Journal: iScience

Article Title: A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs

doi: 10.1016/j.isci.2024.110774

Figure Lengend Snippet:

Article Snippet: NucleoMag NGS beads , Macherey Nagel , Cat# 744970.50.

Techniques: Virus, Recombinant, Software

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 1. The FoxP3 Reporter LV Shows Expression Selective for the Treg Cell Lineage (A) Endogenous human FOXP3 gene shows location of regulatory regions (promoter, CNS1, CNS2, CNS3, and 30 UTR) included in vector. Vector maps show design of CNS123p-mStrawberry and CNS123p-FoxP3-mStrawberry constructs within the pCCL vector backbone. (B) Analysis of human hematopoietic cell lines transduced with CNS123p-mStrawberry. Histograms show endogenous FoxP3 status of each cell line (left) and mStrawberry expression (right) in each cell line transduced with CNS123p-mStrawberry. Plots show mStrawberry expression in human hematopoietic cell lines over a range of vector copy numbers (n = 12 per cell line). (C) Activated human CD4 cells transduced with different doses of CNS123p-mStrawberry. Histograms show mStrawberry expression in viable CD4+ cells analyzed 4 days after activation. (see also Figure S2B). (D) Experimental design to evaluate in vivo lineage-specific expression of CNS123p-mStrawberry. Lin HSPCs were isolated from CD45.2 FoxP3-prom-GFP mice and transduced with CNS123p-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated congenic CD45.1 recipients. CD45.2 donor cells within each hematopoietic lineage were analyzed at 20 weeks post-transplant for mStrawberry reporter LV expression (see also Figure S2A). (E) Histograms depict mStrawberry reporter expression in each hematopoietic lineage in bone marrow, thymus, and spleen of engrafted mice. Each individual histogram line represents mStrawberry expression for an individual mouse (n = 9 mice; see also Figure S2C). (F) y axis represents the percentage of mStrawberry+ cells within each hematopoietic lineage in the BM, spleen, and thymus (n = 9 mice). Data in (E) represent mean ± SD.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Plasmid Preparation, Construct, Transduction, Activation Assay, In Vivo, Isolation, Irradiation

