MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia methods
Aim. Evidence-backed execution summary for MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia methods from MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Methods and Findings
reagent used in the protocol.
- Use
- DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F- negative MF. Expression of MPLW515...
What Did the Researchers Do and Find?
reagent used in the protocol.
- Use
- They decoded the DNA sequence of three genes that are known to be involved in how blood cells develop for 45 patients with MF. They looked at DNA from white blood cells, and also from normal cheek cells for comparison. They found that in four of the 45 patients the DNA in the bone marrow, but not the cheek, carried...
DNA Sequence Analysis and Genotyping for MPLW515L
reagent used in the protocol.
- Use
- PCR amplification and DNA sequencing of select exons of MPL, EPOR, and GCSFR was performed using M13-tailed primers as previously described [ ], and specific primer sequences are listed in. Sequence analysis of bidirectional sequence traces was performed using Mutation Surveyor version 2.28 (SoftGenetics, State Col...
Expression Vectors and Cell Culture
reagent used in the protocol.
- Use
- The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis (Quickchange-XL, Stratagene, La Jolla, California, United States) and confirmed by full-length DNA sequencing. 293T cells were grown in DME...
Western Blot Analysis
reagent used in the protocol.
- Use
- Cells were collected and lysed in lysis buffer and separated by electrophoresis as described previously [ ]. Nitrocellulose membrane was blocked in TBST/5% milk and incubated with one of the following antibodies: anti-pSTAT5 (Cell Signaling, Beverly, Massachusetts, United States), anti-pSTAT3 (Cell Signaling), anti-...
JAK Inhibitor Assays
reagent used in the protocol.
- Use
- For cell-proliferation assays, 32D cells stably expressing FIP1L1-PDGFRA [ ] and MPLW515L were grown in RPMI/10% FBS and then incubated with appropriate concentrations of JAK Inhibitor I (Calbiochem, San Diego, California, United States) for 72 hours. 32D cells expressing MPLWT were grown in RPMI/10%FBS/mTPO in the...
Murine Bone Marrow Transplant Assay
reagent used in the protocol.
- Use
- The murine bone marrow transplant assay was performed as previously described [ ]. MSCV retroviral supernatants were titered by determining the percentage of GFP positive cells 48 hours after infection of Ba/F3 and 32D cells (1 mL supernatant used to infect 1 × 10 6 cells). MPLWT and MPLW515L retroviral superna...
Mouse Analysis
reagent used in the protocol.
- Use
- For flow cytometry, cells were washed in PBS + 1% bovine serum albumin and stained with monoclonal antibodies in PBS + 1% bovine serum albumin for 30 minutes on ice. Antibodies used were allophycocyanin-conjugated Gr-1, B220, Ter119, CD8, cKit; and phycoerythrin-conjugated Mac-1, CD19, CD71, CD4, CD41 (B...
DNA Sequence Analysis and Genotyping for MPLW515L
PCR amplification and DNA sequencing of select exons of MPL, EPOR, and GCSFR was performed using M13-tailed primers as previously described [ ], and specific primer sequences are listed in. Sequence analysis of bidirectional sequence traces was performed using Mutation Surveyor version 2.28 (SoftGenetics, State Col...
- Use
- PCR amplification and DNA sequencing of select exons of MPL, EPOR, and GCSFR was performed using M13-tailed primers as previously described [ ], and specific primer sequences are listed in. Sequence analysis of bidirectional sequence traces was performed using Mutation Surveyor version 2.28 (SoftGenetics, State Col...
Expression Vectors and Cell Culture
The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis (Quickchange-XL, Stratagene, La Jolla, California, United States) and confirmed by full-length DNA sequencing. 293T cells were grown in DME...
- Use
- The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis (Quickchange-XL, Stratagene, La Jolla, California, United States) and confirmed by full-length DNA sequencing. 293T cells were grown in DME...
Murine Bone Marrow Transplant Assay
The murine bone marrow transplant assay was performed as previously described [ ]. MSCV retroviral supernatants were titered by determining the percentage of GFP positive cells 48 hours after infection of Ba/F3 and 32D cells (1 mL supernatant used to infect 1 × 10 6 cells). MPLWT and MPLW515L retroviral superna...
- Use
- The murine bone marrow transplant assay was performed as previously described [ ]. MSCV retroviral supernatants were titered by determining the percentage of GFP positive cells 48 hours after infection of Ba/F3 and 32D cells (1 mL supernatant used to infect 1 × 10 6 cells). MPLWT and MPLW515L retroviral superna...
