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Spleen Focus-Forming Virus

Q: What are the different variations or types of Spleen Focus-Forming Virus?

Spleen Focus-Forming Virus (SFFV) is a specific strain of murine retrovirus that has been widely studied for its unique properties. While there is only one primary type of SFFV, researchers have identified various strains or isolates that exhibit slightly different characteristics. These include differences in their ability to induce rapid erythroid proliferation, spleen enlargement, and the development of erythroleukemia in infected mice. Understanding these nuances can help researchers select the most appropriate SFFV strain for their particular study objectives.

Q: How can PubCompare.ai assist in optimizing Spleen Focus-Forming Virus research protocols?

PubCompare.ai's AI-powered platform can be highly beneficial for researchers working with Spleen Focus-Forming Virus (SFFV). The platform allows you to efficiently screen the latest protocol literature, including published studies, pre-prints, and patents. PubCompare.ai's intelligent comparison tools can help you identify the most effective and reproducible SFFV protocols based on key metrics, such as experimental outcomes, methodology details, and researcher feedback. This enables you to select the best protocols to improve the accuracy and efficiency of your SFFV-related studies.

Q: What are some common usage challenges or considerations when working with Spleen Focus-Forming Virus?

One of the key challenges when working with Spleen Focus-Forming Virus (SFFV) is the need to carefully control and monitor the experimental conditions. SFFV is known to induce rapid and dramatic changes in the hematopoietic system of infected mice, which can make it difficult to maintain consistent results. Researchers must pay close attention to factors like viral titer, route of administration, and the specific mouse strain used, as these variables can significantly impact the observed outcomes. Additionally, the development of erythroleukemia in some SFFV models requires additional safety precautions and ethical considerations. PubCompare.ai can help researchers identify the best practices and protocols to overcome these challanges and optimize their SFFV experiments.

Q: How can PubCompare.ai help identify the latest advancements in Spleen Focus-Forming Virus research?

PubCompare.ai's AI-powered platform is designed to help researchers stay up-to-date with the latest developments in their field of study, including advancements in Spleen Focus-Forming Virus (SFFV) research. The platform continuously scans the literature, pre-printes, and patent databases to identify the newest protocols, methods, and findings related to SFFV. By leveraging PubCompare.ai's intelligent search and analysis tools, you can quickly locate the most relevant and impactful SFFV studies, allowing you to incorporate the latest discoveries into your own research. This helps ensure that your SFFV experiments are aligned with the current state of the art and maximizes the efficiency and impact of your work.

Most cited protocols related to «Spleen Focus-Forming Virus»

CRISPR Transcriptional Regulation Screens

Cited 105 times

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2014

BRAF protein, human Cell Lines Cells Clone Cells Spleen Focus-Forming Virus

Genome-wide CRISPR Deletion Screens for Cell Sensitivity

Cited 55 times

Pooled, genome-wide CRISPR deletion screens were performed in three cell lines: K562 stably expressing SFFV-Cas9-BFP, Ramos cells lentivirally infected with SFFV-Cas9-BFP, and U937 cells lentivirally infected with EF1a-Cas9-Blast34 (link). The library was synthesized, cloned and lentivirally infected into cells as previously described20 (link). Briefly, the parent vector for the libraries was derived from a pSico lentiviral vector which expresses GFP and a puromycin-resistance cassette separated by a T2A sequence45 (link)58 (link); we replaced GFP with mCherry to make the final parent vector, pMCB320. Sublibraries were PCR-amplified from pooled-oligo chips (CustomArray, Agilent), digested with BstXI and BlpI restriction enzymes, and ligated into BstXI/BlpI-cut pMCB320 using T4 ligase. Libraries and vectors will be made available via Addgene. Three days after infection, cells were placed under puromycin selection (0.7 μg ml⁻¹, Sigma) for an additional 3 days after infection, then split at time 0. Throughout the screen, the pooled libraries were maintained at 1,000 cells per guide or a total of ∼250 million cells in large spinner flasks. K562 and U937 were grown for ∼2 weeks, and Ramos cells were growth for ∼3 weeks due to their slower division time. Genomic DNA was extracted following Qiagen's Blood Maxi Kit, and the guide composition was sequenced and compared to the plasmid library using casTLE20 (link) version 1.0 available at https://bitbucket.org/dmorgens/castle. Briefly, casTLE compares each set of gene-targeting guides to the negative controls, selecting the most likely maximum effect size which explains the distribution of targeting guides. It then determines the significance of this maximum effect by permuting the results20 (link). Both safe-targeting and non-targeting controls were used for this analysis. For the ricin sensitivity screen, cells were treated with ricin toxin (Vector Labs) at 0.25 ng ml⁻¹ for 24 h, ricin was removed and then cells were allowed to recover to normal doubling rate. This treatment occurred four times over 2 weeks.

