Journal: Nature Communications

Article Title: Evolution of AF6-RAS association and its implications in mixed-lineage leukemia

doi: 10.1038/s41467-017-01326-5

Figure Lengend Snippet: Insertion of the αN helix between MLL and AF6 disrupts myeloid immortalization and blocks its leukemogenic potential in mice. a Schematic of experimental strategies to measure immortalization of hematopoietic progenitors and induction of leukemogenesis. b Serial replating immortalization assay for progenitor cells transduced with retroviral constructs. Bars represent mean ± SD of the total colonies per 5 × 10 3 cells derived from three replicates. c Typical granulocyte and macrophage colonies from primary methylcellulose cultures of pMSCVneo or MLL-AF6 αN-RA1 and macroscopic blast colonies from MLL-AF6 NCR or MLL-AF6 RA1 cultures. d Representative flow cytometry analysis of donor-derived myeloid cells in the bone marrow of transplanted mice. The red quadrants highlight the immature CD11b + GR1 low/negative myeloid subpopulation which was significantly expanded in MLL-AF6 NCR or MLL-AF6 RA1 expressing bone marrow cells in primary transplants compared to control cells (vector) and MLL-AF6 αN-RA1 expressing bone marrow cells. All mice transplanted with MLL-AF6 NCR or MLL-AF6 RA1 expressing cells were leukemic at 5 weeks ( > 20% blasts in the bone marrow and peripheral blood) while MLL-AF6 αN-RA1 expressing cells did not induce leukemia up to 10 weeks after transplantation. e Donor-derived engraftment in the bone marrow of mice transplanted with 10 5 BM cells transduced with pMSCVneo, MLL-AF6 NCR , MLL-AF6 RA1 or MLL-AF6 αN-RA1 . BM cells were selected for 7 days in G418 prior to transplantation. Shown are the mean ± SD of donor cell engraftment (CD45.2 + ) and of the myeloid (CD11b+, Gr1+) to lymphoid (CD3+, B220+) ratio in donor-derived cells 5 weeks after transplantation (2 × 10 5 /mice, n = 5 or 7 mice). Error bars represent s.d.

Article Snippet: For gene transfer into bone marrow cells, pMSCV constructs consisting of in-frame fusions between MLL and various protein partners (MSCV, -FLAG-AF6 NCR , -MLL-AF6 NCR , -MLL-AF6 RA1 , -MLL-AF6 αN-RA1 ) were used to produce high-titer, helper-free recombinant retroviruses by transduction of the mouse embryonic fibroblast retroviral packaging cell line GP + E86 (ATCC CRL-9642) with VSV-G-pseudotyped MSCV particles .

Techniques: Transduction, Retroviral, Construct, Derivative Assay, Flow Cytometry, Expressing, Control, Plasmid Preparation, Transplantation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Differential susceptibility of human epithelial and monocytic cell lines toward FV Env-containing single-round retrovirus vector particles GFP-expressing retroviral vector supernatants of PFV, HIV, or MLV origin pseudotyped with different viral GPs (PFV Env [PE], SFVmac Env [SE], or VSV-G [VSV]), as indicated, were generated using wild-type 293T packaging cells, and PEI transfection methodology as described in . (A and B) THP-1 (A) and HT1080 (B) target cells were incubated with decreasing serial dilutions of the identical, plain, cell-free vector supernatants pseudotyped with the respective GP as indicated. Three days post-infection (dpi), the percentage of GFP-expressing cells in the individual samples was determined by flow cytometry. Shown are the mean ± SD of three independent titrations on THP-1 cells of separate aliquots of the same vector supernatant production and a single titration on HT1080 cells.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Plasmid Preparation, Expressing, Retroviral, Generated, Transfection, Incubation, Infection, Flow Cytometry, Titration