Mouse Analysis
For flow cytometry, cells were washed in PBS + 1% bovine serum albumin and stained with monoclonal antibodies in PBS + 1% bovine serum albumin for 30 minutes on ice. Antibodies used were allophycocyanin-conjugated Gr-1, B220, Ter119, CD8, cKit; and phycoerythrin-conjugated Mac-1, CD19, CD71, CD4, CD41 (B...
- Use
- For flow cytometry, cells were washed in PBS + 1% bovine serum albumin and stained with monoclonal antibodies in PBS + 1% bovine serum albumin for 30 minutes on ice. Antibodies used were allophycocyanin-conjugated Gr-1, B220, Ter119, CD8, cKit; and phycoerythrin-conjugated Mac-1, CD19, CD71, CD4, CD41 (B...
In Vitro Colony-Forming Assays
To assess megakaryocyte ploidy, bone marrow cells were cultured for four days in RPMI/10% FBS containing 50 ng/mL mTPO and 10 mg/mL SCF. Cells were collected and stained with FITC-rat anti-mouse CD41 antibody (BD Biosciences), followed by 50 ug/mL propidium iodide in 0.1% sodium citrate buffer with 50 ug/mL RNAase,...
- Use
- To assess megakaryocyte ploidy, bone marrow cells were cultured for four days in RPMI/10% FBS containing 50 ng/mL mTPO and 10 mg/mL SCF. Cells were collected and stained with FITC-rat anti-mouse CD41 antibody (BD Biosciences), followed by 50 ug/mL propidium iodide in 0.1% sodium citrate buffer with 50 ug/mL RNAase,...
A Murine Model for MPLW515L-Induced Myeloproliferative Disease
Consistent with the histopathologic findings, flow cytometry analysis of MPLW515L bone marrow cells demonstrated an ˜4-fold increase in Mac1+/Gr1+ mature myeloid cells as compared with bone marrow cells from MPLWT animals ( A, upper panels). These findings were also reflected in the splenocyte populations exami...
- Use
- Consistent with the histopathologic findings, flow cytometry analysis of MPLW515L bone marrow cells demonstrated an ˜4-fold increase in Mac1+/Gr1+ mature myeloid cells as compared with bone marrow cells from MPLWT animals ( A, upper panels). These findings were also reflected in the splenocyte populations exami...
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Methods and Findings
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F- negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 × 10 12 /L), marked splenomegaly due to extramedull...
Expression Vectors and Cell Culture
The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis (Quickchange-XL, Stratagene, La Jolla, California, United States) and confirmed by full-length DNA sequencing. 293T cells were grown in DMEM with 10% fetal bovine serum (FBS). Transient co-transfection of 293T cells and generation of retroviral supernatant were performed using Fugene (Roche, Nutley, New Jersey, United States) according to manufacturer's guidelines. 32D and Ba/F3 cells were grown in RPMI medium 1640 containing 10% FBS and 10% WEHI-3B cell supernatant as a source of IL3. UT7 cells were grown in IMDM medium supplemented with 10% FBS and GMCSF (1 ng/mL). 32D cells, Ba/F3 cells, and murine bone marrow cells were transduced with viral supernatant, and UT7 cells were transduced by electroporation (Am...
JAK Inhibitor Assays
For cell-proliferation assays, 32D cells stably expressing FIP1L1-PDGFRA [ ] and MPLW515L were grown in RPMI/10% FBS and then incubated with appropriate concentrations of JAK Inhibitor I (Calbiochem, San Diego, California, United States) for 72 hours. 32D cells expressing MPLWT were grown in RPMI/10%FBS/mTPO in the presence of JAK Inhibitor I. The number of viable cells was determined using CellTiter 96 Aqueous One Cell Proliferation Assay (Promega, San Luis Obispo, California, United States). For Western blotting, cell lines were incubated in the presence of JAK Inhibitor I for four hours, and phosphorylation of relevant proteins was assessed as described above.