Morgens D.W., Wainberg M., Boyle E.A., Ursu O., Araya C.L., Tsui C.K., Haney M.S., Hess G.T., Han K., Jeng E.E., Li A., Snyder M.P., Greenleaf W.J., Kundaje A, & Bassik M.C. (2017). Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nature Communications, 8, 15178.

Publication 2017

BLOOD Cell Lines Cells Cloning Vectors Clustered Regularly Interspaced Short Palindromic Repeats Deletion Mutation DNA Chips DNA Library DNA Restriction Enzymes Genome Hypersensitivity Infection Ligase Oligonucleotides Parent Plasmids Puromycin Ricin Spleen Focus-Forming Virus U937 Cells

Transduction of Mammalian Cell Lines

Cited 45 times

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2013

Cell Lines Cells Cloning Vectors CXCR4 protein, human Eagle Edetic Acid Flow Cytometry Glutamine HEK293 Cells HeLa Cells Immunoglobulin Isotypes Lentivirus Penicillins Plasmids Retroviridae Simian virus 40 Spleen Focus-Forming Virus Streptomycin TFRC protein, human Transcription, Genetic Transfection Transients

Pooled CRISPR Screen in K562 Cells

Cited 42 times

A previously designed 4 sgRNA/gene CRISPR-Cas9 library was used targeting 5′ ends of conserved exons with sgRNAs varying in length between 19 and 25 base-pairs¹⁴. The library was generated first by infecting K562 cells with a SFFV-Cas9-BFP vector to create a stably expressed Cas9 cell line. We then infected the lentiviral genome-wide sgRNA library into approximately 120 million cells following the same protocol as the genome-wide shRNA library to maintain at least 1,000-fold representation in cells. Infected cells were selected with puromycin (0.7 μg/mL, Sigma) for 3 days. Percentage of mCherry positive cells was measured by flow cytometry (BD Accuri C6). Selected cells were spun out of selection and into normal RPMI 1640 media. At T0, 120 million cells were spun down (300g for 5 min). Cells were then split into two populations and grown for 14 days, maintaining logarithmic growth (500,000 cells/mL) each day. After 14 days of growth, cells were pelleted by centrifugation, and genomic DNA was extracted for all three time samples separately following Qiagen’s Blood Maxi Kit instructions. sgRNA encoding constructs were analyzed by deep sequencing.

Morgens D.W., Deans R.M., Li A, & Bassik M.C. (2016). Systematic comparison of CRISPR-Cas9 and RNAi screens for essential genes. Nature biotechnology, 34(6), 634-636.

Publication 2016

BLOOD Cell Lines Cells Centrifugation Cloning Vectors Clustered Regularly Interspaced Short Palindromic Repeats DNA Library Exons Flow Cytometry Gene Library Genome Genomic Library K562 Cells Population Group Puromycin Short Hairpin RNA Spleen Focus-Forming Virus

Pooled CRISPR Screen in K562 Cells

Cited 35 times

Morgens D.W., Deans R.M., Li A, & Bassik M.C. (2016). Systematic comparison of CRISPR-Cas9 and RNAi screens for essential genes. Nature biotechnology, 34(6), 634-636.

