retrovirus production pbob ef1  (Addgene inc)

Journal: eLife

Article Title: Molecular basis for the role of disulfide-linked αCTs in the activation of insulin-like growth factor 1 receptor and insulin receptor

doi: 10.7554/eLife.81286

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , p-gag/pol , Addgene , #14887 , Retrovirus production.

Techniques: Transfection, Construct, Expressing, Purification, Modification, Plasmid Preparation, Mutagenesis, Recombinant, Sequencing, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Software, Magnetic Beads, In Vitro, Binding Assay, Immunofluorescence

Journal: Nature Communications

Article Title: RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4

doi: 10.1038/s41467-022-34558-1

Figure Lengend Snippet: a Colony-forming units (CFUs) of various MLL fusions per 10,000 cells at third- and fourth-round passages under myeloid conditions ( n = 9: Vector, MLL-ΔFP, MLL-AF4, MLL-mAf4; n = 7: MLL-ENL; n = 6: MLL-AF10). Hoxa9 expression normalized to that of Gapdh of first round colonies is shown as the relative value of the vector control (set to 1) ( n = 12, Vector, MLL-ΔFP, MLL-AF4, MLL-mAf4; n = 8: MLL-ENL; n = 7: MLL-AF10). b Leukemogenic potential of MLL-AF4 and MLL-mAf4 in vivo. Murine HSPCs were transduced with MLL-AF4 constructs and transplanted into syngeneic mice (mock, n = 6; MLL-AF4, n = 10; MLL-mAf4, n = 30; MLL-mAf4 secondary transplantation n = 5). c mRNA expression of the MLL fusion genes in 293 T cells analyzed using a qPCR probe for the EPS region in the pMSCV neo plasmids ( n = 3). d Western blotting of MLL fusions in 293 T cells transfected with the MLL fusion expression vectors in c . Anti-beta tubulin (TUBB) (an internal standard) and neomycin phosphatase II (NPTII) antibodies (a gene transduction control) were included for comparison. e Virus particle production by the MLL fusion expression vectors was quantitated using qRT-PCR with the MSCV-EPS probe and absolute quantification methods ( n = 4). f Transduction of recombinant viruses carrying various MLL fusion genes in murine HSPCs were determined using qPCR probes for MSCV-EPS and the murine Gapdh locus ( n = 7: Vector, MLL-ENL, MLL-AF4, MLL-mAf4, Mock; n = 6: MLL-ΔFP; n = 4: MLL-AF10). g Cell numbers of murine HSPCs infected with various retroviruses carrying MLL fusion genes after 5 days of selection with G418 ( n = 3). h Relative mRNA expression of MLL fusion genes after antibiotic selection using qRT-PCR with the MSCV-EPS probe ( n = 3). i Western blotting of HSPCs transduced with retroviruses carrying MLL fusion genes cultured in G418 for 5 days, as shown in d . Data are presented as the mean ± SD of biologically independent replicates ( a , c , e , f , g , h ). P -value was calculated by one-way ANOVA followed by Tukey’s test ( a , c , e , f , g ). Western blotting was performed on two biological replicates ( d , i ). See also Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Various gene constructs were generated using restriction enzyme digestion or PCR-based mutagenesis and cloned into the pMSCV-neo (for retrovirus production) (Clontech), pCDH-MSCV-MCS-EF1-Hygro (for lentivirus production/transient expression), or the pCMV5 vectors (for transient expression).

Techniques: Plasmid Preparation, Expressing, Control, In Vivo, Transduction, Construct, Transplantation Assay, Western Blot, Transfection, Comparison, Virus, Quantitative RT-PCR, Recombinant, Infection, Selection, Cell Culture