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 2. Lineage-Specific FoxP3 Expression Restores Treg Cell Development from Scurfy (FoxP3 Mutant) HSCs (A) Transplant setup to evaluate Treg cell development. Scurfy (FoxP3mut) mice were rescued with WT CD45.1 splenocytes at birth to allow survival into adulthood to serve as bone marrow donors. Lin HSPCs were isolated from rescued scurfy (FoxP3mut) or wild-type (FoxP3-prom-GFP) donor mice and transduced with CNS123p-mStrawberry or CNS123p-FoxP3-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated WT CD45.1 congenic recipients. After 12 weeks, donor cells from each transplant cohort were evaluated for thymic and splenic reconstitution of Treg cells. Treg cell populations from each group were identified as CD4+mStrawberry+ cells (uncorrected scurfy Treg cells [Sf-Treg cells]; corrected scurfy Treg cells [cSf-Treg cells]) or CD4+GFP+ cells (wild- type Treg cells [WT-Treg cells]). (B) Lineage distribution of total donor thymocytes in mice reconstituted with Sf, cSf, or WT BM lin cells (n = 3–5 mice/arm). (C) Thymic Treg cell reconstitution. FACS plots show donor CD45.2+CD4SP cells in the thymus of transplant recipients. Gates delineate thymic Sf-Treg cells, cSf-Treg cells, and WT-Treg cells. Bottom panel shows expression of the Treg cell surface markers CD25, GITR, and CTLA4 within each putative Treg cell population (surface marker expression for Sf-Treg cells and cSf-Treg cells is shown for mStrawberryhigh cells or cells expressing the top 50% of mStrawberry expression). Histograms depict one representative experiment of 3 (see also Figure S2D). (D) FACS sort for splenic Treg and Tconv cells. Splenic Treg cell populations (mStrawberryhigh or GFP+ gates) were FACS sorted for a Treg cell suppression assay. Tconv cell populations (mStrawberry or GFP gates) were sorted for an iTreg cell induction assay. (E) In vitro Treg cell suppression assay. Sorted Treg cells (shown in D) were co-cultured with responder T cells (Tresp cells) (congenic WT CD4+ cells labeled with a fluorescent proliferation dye) at a 1:1 ratio in the presence of bead-bound CD3 and CD28 antibodies. Histograms depict Tresp cell proliferation in one of three representative experiments. Bar graph shows proliferation index for each Treg cell culture condition (n = 6–9 Tresp cell cultures per arm from 3 different Treg cell sources per arm; data normalized to internal ‘‘no Treg cell’’ control for each experiment). (F) Sorted splenic Tconv cells (shown in D) were activated with CD3 and CD28 antibodies in the presence of 20 ng/mL interleukin-2 (IL-2) and 20 ng/mL TGF-b for 4 days to induce iTreg cells. FACS plots show mStrawberry or GFP expression from each group (n = 3 mice/group). Data in (B), (C), (E), and (F) are presented as mean ± SD. Data in (B) were analyzed by Kruskal-Wallis test for each lineage. Data in (E) are analyzed by Kruskal- Wallis test for overall comparison for all groups and Mann-Whitney U test for pairwise comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Mutagenesis, Isolation, Transduction, Irradiation, Marker, Suppression Assay, In Vitro, Cell Culture, Labeling, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 3. The Lineage-Specific FoxP3 cDNA Vector Generates Functional Treg Cells Capable of Rescuing the Scurfy Mouse (A) Assay for in vivo Treg cell function: three groups of CD4 cells containing putative Treg cells were generated by congenic bone marrow transplants: uncorrected scurfy CD4 (Sf-CD4; no. 1); corrected scurfy CD4 (cSf-CD4; no. 2); and wild-type CD4 (WT-CD4; no. 3). To obtain Sf-CD4 and cSf-CD4, CD45.2 Lin HSPCs were isolated from scurfy donors (rescued at birth by CD45.1 splenocytes) and transduced with either CNS123p-mStrawberry (Sf-CD4; no. 1) or CNS123p-FoxP3- mStrawberry (cSf-CD4; no. 2). To obtain WT-CD4, CD45.2 Lin HSPCs were isolated from FoxP3-prom-GFP donors and transduced with CNS123p- mStrawberry (no. 3). Transduced cells were transplanted into lethally irradiated congenic (CD45.1) recipients. 