Publication 2016

Most recents protocols related to «Spleen Focus-Forming Virus»

CD19+ T Cell Cytotoxicity Assay

CD19⁺ mKate2+ A549 cells were produced by lentiviral transduction using pCDH-CMV-CD19 puro and ILV-EF1a-mKate-9-X01(Flash Therapeutics) lentivectors. 1,000 cells were plated 4 d prior to coculture in low-cluster 96-well plates (#7007; Costar) and allowed to form spheroids in RPMI, 10% FBS, and 5% PS. Total CD3⁺ T cells were isolated by negative selection from peripheral blood mononuclear cells (#17951; Stem Cell) and stimulated with Dynabeads at a ratio of 1:3 (beads:cell). Cells were cultured at 1 million per ml in X-VIVO 15 with 5% PS, 5% decomplemented human serum (Sigma-Aldrich), and 50 mM 2-mercaptoethanol (#31350-010; GIBCO). 24 h following TCR stimulation, 100,000 cells in 100 μl were cotransduced with 100 μl of lentivector viral particles (pTRIP-SFFV-GFP or pTRIP-SFFV-GFPRELAK5R) combined with the CAR lentivirus (rLV.EFA.19BBz; Flash Therapeutics) at a multiplicity of infection (MOI) of 10 (corresponding usually to <1 µl). Spinoculation was carried out at 1,200 g for 2 h at 25°C. Cells were expanded for 48 h with fresh media and 300 U/ml IL-2. 3 d following transduction, T cells were stained with CD19 CAR detection reagent (130-115-965; Miltenyi) for 15 min at 4°C, washed twice, and subsequently with streptavidin A647 conjugate (#S21374; Invitrogen) for 30 min at 4°C. Cells were fixed and acquired on a FACSVerse (BD). The next day, Dynabeads were removed by magnetic separation, and 250, 500, or 1,000 CAR+ cells were added per well of A549 spheroids in quadruplicates. To evaluate the impact of IFN-I on CART function, IFNα2a (10,000 U/ml or 1,000 U/ml) was added at the time of coculture. Plates were then placed in an IncuCyteS3 and images were acquired using a 10× objective (1.24 μm/pixel) every 3 h for 5–6 d. Images were analyzed using the IncuCyte S3 software. At the end of the culture, supernatants were harvested and assayed for IFN concentrations.

Jeremiah N., Ferran H., Antoniadou K., De Azevedo K., Nikolic J., Maurin M., Benaroch P, & Manel N. (2023). RELA tunes innate-like interferon I/III responses in human T cells. The Journal of Experimental Medicine, 220(5), e20220666.

Publication 2023

2-Mercaptoethanol A549 Cells CART protein, human Cells Coculture Techniques Homo sapiens Infection Interferon alfa 2a Interferon Type I Lentivirus mKate PBMC Peripheral Blood Mononuclear Cells Serum Spleen Focus-Forming Virus Stem Cells Streptavidin T-Lymphocyte Therapeutics Virion

Molecular Cloning of Engineered Lentiviral Constructs

pSIV3⁺, psPAX2, HXB2 env, CMV-VSVG, pTRIP-SFFV-GFP, and pTRIP-SFFV-GFPIRF3 were previously described (Manel et al., 2010 (link)). pTRIP-SFFV-GFP-RELA was obtained by PCR cloning of RELA (#23255; Addgene) into the pTRIP-SFFV-GFP backbone. RELA K5R and RELA K5Q mutants were generated by subcloning DNA fragments (Twist Biosciences) into the pTRIP-SFFV-GFP-RELA plasmid, resulting in pTRIP-SFFV-GFP-RELA K5R and pTRIP-SFFV-GFP-RELA K5Q. RELA K310R mutant was generated by overlapping PCR mutagenesis in pTRIP-SFFV-GFPRELA plasmid resulting in pTRIP-SFFV-GFP-RELA K310R. pLKO.1-puro-IRF7sh1 (IRF7sh1, 5′-CCCGAGCTGCACGTTCCTATA-3′), pLKO.1-puroIRF7sh5 (IRF7sh5, 5′-CGCAGCGTGAGGGTGTGTCTT-3′), and pLKO.1puro-shLacZ (LacZsh) were previously described (Döring et al., 2021 (link)). HIV-mTagBFP2 and HIV-2 ROD9 ∆env∆nef mTagBFP2⁺ were previously described (Bhargava et al., 2021 (link)). pTRIP-SFFV-GFP (control), pTRIP-SFFV-EGFP-FLAG-cGAS (cGAS), pTRIP-SFFV-EGFP-NLS (NLS), and pTRIP-SFFV-EGFP-NLS-FLAG-cGAS (NLS-cGAS) were previously described (Gentili et al., 2019 (link)). pTRIP-SFF-tagBFP-2A, pTRIP-SFFV–tagBFP-2A-STING, IFNB-pGL3, and pTRH1-NFkB-dscGFP have been previously described (Cerboni et al., 2017 (link)). pTRIP-SFFV-tagBFP-2A-ZebrafishSTING was subcloned from pCOM37_pcDNA4 ZFish STING (de Oliveira Mann et al., 2019 (link)). Human CD19 cDNA (NCBI accession no. NM_001178098; Genescript) was cloned into the pCDH-CMV-MCS-EF1-Puro plasmid (System Biosciences) to create pCDH-CMV-CD19 puro.