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Viral glycoprotein-dependent influence of exogenous PEI addition on retroviral vector transduction efficiency of different target cells GFP-expressing LV vector supernatants pseudotyped with VSV-G (HIV-VSV) or SFVmac Env (HIV-SE) were generated using wild-type 293T packaging cells and PEI transfection methodology, as described in . (A–E) Different target cells of (A) myeloid, (B) lymphoid, or (C–E) fibroblast origin, as indicated, were incubated with decreasing serial dilutions of the identical LV supernatants, pseudotyped with the respective GP, as indicated, that contained fixed concentrations of exogenously added PEI, as indicated. Shown are the mean ± SD (n = 3) of the percentage of GFP-expressing cells in the individual samples determined 3 dpi by flow cytometry.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Retroviral, Plasmid Preparation, Transduction, Expressing, Generated, Transfection, Incubation, Flow Cytometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Comparison of PFV and LV vector titers obtained by transient transfection of various 293T packaging cell lines Parental 293T (wild-type [WT]) or PG-deficient 293T variant (25A, 306) packaging cell lines were transiently transfected with expression plasmids encoding for either a replication-deficient, expression optimized 4-component PFV (PFV) vector system or a replication-deficient 3-component LV (HIV) vector system using PEI or CaP transfection reagents. GP packaging plasmids employed encoded PFV Env protein (PE, orange bars), macaque SFV Env protein (SE, blue bars), or the VSV glycoprotein (VSV, black bars). Subsequently, infectivity of supernatants was determined by titration on HT1080 target cells using a flow cytometric EGFP transfer assay. (A) Mean values ± SD (n = 3) of absolute vector titers of plain supernatants on HT1080 target cells are shown. (B and C) The infectivity data of (A) are regrouped so that for each vector type, the infectivity obtained by production in 293T WT (black bars), 25A (bright red bars), or 306 (dark red bars) packaging cells are compared with each other. (B) Mean values ± SD of fold difference of vector titers of individual samples relative to PFV vector supernatants containing PFV Env and derived by CaP-mediated transient transfection of parental 293T (WT) packaging cells are shown. (C) Mean values ± SD of fold difference of vector titers of the individual samples, grouped by vector type, relative to the respective type of vector derived by transient transfection of parental 293T (WT) packaging cells . For each vector type, the respective vector titer obtained by transfection of 293T WT packaging cells was set as reference to 1, and the vector titers obtained by transfection of 293T-25A or -306 packaging cells are expressed as relative values to the respective reference. Two-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns: not significant (p ≥ 0.05).

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Comparison, Plasmid Preparation, Transfection, Variant Assay, Expressing, Infection, Titration, Derivative Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Comparison of physical particle release obtained by transient transfection of various 293T-based packaging cell lines with components of different FV vector systems Parental 293T (WT) or PG-deficient 293T variant (25A) packaging cell lines were transiently transfected with PFV proviral expression constructs for production of replication-competent PFV (RCV) or expression constructs for production of EGFP encoding replication-deficient PFV single-round vector particles (SRVs) or expression constructs for production of EGFP encoding replication-deficient PFV RNA-transfer vector particles (TraFo) harboring PE, SE, or VSV as indicated, using PEI or CaP transfection methodologies. Physical particle release was determined by western blot analysis of equal volumes (100% sample) of samples of PFV particles concentrated and purified by ultracentrifugation followed by SDS-PAGE and immunoblotting using PFV Gag and PFV Env LP subunit-specific polyclonal antisera. A 3-fold serial dilution of the PFV SRV PE sample is shown in lanes 1 to 4. Shown are data from a representative experiment (n = 2). The infectivity data of the respective viral supernatants are shown in Figure S8 .

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Comparison, Transfection, Plasmid Preparation, Variant Assay, Expressing, Construct, Western Blot, Purification, SDS Page, Serial Dilution, Infection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Comparison of replication-competent PFV titers and physical particle release obtained by transient transfection of various 293T-based packaging cell lines Parental 293T (WT) or PG-deficient 293T variant (25A) packaging cell lines were transiently transfected with PFV proviral expression constructs using PEI or CaP transfection methods with (+) or without (−) sodium butyrate (NaBu) induction. (A) Physical particle release was determined by western blot analysis of equal volumes (100% sample) of samples of PFV particles concentrated and purified by ultracentrifugation followed by SDS-PAGE and immunoblotting using PFV Gag and PFV Env LP subunit-specific polyclonal antisera. A 3-fold serial dilution of the CaP 25A sample is shown in lanes 1 to 4. (B) Infectivity of supernatants was determined on HT1080 PLNE target cells harboring a PFV transactivator Tas-dependent EGFP reporter protein expression cassette using a flow cytometric assay at 24 h post-infection (hpi). Mean values ± SD (n = 4–6) of absolute viral titers on HT1080 PLNE target cells as well as relative infectivity in comparison to virus supernatants produced by CaP-mediated transient transfection of PG-deficient 293T-25A (25A) packaging cells are shown. Two-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns: not significant (p ≥ 0.05).