8 weeks post-transplant, donor CD45.2+CD4+ cells were purified with magnetic beads from the spleens of transplant recipients and injected intraperitoneally into scurfy neonates. Scurfy neonates and age-matched WT receiving PBS injection were also analyzed as control conditions no. 4 and no. 5. The autoimmune phenotype of all groups was evaluated at 21 days of life (see also Figure S3). (B) Photographs of scurfy mice or WT controls at 21 days. White arrows highlight ear skin phenotype in mice from each group. Ear skin inflammation (char- acterized by small, thickened, scaly ears) is seen in untreated scurfy mice (Sf + PBS) and scurfy mice receiving uncorrected scurfy CD4 cells (‘‘Sf + Sf-CD4’’). Normal ear skin without inflammation is seen in WT controls (‘‘WT + PBS’’) and scurfy mice receiving wild-type or corrected scurfy CD4 cells (‘‘Sf + WT-CD4’’ and ‘‘Sf + cSf-CD4’’). (C) Spleen-to-body-weight ratio for rescued scurfy mice or WT littermate controls. (D) Activated (CD44+CD62L) CD4 T cells (expressed as a percentage of total CD4 splenocytes) in the spleens of rescued scurfy mice or WT littermate controls. (E) Serum cytokine levels in rescued scurfy mice or WT littermate controls. Data in (C)–(E) are presented as mean ± SD. Data on (C)–(E) represent n = 3 independent experiments pooled for analysis with a total of 3–6 mice/arm. Data in (C)–(E) were analyzed by Kruskal-Wallis test for overall comparison for all groups, and Mann-Whitney U test was performed for pairwise comparisons. *p < 0.05; **p < 0.01.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Functional Assay, In Vivo, Cell Function Assay, Generated, Isolation, Transduction, Irradiation, Magnetic Beads, Injection, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 4. The FoxP3 Reporter Vector CNS123p-mStrawberry Shows Treg Cell Lineage-Selective Expression in a Humanized Mouse Model (A) Experimental setup for humanized mouse models. Cord blood CD34+ HSPCs were transduced with CNS123p-mStrawberry and transplanted into neonatal NSG mice (B, C, E, and F) or NSG-SGM3 mice (D). 12–16 weeks post-transplant, engrafted hCD45+ cells were analyzed for mStrawberry expression. (B) mStrawberry reporter expression in each hematopoietic lineage. Each overlaid histogram represents mStrawberry expression in an individual mouse (n = 10–14 mice humanized with 2 different cord blood CD34+ donors; see also Figures S4A–S4C). (C) Percentage of mStrawberry+ cells in each lineage shown in (B). (D) Co-expression of FoxP3 and mStrawberry in humanized mice. Splenic human CD4+ cells were FACS sorted into mStrawberry+ and mStrawberry pop- ulations followed by intracellular staining for FoxP3 expression. Left panel shows sorting of human CD4+ cells by mStrawberry expression, and right panel shows FoxP3 expression in sorted populations. Results are representative of 2 independent experiments. (E) CNS2 methylation analysis of T cell populations from humanized mice. Figure shows the locations of CNS2 within the endogenous FOXP3 gene and CNS2 within the viral genome. Red arrows indicate differential primer binding sites for amplification of endogenous or viral CNS2. FACS plot shows sorting gates used to define Treg cell (CD4+CD25+) and Tconv cell (CD4+CD25) populations in CD4-enriched cells isolated from the pooled spleens of 3–5 humanized mice. Heatmap represents the percentage of methylated reads detected at each of the 9 CpG sites within endogenous and viral CNS2. Results are representative of 2 independent experiments using pooled NSG cohorts humanized from 2 different CB CD34+ donors (see also Figures S4D–S4F). (F) Experimental setup for NSG competitive repopulation assay. ‘‘Test’’ CB CD34+ cells were transduced with either CNS123p-mStrawberry or CNS123p-FoxP3- mStrawberry, and ‘‘competitor’’ CD34+ cells were transduced with a UBC-mCitrine vector. Test and competitor cells were co-transplanted at a 1:1 ratio into NSG neonates, and the percentage of competitor (mCitrine+) CD45+ cells engrafted in the BM at 12 weeks was determined for each group (n = 6 mice per group; humanized from 2 different CB CD34+ donors). Data in (F) represent mean ± SD. Data in (F) were analyzed by Mann-Whitney U test.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Expressing, Transduction, Staining, Methylation, Binding Assay, Isolation, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Identification of Actionable Gene Variants in Pulmonary Large-Cell Neuroendocrine Carcinoma: A Real-World Analysis of a Polish Cohort

doi: 10.3390/ijms27072939

Figure Lengend Snippet: The graphic representation of TMEM79::NTRK1 fusion, including exonic (TMEM79 NM_032323.3 exon 3; NTRK1 NM_002529.3 exon 2) and chromosomal (chr1:156256264, chr1:156834146) breakpoint positions based on the output data from the Archer Analysis software version 7.2 (ArcherDX, Inc., Boulder, CO, USA; ( A )) and on the variant visualization by the Alamut Visual Plus v1.12 software (SOPHiA GENETICS, Inc., Boston, MA, USA; ( B )).

Article Snippet: Gene fusion variants were further evaluated using Alamut Visual Plus (v1.12; SOPHiA GENETICS, Inc., Boston, MA, USA).

Techniques: Software, Variant Assay