Murine Bone Marrow Transplant Assay
The murine bone marrow transplant assay was performed as previously described [ ]. MSCV retroviral supernatants were titered by determining the percentage of GFP positive cells 48 hours after infection of Ba/F3 and 32D cells (1 mL supernatant used to infect 1 × 10 6 cells). MPLWT and MPLW515L retroviral supernatants were able to reproducibly infect 40%-60% of Ba/F3 and 32D cells as assessed by flow cytometry. Balb/C donor mice were treated with 5-Florouracil (150 mg/kg) seven days prior to bone marrow harvest. Bone marrow cells were harvested from donor mice, treated with red blood cell lysis buffer and cultured for 24 hours in transplantation medium (RPMI + 10% FBS + 6 ng/mL IL-3, 10 ng/mL IL-6, and 10 ng/mL stem-cell factor (SCF)). Cells were treated by spin infection with retroviral supernatants (1 mL supernatant per 4 × 10 6 cells, plus polybrene) and centrifuged a...
Mouse Analysis
For flow cytometry, cells were washed in PBS + 1% bovine serum albumin and stained with monoclonal antibodies in PBS + 1% bovine serum albumin for 30 minutes on ice. Antibodies used were allophycocyanin-conjugated Gr-1, B220, Ter119, CD8, cKit; and phycoerythrin-conjugated Mac-1, CD19, CD71, CD4, CD41 (BD Pharmingen, San Diego, California, United States). Flow cytometry analysis was performed on a FACSCalibur instrument (BD Biosciences, San Jose, California, United States) and analyzed with CellQuest software (BD Biosciences). Viability was assessed by incubating cells with 7-AAD (7-amino-actinomycin D) (BD Pharmingen) for five minutes prior to flow cytometry. Cells were gated for viability (using forward/side scatter and 7-AAD) and GFP positivity, and 10,000 events were analyzed from this subset for marker expression.
In Vitro Colony-Forming Assays
To assess megakaryocyte ploidy, bone marrow cells were cultured for four days in RPMI/10% FBS containing 50 ng/mL mTPO and 10 mg/mL SCF. Cells were collected and stained with FITC-rat anti-mouse CD41 antibody (BD Biosciences), followed by 50 ug/mL propidium iodide in 0.1% sodium citrate buffer with 50 ug/mL RNAase, as previously described [ ]. Data was acquired on a FACSCalibur (BD Biosciences) and analyzed with CellQuest software.
A Murine Model for MPLW515L-Induced Myeloproliferative Disease
To assess the in vivo effects of the MPLW515L mutant receptor, we developed a murine model of MPLW515L-induced myeloproliferative disease. Bone marrow cells were transduced with MSCV-IRES-EGFP vectors containing MPLWT or MPLW515L and transplanted into lethally irradiated Balb/C mice. MPLW515L-transduced animals, but not MPLWT-transduced animals, developed a short latency (median latency = 18 days post BMT), fully penetrant, lethal MPD ( A) notable for marked thrombocytosis (mean platelet count = 3.414 × 10 12 /L) as well as leukocytosis (mean WBC = 201 × 10 9 /L) ( B). There was no significant difference in the hematocrit of MPLW515L mice compared with MPLWT mice. At the time of sacrifice, MPLW515L-expressing mice showed splenomegaly and hepatomegaly ( C and D), and staining of bone marrow demonstrated increased reticulin fibrosis in MPLW515L, but not MPLWT-expressing mice (...
A Murine Model for MPLW515L-Induced Myeloproliferative Disease
(A) Kaplan-Meier survival plot of Balb/C mice transduced with MPLW515L ( n = 9) and MPLWT ( n = 6) showing death of all MPLW515L mice between 17 and 32 days post-BMT compared with MPLWT ( n = 6), which were sacrificed for endpoint analysis without evidence of disease.
Measurement outputs
What raw and processed outputs should exist?
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, iden...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The study protocol was reviewed and approved by the Boston (Massachusetts, United States) Children's Hospital Animal Care and Use Committee.
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
PCR amplification and DNA sequencing of select exons of MPL, EPOR, and GCSFR was performed using M13-tailed primers as previously described [ ], and specific primer sequences ar...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
To assess the in vivo effects of the MPLW515L mutant receptor, we developed a murine model of MPLW515L-induced myeloproliferative disease.
from paperScoring or quantification
Quantify the primary readouts for this experiment: DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, iden...; The study protocol was reviewed and approved by the Boston (Massachusetts, United States) Children's Hospital Animal Care and Use Committee.; PCR amplification and DNA sequencing of select exons of MPL, EPOR, and GCSFR was performed using M13-tailed primers as previously described [ ], and specific primer sequences ar...; The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
To assess the in vivo effects of the MPLW515L mutant receptor, we developed a murine model of MPLW515L-induced myeloproliferative disease. Bone marrow cells were transduced with...; Consistent with the histopathologic findings, flow cytometry analysis of MPLW515L bone marrow cells demonstrated an ˜4-fold increase in Mac1+/Gr1+ mature myeloid cells as c...