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Comparison, Transfection, Variant Assay, Expressing, Construct, Western Blot, Purification, SDS Page, Serial Dilution, Infection, Flow Cytometry, Virus, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Reduced permissiveness of PG-deficient 293T cells toward FV GP-containing retroviral vector particles Wildtype 293T cells (WT) or PG-deficient variants (25A, 306) were incubated with decreasing serial dilutions of GFP-expressing PFV (PFV) or LV (HIV) vector particles harboring VSV-G (VSV), PFV (PE), or SFVmac (SE) glycoproteins, as indicated. PFV and LV vector supernatants were generated by transient transfection of 293T-25A packaging cells using CaP transfection method. Three dpi, the percentage of GFP-expressing cells in the individual samples was determined by flow cytometry. Shown are the mean values ± SD (n = 3) of the percentage of GFP-expressing cells.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Retroviral, Plasmid Preparation, Incubation, Expressing, Generated, Transfection, Flow Cytometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Viral glycoprotein-dependent influence of exogenous PEI addition on retroviral vector transduction efficiency of different target cells WT 293T cells (293 WT) or PG-deficient (293T-25A) target cells were incubated with decreasing serial dilutions of GFP-expressing LV (HIV) vector particles pseudotyped with (A) VSV-G (VSV), (B) PFV (PE), or (C) SFVmac (SE) glycoproteins, derived by transient transfection of 293T-25A packaging cells using CaP transfection technique, that contained fixed concentrations of exogenously added PEI, as indicated. Three dpi, the percentage of GFP-expressing cells in the individual samples was determined by flow cytometry. Shown are the mean values ± SD (n = 3) of the percentage of GFP-expressing cells.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Retroviral, Plasmid Preparation, Transduction, Incubation, Expressing, Derivative Assay, Transfection, Flow Cytometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

doi: 10.1016/j.omtm.2022.07.004

Figure Lengend Snippet: Titration of PFV and HIV-1 retroviral vector supernatants harboring various viral glycoproteins and produced by calcium phosphate or polyethylene imine transfection using 293T-25A cells on THP-1 target cells Cell-free PFV (PFV) and LV (HIV) SRV supernatants harboring various viral GPs (VSV, PE, SE) were produced by transient transfection of PG-deficient 293T 25A packaging cells using the respective EGFP encoding transfer vector plasmid and packaging construct combination by PEI or CaP transfection methodologies, as indicated. Subsequently, THP-1 target cells were incubated with decreasing serial dilutions of the respective vector supernatants as indicated. Six dpi, the percentage of GFP-expressing cells in the individual samples was determined by flow cytometry. Shown are the mean values ± SD (n = 3).

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573, ) as well as its HS-deficient variants 293T-25A and 293T-306 generated in this study and described below (see ); the human epithelial fibrosarcoma cell line HT1080 (ATCC CCL-121 ) as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR-driven EGFP reporter gene expression cassette; and mouse fibroblast L cells (ATCC CRL-264, ) as well as its variant Sog9 with a defect in initiation of GAG assembly so that no surface HS is being produced (gift of F. Neipel, Erlangen ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Titration, Retroviral, Plasmid Preparation, Produced, Transfection, Construct, Incubation, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Cold-Induced Thermogenesis Increases Acetylation on the Brown Fat Proteome and Metabolome

doi: 10.1101/445718

Figure Lengend Snippet: A. Outline of UCP1 acetylation mutant constructs design. Note that all four acetylation sites are predicted to be mitochondrial matrix-facing area. B. Immunoblots of UCP1 wild-type or mutant constructs expressed in 293T cells. C. mRNA expression levels, measured by qPCR, of UCP1 wild type or mutant constructs expressed in 293T cells. Data points are biological replicates, and error bars represent standard error of the mean. D. Immunoblot (left) and quantification (right) showing protein expression of UCP1 wild-type or mutant constructs in a time series after cycloheximide (CHX) treatment. Half-lives of UCP1 proteins are calculated in the graph. In all immunoblot analysis, expression of alpha-tubulin was used as a loading control.

Article Snippet: 293T cells (ATCC) were transfected with pMSCV-puro retroviral vectors expressing UCP1-Flag WT, UCP1-Flag 3KR, UCP1-Flag 3KQ, UCP1-Flag 4KR, UCP1-Flag 4KQ in combination with the retroviral packaging plasmids pCMV-VSVG (Addgene) and pCMV-Gag-Pol (Addgene).

Techniques: Mutagenesis, Construct, Western Blot, Expressing, Control