Publication 2023

Chromogranin A DNA, Complementary HIV-2 Homo sapiens Interferon, beta Mutagenesis NF-kappa B Paragangliomas 3 Plasmids RELA protein, human Spleen Focus-Forming Virus Vertebral Column

Transcriptional Analysis of RELA Mutant CD4+ T Cells

CD4⁺ T cells transduced with either pTRIP-SFFV-GFP or pTRIP-SFFV-GFP-RELA or pTRIP-SFFV-GFP-RELA K5R and RNA was isolated 4 d after transduction. The analysis was performed by GenoSplice. Analysis of sequencing data quality, reads repartition (e.g., for potential ribosomal contamination), inner distance size estimation, genebody coverage, and strand-specificity of the library were performed using FastQC v0.11.2, Picard-Tools v1.119, Samtools v1.0, and RSeQC v2.3.9. Reads were mapped using STAR v2.7.5a on the human hg38 genome assembly and read count was performed using featureCount from SubRead v1.5.0. Gene expression was estimated as described previously (Paillet et al., 2021 (link)) using Human FAST DB v2022_1 annotations. Only genes expressed in at least one of the two compared conditions were analyzed further. Genes were considered as expressed if their FPKM value was greater than the FPKM of 96% of the intergenic regions (background). Analysis at the gene level was performed using DESeq2 using experiment ID in the DESeq2 GLM model. Genes were considered differentially expressed for fold changes ≥1.5 and P values ≤0.05. Pathway analyses were performed using WebGestalt v0.4.4 merging results from upregulated and downregulated genes only, as well as all regulated genes. Pathways and networks were considered significant with P values ≤0.05. The results of this analysis were compared to those from Zhao et al. (2015) (link) corresponding to the GSE68329 GEO dataset ID. Regulated genes from Zhao et al. (2015) (link) were retrieved using GEO2R using adjusted P value ≤0.05 and fold change ≥ 1.5. Gene expression data have been deposited at GEO (accession no. GSE182647).

Publication 2023

CD4 Positive T Lymphocytes DNA Library Gene Expression Genes Genome, Human Homo sapiens Intergenic Region RELA protein, human Ribosomes Sequence Analysis Spleen Focus-Forming Virus

Lentivirus and SIV-VLP Production Protocols

293FT cells were cultured in DMEM (#61965026; Thermo Fisher Scientific) with 5% PS and 10% FBS. 293FT cells were plated at 0.8 million cells in a 6-well plate and transfected with 3 μg of DNA complexed in 8 μl of TransIT-293 (#MIR2706; Mirus Bio) per well. The ratio of DNA used for transfections was as follows: 0.2 μg HXB2 env, 0.2 μg CMV-VSVG, 1 μg psPAX2, and 1.6 μg of pTRIP-SFFV or pLKO.1 lentivector. The ratio of plasmids for the production of HIV-1 and HIV-2 BFP single-round reporter viruses was 0.2 μg HXB2 env, 0.2 μg CMV-VSVG, and 2.6 μg HIV plasmid. SIV-VLPs were produced using 0.4 μg CMV-VSVG with 2.6 μg pSIV3⁺. Lentiviruses for MDMs were prepared by plating 7 million 293FT cells in T75 flasks and transfected with 8.35 μg DNA complexed in 116 µl PEImax (0.75 mM; #24765; Polyscience) per flask. The ratio of DNA used for transfection includes 3 μg psPAX2, 1.25 μg CMV-VSVG, and 4.10 µg pTRIP-SFFV-GFP. For SIV-VLP production for MDMs, 2.5 μg CMV-VSVG with 8.2 μg pSIV3⁺ was used. 18 h following transfection, media was removed and replenished with fresh media (3 ml for T cells and MDDCs or 8.5 ml for MDMs). 24–26 h later, viral supernatants were harvested, filtered using 0.45 μM filters, and used fresh or stored at −80°C.
For CAR expression, rLV.EFA.19BBz CAR lentivirus was produced using pLV, pHIV-GagPol, and pEnv plasmids and concentrated by ultracentrifugation (Flash Therapeutics). Titer was determined by serial dilution on activated human T cells.