from paperReporting output
Report representative outputs alongside summary comparisons for DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, iden..., The study protocol was reviewed and approved by the Boston (Massachusetts, United States) Children's Hospital Animal Care and Use Committee., PCR amplification and DNA sequencing of select exons of MPL, EPOR, and GCSFR was performed using M13-tailed primers as previously described [ ], and specific primer sequences ar..., The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis....
inferred from protocolStructured statistical methods
To assess the in vivo effects of the MPLW515L mutant receptor, we developed a murine model of MPLW515L-induced myeloproliferative disease. Bone marrow cells were transduced with...; Consistent with the histopathologic findings, flow cytometry analysis of MPLW515L bone marrow cells demonstrated an ˜4-fold increase in Mac1+/Gr1+ mature myeloid cells as c...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F- negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 × 10 12 /L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.
The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis (Quickchange-XL, Stratagene, La Jolla, California, United States) and confirmed by full-length DNA sequencing. 293T cells were grown in DMEM with 10% fetal bovine serum (FBS). Transient co-transfection of 293T cells and generation of retroviral supernatant were performed using Fugene (Roche, Nutley, New Jersey, United States) according to manufacturer's guidelines. 32D and Ba/F3 cells were grown in RPMI medium 1640 containing 10% FBS and 10% WEHI-3B cell supernatant as a source of IL3. UT7 cells were grown in IMDM medium supplemented with 10% FBS and GMCSF (1 ng/mL). 32D cells, Ba/F3 cells, and murine bone marrow cells were transduced with viral supernatant, and UT7 cells were transduced by electroporation (Amaxa, Gaithersburg, Maryland, United States). Cells were then selected in G418 (1mg/mL). To assess for factor-independent growth and for thrombopoietin hypersensitivity, cells were first washed three times in PBS, and then 1 × 10 5 viable cells/mL were resuspended in appropriate cytokine-free me...
For cell-proliferation assays, 32D cells stably expressing FIP1L1-PDGFRA [ ] and MPLW515L were grown in RPMI/10% FBS and then incubated with appropriate concentrations of JAK Inhibitor I (Calbiochem, San Diego, California, United States) for 72 hours. 32D cells expressing MPLWT were grown in RPMI/10%FBS/mTPO in the presence of JAK Inhibitor I. The number of viable cells was determined using CellTiter 96 Aqueous One Cell Proliferation Assay (Promega, San Luis Obispo, California, United States). For Western blotting, cell lines were incubated in the presence of JAK Inhibitor I for four hours, and phosphorylation of relevant proteins was assessed as described above.
The murine bone marrow transplant assay was performed as previously described [ ]. MSCV retroviral supernatants were titered by determining the percentage of GFP positive cells 48 hours after infection of Ba/F3 and 32D cells (1 mL supernatant used to infect 1 × 10 6 cells). MPLWT and MPLW515L retroviral supernatants were able to reproducibly infect 40%-60% of Ba/F3 and 32D cells as assessed by flow cytometry. Balb/C donor mice were treated with 5-Florouracil (150 mg/kg) seven days prior to bone marrow harvest. Bone marrow cells were harvested from donor mice, treated with red blood cell lysis buffer and cultured for 24 hours in transplantation medium (RPMI + 10% FBS + 6 ng/mL IL-3, 10 ng/mL IL-6, and 10 ng/mL stem-cell factor (SCF)). Cells were treated by spin infection with retroviral supernatants (1 mL supernatant per 4 × 10 6 cells, plus polybrene) and centrifuged at 1,800 g for 90 minutes. The spin infection was repeated 24 hours later. The cells were then washed and resuspended in Hank's balanced salt solution, and injected into lateral tail veins of lethally irradiated (2 × 4.5 Gy [450 rad]) Balb/C recipient mice (Taconic, Germantown, New York, United...
