Journal: Journal of virological methods

Article Title: ANALYSIS OF TWO MONOCLONAL ANTIBODIES REACTIVE WITH ENVELOPE PROTEINS OF MURINE RETROVIRUSES: ONE PAN SPECIFIC ANTIBODY AND ONE SPECIFIC FOR MOLONEY LEUKEMIA VIRUS

doi: 10.1016/j.jviromet.2014.02.006

Figure Lengend Snippet: Reactivity of MAbs 538, 573 with ecotropic, amphotropic and xenotropic MuLVs

Article Snippet: The MuLVs used in these studies are described in the citations included in and . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 MAb Reference Class Strain 538 573 Ecotropic F-MuLV a − + R-MuLV − + ( Rauscher, 1962 ) AKR 2A − + ( Cloyd et al., 1979 ) M-MuLV 8.2 + + ( Shoemaker et al., 1980 ) M-MuLV 1387 + + ( Evans and Cloyd, 1984 ) WN1802N − + ( Cloyd et al., 1980 ) CasBr-E − + ( Hartley and Rowe, 1976 ) 1504E − + ( Hartley and Rowe, 1976 ) Xenotropic b AKR 6 − + ( Cloyd et al., 1979 ) NIH AT124 − + ( Todaro et al., 1973 ) Balb IU-1 − + ( Hartley and Rowe, 1976 ) C58L1 xeno − + ( Chattopadhyay et al., 1981 ) Cas E no. 1 − + ( Cloyd et al., 1979 ) NFS-Th1 − + ( Chattopadhyay et al., 1981 ) XMRV VP62 − + ( Urisman et al., 2006 ) NZB-8882 − + ( Rein and Schultz, 1984 ) NZB CI.

Techniques:

Journal: Journal of virological methods

Article Title: ANALYSIS OF TWO MONOCLONAL ANTIBODIES REACTIVE WITH ENVELOPE PROTEINS OF MURINE RETROVIRUSES: ONE PAN SPECIFIC ANTIBODY AND ONE SPECIFIC FOR MOLONEY LEUKEMIA VIRUS

doi: 10.1016/j.jviromet.2014.02.006

Figure Lengend Snippet: Detection of cell surface expression of MuLV SU proteins by MAb 573 and MAb 538 by flow cytometry. (A). 3T3 cells chronically infected with the xenotropic MuLV, CasE no.1, the amphotropic MuLV, 4070A, the ecotropic MuLV, Fr-MuLV or the polytropic MuLV, M-RV 7A, were treated with MAb 573 and a secondary FITC goat Ab directed at mouse Ig (white) or with the secondary FITC antibody alone (grey) and analyzed by FACS. (B). 3T3 cells chronically infected with Mo-MuLV were treated with MAb 538 and the secondary goat FITC Ab (white) or with the secondary antibody alone (grey).

Article Snippet: The MuLVs used in these studies are described in the citations included in and . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 MAb Reference Class Strain 538 573 Ecotropic F-MuLV a − + R-MuLV − + ( Rauscher, 1962 ) AKR 2A − + ( Cloyd et al., 1979 ) M-MuLV 8.2 + + ( Shoemaker et al., 1980 ) M-MuLV 1387 + + ( Evans and Cloyd, 1984 ) WN1802N − + ( Cloyd et al., 1980 ) CasBr-E − + ( Hartley and Rowe, 1976 ) 1504E − + ( Hartley and Rowe, 1976 ) Xenotropic b AKR 6 − + ( Cloyd et al., 1979 ) NIH AT124 − + ( Todaro et al., 1973 ) Balb IU-1 − + ( Hartley and Rowe, 1976 ) C58L1 xeno − + ( Chattopadhyay et al., 1981 ) Cas E no. 1 − + ( Cloyd et al., 1979 ) NFS-Th1 − + ( Chattopadhyay et al., 1981 ) XMRV VP62 − + ( Urisman et al., 2006 ) NZB-8882 − + ( Rein and Schultz, 1984 ) NZB CI.