Publication 2023

Cells HIV-1 HIV-2 Homo sapiens Lentivirus Macular Edema, Cystoid methylene dimethanesulfonate Plasmids Spleen Focus-Forming Virus T-Lymphocyte Technique, Dilution Therapeutics Transfection Ultracentrifugation Virus

Lentiviral Transduction of Immune Cells

CD14⁺ cells were plated at 1 million cells/ml and transduced with equal volumes of freshly harvested SIV-VLPs and pTRIP-SFFV vectors in the presence of 8 μg/ml protamine (#P4020; Sigma-Aldrich). CD4⁺ T cells were transduced with lentivectors 24 h post-TCR stimulation in 100 μl of cells (0.2 million cells) and 100 μl of freshly harvested lentivirus in the presence of 8 μg/ml protamine. T cells were spinoculated at 1,200 g for 2 h at 25°C. 0.5 million THP-1 cells in 500 μl media (RPMI, 10% FBS, and 1% PS) were transduced with 500 μl freshly harvested lentivirus in the presence of 8 μg/ml protamine. 1 μg/ml Puromycin (#ant-pr-1; Invivogen) was added 2 d after transduction when cells were transduced with pLKO.1-puro plasmid.

Publication 2023

CD4 Positive T Lymphocytes Cells Cloning Vectors Lentivirus Plasmids Protamines Puromycin Spleen Focus-Forming Virus T-Lymphocyte THP-1 Cells

Top products related to «Spleen Focus-Forming Virus»

Phr sffv dcas9 bfp krab by Addgene

The PHR-SFFV-dCas9-BFP-KRAB is a plasmid construct that expresses a catalytically inactive CRISPR-associated protein 9 (dCas9) fused with a blue fluorescent protein (BFP) and a Krüppel-associated box (KRAB) domain. The dCas9 protein binds to target DNA sequences specified by a guide RNA but does not cleave the DNA.

Pspax2 by Addgene

Sourced in United States, Switzerland, China, Japan, Germany, United Kingdom, France, Canada

PsPAX2 is a packaging plasmid used for the production of lentiviral particles. It contains the necessary genes for lentiviral packaging, but does not contain the viral genome. PsPAX2 is commonly used in lentiviral production workflows.

Phr sffv krab dcas9 p2a mcherry by Addgene

Sourced in United States

PHR-SFFV-KRAB-dCas9-P2A-mCherry is a plasmid construct containing a catalytically-dead Cas9 (dCas9) fused to the KRAB repressor domain and mCherry fluorescent protein. The construct is driven by the SFFV promoter.

Pmd2 g by Addgene

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The PMD2.G is a lab equipment product. It is a plasmid that can be used for various research applications.

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Lentiguide puro by Addgene

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LentiGuide-Puro is a plasmid vector used for CRISPR-Cas9 gene editing. It contains a puromycin resistance gene for selection of transduced cells. The vector is designed for the expression of single guide RNAs (sgRNAs) to direct the Cas9 enzyme to target genomic sequences.

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Puromycin is a laboratory reagent used for selection of mammalian cells expressing a puromycin resistance gene. It acts as an antibiotic that inhibits protein synthesis, leading to cell death in cells that do not express the resistance gene.

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Fetal bovine serum (fbs) by Thermo Fisher Scientific

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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.