For flow cytometry, cells were washed in PBS + 1% bovine serum albumin and stained with monoclonal antibodies in PBS + 1% bovine serum albumin for 30 minutes on ice. Antibodies used were allophycocyanin-conjugated Gr-1, B220, Ter119, CD8, cKit; and phycoerythrin-conjugated Mac-1, CD19, CD71, CD4, CD41 (BD Pharmingen, San Diego, California, United States). Flow cytometry analysis was performed on a FACSCalibur instrument (BD Biosciences, San Jose, California, United States) and analyzed with CellQuest software (BD Biosciences). Viability was assessed by incubating cells with 7-AAD (7-amino-actinomycin D) (BD Pharmingen) for five minutes prior to flow cytometry. Cells were gated for viability (using forward/side scatter and 7-AAD) and GFP positivity, and 10,000 events were analyzed from this subset for marker expression.
To assess megakaryocyte ploidy, bone marrow cells were cultured for four days in RPMI/10% FBS containing 50 ng/mL mTPO and 10 mg/mL SCF. Cells were collected and stained with FITC-rat anti-mouse CD41 antibody (BD Biosciences), followed by 50 ug/mL propidium iodide in 0.1% sodium citrate buffer with 50 ug/mL RNAase, as previously described [ ]. Data was acquired on a FACSCalibur (BD Biosciences) and analyzed with CellQuest software.
To assess the in vivo effects of the MPLW515L mutant receptor, we developed a murine model of MPLW515L-induced myeloproliferative disease. Bone marrow cells were transduced with MSCV-IRES-EGFP vectors containing MPLWT or MPLW515L and transplanted into lethally irradiated Balb/C mice. MPLW515L-transduced animals, but not MPLWT-transduced animals, developed a short latency (median latency = 18 days post BMT), fully penetrant, lethal MPD ( A) notable for marked thrombocytosis (mean platelet count = 3.414 × 10 12 /L) as well as leukocytosis (mean WBC = 201 × 10 9 /L) ( B). There was no significant difference in the hematocrit of MPLW515L mice compared with MPLWT mice. At the time of sacrifice, MPLW515L-expressing mice showed splenomegaly and hepatomegaly ( C and D), and staining of bone marrow demonstrated increased reticulin fibrosis in MPLW515L, but not MPLWT-expressing mice ( E).
(A) Kaplan-Meier survival plot of Balb/C mice transduced with MPLW515L ( n = 9) and MPLWT ( n = 6) showing death of all MPLW515L mice between 17 and 32 days post-BMT compared with MPLWT ( n = 6), which were sacrificed for endpoint analysis without evidence of disease.
Machine-readable layer
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"name": "MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia methods",
"description": "Evidence-backed execution summary for MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia methods from MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia.",
"totalTime": "PT31230M",
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{
"@type": "HowToStep",
"position": 1,
"name": "Methods and Findings",
"text": "DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F- negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 × 10 12 /L), marked splenomegaly due to extramedull..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Expression Vectors and Cell Culture",
"text": "The MSCV-MPL-Neo and MSCV-MPL-IRES-EGFP retroviral vectors were generously provided by W. Tong and H. Lodish. The MPLW515L mutation was generated using site-directed mutagenesis (Quickchange-XL, Stratagene, La Jolla, California, United States) and confirmed by full-length DNA sequencing. 293T cells were grown in DMEM with 10% fetal bovine serum (FBS). Transient co-transfection of 293T cells and generation of retroviral supernatant were performed using Fugene (Roche, Nutley, New Jersey, United States) according to manufacturer's guidelines. 32D and Ba/F3 cells were grown in RPMI medium 1640 containing 10% FBS and 10% WEHI-3B cell supernatant as a source of IL3. UT7 cells were grown in IMDM medium supplemented with 10% FBS and GMCSF (1 ng/mL). 32D cells, Ba/F3 cells, and murine bone marrow cells were transduced with viral supernatant, and UT7 cells were transduced by electroporation (Am..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "JAK Inhibitor Assays",
"text": "For cell-proliferation assays, 32D cells stably expressing FIP1L1-PDGFRA [ ] and MPLW515L were grown in RPMI/10% FBS and then incubated with appropriate concentrations of JAK Inhibitor I (Calbiochem, San Diego, California, United States) for 72 hours. 32D cells expressing MPLWT were grown in RPMI/10%FBS/mTPO in the presence of JAK Inhibitor I. The number of viable cells was determined using CellTiter 96 Aqueous One Cell Proliferation Assay (Promega, San Luis Obispo, California, United States). For Western blotting, cell lines were incubated in the presence of JAK Inhibitor I for four hours, and phosphorylation of relevant proteins was assessed as described above."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Murine Bone Marrow Transplant Assay",