Techniques: Expressing, Flow Cytometry, Infection

Journal: bioRxiv

Article Title: Non-destructive transcriptomics via vesicular export

doi: 10.1101/2024.11.11.622832

Figure Lengend Snippet: (A) Inducible expression of cellular RNA-export machinery enables repeated sampling of vesicle-enclosed transcripts from the supernatant of the same cell population. In contrast, conventional transcriptomics requires cell lysis at each time point. (B) Schematic of the transfer vector for generating stable NTVE cell lines via ecotropic lentiviral delivery of a modular cassette with a PGK promoter driving the expression of the TetON3G transactivator for doxycycline-dependent expression of non-infectious Gag chimeras leading to membrane budding. The cassette is flanked by two Long terminal repeats (LTR) and stabilized by the Woodchuck Posttranscriptional Response Element (WPRE), a triple helix, and a tRNA. For selection, expression of the puromycin N-acetyltransferase is coupled via a 2A peptide. The NTVE budding module can be equipped with different adapters for packaging RNAs, and affinity handles for purification. A HiBiT tag allows for bioluminescence-based quantification of NTVE vesicles. ( C ) A PP7-coat protein (PCP) serves as a specific adapter to export PP7-tagged barcode RNAs. ( D ) NTVE vesicles can be decorated with purification tags mounted on membrane-anchored deactivated VSV-G (W72A) to enable the separation of distinct VLPs from different cell populations. ( E ) The first two RNA recognition motifs (RRM) from poly(A)-binding protein PABPC1 enable the export of polyadenylated mRNAs, while the GCN4 leucine-zipper domain mediates dimerization. (F) To enhance the accumulation of otherwise unstable intronic RNA, e . g ., PP7-tagged introns via NTVE PCP , intronic RNA was stabilized by either co-expression of a catalytically inactive DBR1 or via the Tornado system, enabling NTVE export of introns. RNA levels were determined via RT-qPCR and normalized to a control condition lacking NTVE PCP expression. (G) HEK293T cells with integrated NTVE PCP expression cassette were transfected with plasmids encoding either mScarlet-I (mSL) or mGreenLantern (mGL) with PP7-tagged 3’UTRs. 24 h post-transfection, cells were reseeded in the ratios 10:1, 1:1, and 1:10. The different co-cultured cells were subsequently quantified by fluorescence microscopy (left y -axis). In addition, the mGL and mSL mRNA abundance in the supernatant was quantified via RTqPCR (right x -axis). Both measures were normalized to separate mono-cultures, exclusively expressing only one of the reporters. (H) For purification using the epitope-tagged dVSV-G (W72A), stable NTVE PCP HEK293T cells were transfected with plasmids encoding either mSL or mGL with PP7-tagged 3’UTRs. 24 h post-transfection, cells were reseeded in a ratio of 1:1 and subsequent NTVE induction (500 ng/ml doxycycline). The tagged VLPs of the co-cultured cell lines were pulled down with either anti-FLAG or anti-HA magnetic beads, and mGL/mSL abundance was analyzed via RT-qPCR. ( I ) Normalized distribution of the hydrodynamic radii of NTVE vesicles after doxycycline induction and enrichment by ultracentrifugation from the supernatant compared to a DMSO vehicle control. ( J ) Cryo-electron micrograph of NTVE PABP particles enriched by ultracentrifugation. Scalebar 50 nm. (K) Accumulation of NTVE vesicles in the supernatant after doxycycline induction (Continuous line: 500 ng/ml; dashed line: 50 ng/ml) monitored by bioluminescence signals from a HiBiT tag fused to PABPC1. Two stable cell lines that drive the TetON3g transactivator either via PGK or a CAG-promoter are compared. Points indicate the mean of three biological replicates with bounds for s.d.. ( L ) Distribution of expression levels for nuclear-encoded genes (box and violin plot) and mitochondrially-encoded genes (orange dots) from a stable cell line with inducible expression of NTVE PABP (top) and with transient overexpression of the same construct (bottom). Elevated mitochondrial species in the supernatant indicate potential cytotoxicity. (M) Normalized counts per million (CPM) identified in NTVE vesicles collected in the supernatant of HEK293T cells 72 hours after doxycycline induction plotted against those detected from the corresponding lysates. Genes encoded on the mitochondrial genome are labeled with their gene symbol and plotted in gray. Genes with less than 1% of the CPM in NTVE compared to the lysate are labeled in black. (N) Mean transcripts per million (TPM) of expressed coding transcripts (TPM >1) for bins of transcript lengths exported by NTVE PABP compared to that in the corresponding lysates. Data are plotted as the mean ±s.d. of triplicates.

Article Snippet: For ecotropic pseudotyping of the lentiviral delivery, the ecotropic MLV glycoprotein (Addgene #35617) was used in combination with psPAX2 (Addgene #12260) and a transfer vector encoding for NTVE.