What are the different variations or types of Spleen Focus-Forming Virus?

Spleen Focus-Forming Virus (SFFV) is a specific strain of murine retrovirus that has been widely studied for its unique properties. While there is only one primary type of SFFV, researchers have identified various strains or isolates that exhibit slightly different characteristics. These include differences in their ability to induce rapid erythroid proliferation, spleen enlargement, and the development of erythroleukemia in infected mice. Understanding these nuances can help researchers select the most appropriate SFFV strain for their particular study objectives.

How can PubCompare.ai assist in optimizing Spleen Focus-Forming Virus research protocols?

PubCompare.ai's AI-powered platform can be highly beneficial for researchers working with Spleen Focus-Forming Virus (SFFV). The platform allows you to efficiently screen the latest protocol literature, including published studies, pre-prints, and patents. PubCompare.ai's intelligent comparison tools can help you identify the most effective and reproducible SFFV protocols based on key metrics, such as experimental outcomes, methodology details, and researcher feedback. This enables you to select the best protocols to improve the accuracy and efficiency of your SFFV-related studies.

What are some common usage challenges or considerations when working with Spleen Focus-Forming Virus?

One of the key challenges when working with Spleen Focus-Forming Virus (SFFV) is the need to carefully control and monitor the experimental conditions. SFFV is known to induce rapid and dramatic changes in the hematopoietic system of infected mice, which can make it difficult to maintain consistent results. Researchers must pay close attention to factors like viral titer, route of administration, and the specific mouse strain used, as these variables can significantly impact the observed outcomes. Additionally, the development of erythroleukemia in some SFFV models requires additional safety precautions and ethical considerations. PubCompare.ai can help researchers identify the best practices and protocols to overcome these challanges and optimize their SFFV experiments.

How can PubCompare.ai help identify the latest advancements in Spleen Focus-Forming Virus research?

PubCompare.ai's AI-powered platform is designed to help researchers stay up-to-date with the latest developments in their field of study, including advancements in Spleen Focus-Forming Virus (SFFV) research. The platform continuously scans the literature, pre-printes, and patent databases to identify the newest protocols, methods, and findings related to SFFV. By leveraging PubCompare.ai's intelligent search and analysis tools, you can quickly locate the most relevant and impactful SFFV studies, allowing you to incorporate the latest discoveries into your own research. This helps ensure that your SFFV experiments are aligned with the current state of the art and maximizes the efficiency and impact of your work.

More about "Spleen Focus-Forming Virus"

Spleen Focus-Forming Virus (SFFV) is a murine retrovirus that induces rapid erythroid proliferation and spleen enlargement in infected mice.
SFFV primarily targets erythroid progenitor cells, leading to the formation of distinct foci in the spleen.
This unique property has made SFFV a valuable model for studying hematopoietic cell transformation and the molecular mechanisms underlying erythroid differentiation.
SFFV infection can also result in the development of erythroleukemia, providing insights into the genetic and epigenetic factors that contribute to malignant transformation.
Researchers can leverage PubCompare.ai's AI-powered platform to optimize their SFFV research protocols, locate the latest literature, pre-prints, and patents, and identify the best methods and products to improve reproducibility and efficiency.
The platform can help researchers find information on related tools and reagents, such as PHR-SFFV-dCas9-BFP-KRAB, PsPAX2, PHR-SFFV-KRAB-dCas9-P2A-mCherry, PMD2.G, Polybrene, Lipofectamine 2000, LentiGuide-Puro, Puromycin, GeneArt, and FBS.
By utilizing PubCompare.ai's intelligent comparison tools, researchers can compare different SFFV-related protocols and methodologies, identify the most effective approaches, and enhance the quality and reproducibility of their studies.
This can lead to a better understanding of the genetic and epigenetic mechanisms underlying erythroid differentiation and malignant transformation, ultimately advancing the field of hematopoietic research.
The Spleen Focus-Forming Virus (SFFV) is a fasinating model system that continues to provide valuable insights into the complexities of cell transformation and blood cell development.
With the help of PubCompare.ai's AI-powered platform, researchers can streamline their SFFV-focused investigations and make significant progress in this important area of study.