Journal: Nature Communications

Article Title: RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4

doi: 10.1038/s41467-022-34558-1

Figure Lengend Snippet: a Domain swapping mutants of various MLL-AF4 mutants are shown in blue (mouse), white (human), and green (synonymous mutations). RNA and amino acid sequences of the post-transcriptional regulatory sequence (PTRS) of human AF4 (hPTRS) and its corresponding sequences of mouse AF4 (mPTRS) and the synonymous mutant (sPTRS). The minimum PTRS is indicated with a red rectangle. b Western blotting of MLL-AF4 mutants in 293 T cells, as described in Fig. . c Transforming ability of MLL-AF4 mutants under myeloid conditions, as described in Fig. ( n = 8: Vector, MLL-ΔFP, MLL-AF4, MLL-mAf4; n = 5: MLL-AF4 sPTRS; n = 4: MLL-AF4 mPTRS, MLL-mAf4 hPTRS). Hoxa9 expression normalized to that of Gapdh of first round colonies is shown as the relative value of the MLL-mAf4 (set to 100) ( n = 9, Vector, MLL-AF4, MLL-mAf4; n = 8: MLL-ΔFP; n = 6: MLL-AF4 sPTRS; n = 5: MLL-AF4 mPTRS, MLL-mAf4 hPTRS). d Virus particle production by the MLL-AF4 mutant expression vectors. Virus particle production was quantitated as described in Fig. ( n = 4). e Relative transduction units of retroviruses carrying various MLL-AF4 mutant genes in murine HSPCs were determined as described in Fig. ( n = 7, Vector, MLL-AF4, MLL-mAf4; n = 6: MLL-ΔFP, Mock; n = 3: MLL-AF4 sPTRS). f Cell numbers of MLL-AF4 -transduced murine HSPCs after 5 days of G418 selection ( n = 3). g Transforming ability of MLL-AF4 mutants under an ex vivo lymphoid culture condition. Cell numbers per 10,000 cells at the third passage is shown ( n = 3). h Western blotting of the various MLL-AF4 synonymous mutants in 293 T cells, as described in Fig. . i Transforming ability of various MLL-AF4 synonymous mutants under myeloid conditions. CFUs per 10,000 cells in third- and fourth-round culture is shown ( n = 4). Hoxa9 expression normalized to Gapdh is shown as the relative value of MLL-mAF4 (set to 100) ( n = 4). Data are presented as the mean ± SD of indicated biologically independent replicates ( c , d , e , f , g , i P -value was calculated by one-way ANOVA followed by Tukey’s test ( c , d , e , f , i ). Western blotting was performed on two biological replicates ( b , h ). See also Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Various gene constructs were generated using restriction enzyme digestion or PCR-based mutagenesis and cloned into the pMSCV-neo (for retrovirus production) (Clontech), pCDH-MSCV-MCS-EF1-Hygro (for lentivirus production/transient expression), or the pCMV5 vectors (for transient expression).

Techniques: Sequencing, Mutagenesis, Western Blot, Plasmid Preparation, Expressing, Virus, Transduction, Selection, Ex Vivo

Journal: Nature Communications

Article Title: RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4

doi: 10.1038/s41467-022-34558-1

Figure Lengend Snippet: a Leukemogenic potential of MLL-AF4 sPTRS in vivo. Murine HSPCs were transduced with retrovirus for the MLL-AF4 sPTRS construct and transplanted into syngeneic mice. MLL-AF4 sPTRS, n = 30. The B/M-MPAL and T-ALL cases were excluded as retrovirus genome integration was not detected (Supplementary Fig. ). The MLL-AF4 and MLL-mAf4 data (Fig. ) are shown for comparison. b The immunophenotype of MLL-AF4 sPTRS-mediated leukemia. Bone marrow cells from mice with MLL-AF4 sPTRS-mediated leukemia were analyzed via flow cytometry for the indicated markers. Unstained control is indicated in blue and stained sample indicated in red. c The expression of myeloid markers, Gr1 and Cd11b, in AML cells of MLL-AF4 sPTRS-leukemia. MLL-mAF4- and MLL-ENL-mediated AMLs were included for comparison. d qRT-PCR using bone marrow cells from mice with MLL-AF4 sPTRS-mediated leukemia. Expression levels normalized to Gapdh are shown relative to those of HSPCs. Data are presented as the mean ± SD of PCR triplicates. See also Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Various gene constructs were generated using restriction enzyme digestion or PCR-based mutagenesis and cloned into the pMSCV-neo (for retrovirus production) (Clontech), pCDH-MSCV-MCS-EF1-Hygro (for lentivirus production/transient expression), or the pCMV5 vectors (for transient expression).

Techniques: In Vivo, Transduction, Construct, Comparison, Flow Cytometry, Control, Staining, Expressing, Quantitative RT-PCR