indicates that T cells were subjected to transient cold-shock after electroporation, while control cells were constantly cultured at 37°C. * and ** specify p -values of <0.05 or <0.01 (Student’s t-test, n = 3–5). (C) TALEN expression. Percentage of TALEN-expressing T cells upon mRNA transfer as determined by flow cytometry (n = 1). (D,E) Structural variations. Circos plots illustrate T-CAST results with enlargement of the chromosome three region encompassing CCR5 and CCR2 loci. Lines represent chromosomal rearrangements with the CCR5 target site: OMTs with >20 hits in red, ambiguous classification (OMT/HMT) in yellow. (F,G) Genotyping. NGS at denoted sites was performed on untreated (UT) T cells, and T cells edited with WT-TALENs or OH-TALENs as indicated. The group in which the samples were originally identified is indicated on the bottom. *** specifies p -value<0.001 (Fisher’s exact test, Bonferroni corrected p -values).

Article Snippet: Cells were resuspended in 50 μl CliniMACS ® Electroporation Buffer (Miltenyi Biotec).

Techniques: Activity Assay, TALENs, Electroporation, Control, Cell Culture, Expressing, Flow Cytometry

Journal: Frontiers in Genome Editing

Article Title: T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

doi: 10.3389/fgeed.2023.1130736

Figure Lengend Snippet: T-CAST analysis for TRAC -targeting TALENs (A) Genotyping. Results of T7E1 assay of WT-TALEN and OH-TALEN (KKR-ELD and KVR-EAD configuration) are shown. 32°C indicates that T cells were subjected to transient cold-shock after electroporation, while control cells were constantly cultured at 37°C. * specifies p -value<0.05 (Student’s t-test, n = 3–6). (B) TCR expression. Displayed is the fraction of TCRα/β-negative T cells upon transfer of TALEN-encoding mRNA as determined by flow cytometry. Where indicated (32°C) T cells were subjected to a transient cold-shock. *** specifies p -value<0.001 (Student’s t-test; n = 3–7). (C–E) Structural variations. Circos plots illustrate T-CAST results with enlargement of the chromosome 14 region encompassing TRAC . Red lines represent chromosomal rearrangements (OMTs with >20 hits) with the TRAC target site. (F) Genotyping. NGS at denoted sites was performed on untreated (UT) T cells, and T cells edited with WT-TALENs or OH-TALENs as indicated. The group in which the samples were originally identified is indicated on the bottom. *** specifies p -value<0.001 (Fisher’s exact test, Bonferroni corrected p -values).

Article Snippet: Cells were resuspended in 50 μl CliniMACS ® Electroporation Buffer (Miltenyi Biotec).

Techniques: TALENs, Electroporation, Control, Cell Culture, Expressing, Flow Cytometry

Journal: Experimental Hematology

Article Title: Expanded potential stem cell media as a tool to study human developmental hematopoiesis in vitro

doi: 10.1016/j.exphem.2019.07.003

Figure Lengend Snippet: Gene targeting in EPSCM PSCs, related to . ( A ) Map of the ROSA26 gene targeting vector used in this study. The plasmid was linearized with AsiSI before electroporation. ( B ) Map of the SPI1 gene targeting vector used in this study. ( C ) 5′ and 3′ PCR genotyping of SPI1-T2A-H2B-Venus cassette knock-in in clonally derived EPSCM PSC lines. ( D ) Representative flow cytometry plots for Venus expression for SPI1-T2A-H2B-Venus clone 4 and clone 8 EPSCM PSC-derived EBs at day 20. ( E ) DNA sequence of the untargeted SPI1 allele in clone 4 and clone 8 as determined by Sanger sequencing, with SPI1 sgRNA-target site highlighted.

Article Snippet: After dissociation, cells were collected, counted, and resuspended in EmbryoMax ES Cell Electroporation Buffer (Merck Millipore).

Techniques: Plasmid Preparation, Electroporation, Knock-In, Derivative Assay, Flow Cytometry, Expressing, Sequencing

Journal: Scientific Reports

Article Title: Biomimetic cellular vectors for enhancing drug delivery to the lungs

doi: 10.1038/s41598-019-55909-x

Figure Lengend Snippet: CELVEC as a drug delivery vehicle. ( A ) Loading of doxorubicin (DOX) into CELVEC as a function of initial DOX concentration used. ( B ) Release of DOX from macrophages over 24 h when loading was performed passively (left) or supplemented with electroporation (right). Inset graphs depict DOX release over 6 h as determined using a one-phase association ordinary fit line. Dotted black line designated 50% release of payload. ( C ) Cellular viability of human MDA-231 triple negative breast cancer cells following treatment with free DOX and DOX-loaded CELVEC at low (325 nM) and high (975 nM) dosages. The data is plotted as the mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Electroporation Buffer containing doxorubicin (DOX, LC Laboratories) was created by slowly adding hydrochloric acid to obtain a final pH of 5.2.

Techniques: Concentration Assay, Electroporation

Journal: Scientific Reports

Article Title: Biomimetic cellular vectors for enhancing drug delivery to the lungs

doi: 10.1038/s41598-019-55909-x

Figure Lengend Snippet: Effect of DOX loading in CELVEC performance. ( A ) Flow cytometry dot plot depicting PS on CELVEC (blue) and DOX@CELVEC (green) surface 4 h post-generation. ( B ) Quantitative assessment of flow cytometry dot plot data exhibiting DOX and Annexin V positive cells using gates established in (A). ( C ) Cytotoxic evaluation of murine macrophage cells following electroporation (CELVEC, blue) and electroporation supplemented with DOX (DOX@CELVEC, green) to create DOX@CELVEC. Neutral red uptake was employed to evaluate cellular viability and proliferation over 72 h. Doxorubicin loading was performed at 20 mg/mL. Values normalized to untreated murine macrophages (CTRL). ( D ) Quantification of CELVEC adhered to TNF-α-activated HUVEC following 30 min of in vivo -like flow conditions of 0.1 dyn/cm 2 . ( E ) Representative fluorescent microscope images used for ( D,E ) depicting CELVEC adhesion following 30 min. of flow. Scale bar, 100 μm. The data is plotted as the mean ± SEM. ***p < 0.001.

Article Snippet: Electroporation Buffer containing doxorubicin (DOX, LC Laboratories) was created by slowly adding hydrochloric acid to obtain a final pH of 5.2.

Techniques: Flow Cytometry, Electroporation, In Vivo, Microscopy