Techniques: Expressing, Sampling, Lysis, Plasmid Preparation, Membrane, Selection, Purification, Binding Assay, Quantitative RT-PCR, Control, Transfection, Cell Culture, Fluorescence, Microscopy, Magnetic Beads, Stable Transfection, Over Expression, Construct, Labeling

Journal: bioRxiv

Article Title: Non-destructive transcriptomics via vesicular export

doi: 10.1101/2024.11.11.622832

Figure Lengend Snippet: ( A ) FLAG- or HA-tags mounted on non-fusogenic dVSV-G(W72A) coupled to TRE3G-driven NTVE PABP expression via 2A skipping peptide. The murine Neuro-2a and the human HEK293T cell lines were co-cultured, and mRNA secretion was induced by adding 500 ng/ml doxycycline. ( B ) NTVE vesicles were purified by the respective anti-Flag or anti-HA magnetic beads, followed by RNA preparation and NGS. Reads were mapped on human and mouse cDNA to quantify the proportion of human and murine transcripts. ( C , D ) Engineered cell-cell communication via vesicular mRNA export from a sender cell encoding either a recombinase (C) or a prime editor ribonucleoprotein (D) and uptake into HEK293T receiver cells. The vesicles are pseudotyped via glycoproteins, for which the receiver cells express the respective receptors. The receiver cell undergoes a genetic edit of a fluorescent reporter locus. ( C ) Vesicular export of a PP7-tagged mRNA coding for the recombinase pa01 NLS that activates an mGreenLantern reporter system in receiver cells by site-specific inversion. Vesicles present MLV-Env(ΔC16) glycoproteins for selective entry into receiver cells via a murinized SLC7A1 acting as a receptor. FACS scatter plots show the co-culture population of sender and receiver cells 3 days post (mock) induction of NTVE expression with doxycycline. ( D ) Co-culture from (C) captured with confocal microscopy after 72 hours, scale bar 50μm. ( E ) Delivery of a prime editor as a ribonucleoprotein (RNP) complex, facilitating a shift from blue to green fluorescence upon editing of a reporter locus. In contrast to (C), sender cells co-express the VSV-G glycoprotein to facilitate uptake on the recipient cells via LDL receptors. FACS scatter plots show the co-culture population of sender and receiver cells 3 days post (mock) induction of NTVE expression with doxycycline.

Article Snippet: For ecotropic pseudotyping of the lentiviral delivery, the ecotropic MLV glycoprotein (Addgene #35617) was used in combination with psPAX2 (Addgene #12260) and a transfer vector encoding for NTVE.

Techniques: Expressing, Cell Culture, Purification, Magnetic Beads, Co-Culture Assay, Confocal Microscopy, Fluorescence

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Accelerated appearance of multiple B cell lymphoma types in NFS/N mice congenic for ecotropic murine leukemia viruses.

doi: 10.1038/labinvest.3780020

Figure Lengend Snippet: Figure 2. Southern blot analyses for identification of somatic MuLV integrations in lymphomas of NFS. V1 mice. DNA samples of spleens (S) and corresponding tail biopsies (T) from NFS.Akv2 mice were digested with EcoRI and hybridized with the ecotropic-specific probe EcoSp. Germline integrations are those also present in tail DNA. One to two somatic integrations are seen in the lymphoma DNAs, whereas the spleen with reactive (R) changes had none.

Article Snippet: Tests for expression of ecotropic MuLV used XC cell (ATCC CCL 165) plaque assays (Rowe et al, 1970) on SC-1 cells (ATCC CRL 1404) of tail extracts from 6- to 10-week-old mice or mitomycin C-treated spleen cell infectious centers (Cloyd et al, 1981).

Techniques: Southern Blot

Journal: Gene Therapy

Article Title: The stability of envelope-pseudotyped lentiviral vectors

doi: 10.1038/s41434-020-00193-y

Figure Lengend Snippet: Envelope-pseudotyped lentiviral vectors studied in this project.

Article Snippet: Plasmids containing the gene for VSV-G glycoprotein (pCMV-VSV-G, #8454), rabies virus (strain SAD B19) glycoprotein (pHCMV-RabiesG, #15785), ecotropic Moloney MULV-envelope gene (pHCMV-EcoEnv, #15802), Moloney MULV strain 10A1 envelope gene (pHCMV-10A1, #15805), and amphotropic 4070A gene (pHCMV-AmphoEnv, #15799) were purchased from Addgene ( www.addgene.org ) with their respective plasmid numbers.

Techniques: Virus