"text": "The murine bone marrow transplant assay was performed as previously described [ ]. MSCV retroviral supernatants were titered by determining the percentage of GFP positive cells 48 hours after infection of Ba/F3 and 32D cells (1 mL supernatant used to infect 1 × 10 6 cells). MPLWT and MPLW515L retroviral supernatants were able to reproducibly infect 40%-60% of Ba/F3 and 32D cells as assessed by flow cytometry. Balb/C donor mice were treated with 5-Florouracil (150 mg/kg) seven days prior to bone marrow harvest. Bone marrow cells were harvested from donor mice, treated with red blood cell lysis buffer and cultured for 24 hours in transplantation medium (RPMI + 10% FBS + 6 ng/mL IL-3, 10 ng/mL IL-6, and 10 ng/mL stem-cell factor (SCF)). Cells were treated by spin infection with retroviral supernatants (1 mL supernatant per 4 × 10 6 cells, plus polybrene) and centrifuged a..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Mouse Analysis",
"text": "For flow cytometry, cells were washed in PBS + 1% bovine serum albumin and stained with monoclonal antibodies in PBS + 1% bovine serum albumin for 30 minutes on ice. Antibodies used were allophycocyanin-conjugated Gr-1, B220, Ter119, CD8, cKit; and phycoerythrin-conjugated Mac-1, CD19, CD71, CD4, CD41 (BD Pharmingen, San Diego, California, United States). Flow cytometry analysis was performed on a FACSCalibur instrument (BD Biosciences, San Jose, California, United States) and analyzed with CellQuest software (BD Biosciences). Viability was assessed by incubating cells with 7-AAD (7-amino-actinomycin D) (BD Pharmingen) for five minutes prior to flow cytometry. Cells were gated for viability (using forward/side scatter and 7-AAD) and GFP positivity, and 10,000 events were analyzed from this subset for marker expression."
},
{
"@type": "HowToStep",
"position": 6,
"name": "In Vitro Colony-Forming Assays",
"text": "To assess megakaryocyte ploidy, bone marrow cells were cultured for four days in RPMI/10% FBS containing 50 ng/mL mTPO and 10 mg/mL SCF. Cells were collected and stained with FITC-rat anti-mouse CD41 antibody (BD Biosciences), followed by 50 ug/mL propidium iodide in 0.1% sodium citrate buffer with 50 ug/mL RNAase, as previously described [ ]. Data was acquired on a FACSCalibur (BD Biosciences) and analyzed with CellQuest software."
},
{
"@type": "HowToStep",
"position": 7,
"name": "A Murine Model for MPLW515L-Induced Myeloproliferative Disease",
"text": "To assess the in vivo effects of the MPLW515L mutant receptor, we developed a murine model of MPLW515L-induced myeloproliferative disease. Bone marrow cells were transduced with MSCV-IRES-EGFP vectors containing MPLWT or MPLW515L and transplanted into lethally irradiated Balb/C mice. MPLW515L-transduced animals, but not MPLWT-transduced animals, developed a short latency (median latency = 18 days post BMT), fully penetrant, lethal MPD ( A) notable for marked thrombocytosis (mean platelet count = 3.414 × 10 12 /L) as well as leukocytosis (mean WBC = 201 × 10 9 /L) ( B). There was no significant difference in the hematocrit of MPLW515L mice compared with MPLWT mice. At the time of sacrifice, MPLW515L-expressing mice showed splenomegaly and hepatomegaly ( C and D), and staining of bone marrow demonstrated increased reticulin fibrosis in MPLW515L, but not MPLWT-expressing mice (..."
},
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"position": 8,
"name": "A Murine Model for MPLW515L-Induced Myeloproliferative Disease",
"text": "(A) Kaplan-Meier survival plot of Balb/C mice transduced with MPLW515L ( n = 9) and MPLWT ( n = 6) showing death of all MPLW515L mice between 17 and 32 days post-BMT compared with MPLWT ( n = 6), which were sacrificed for endpoint analysis without evidence of disease."
}
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"name": "DNA Sequence Analysis and Genotyping for MPLW515L"
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"name": "Murine Bone Marrow Transplant Assay"
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{
"@type": "HowToTool",
"name": "A Murine Model for MPLW515L-Induced Myeloproliferative Disease"
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"name": "What Did the Researchers Do and Find?"
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"name": "DNA Sequence Analysis and Genotyping for MPLW515L"
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"@type": "HowToSupply",
"name": "Expression Vectors and Cell Culture"
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"name": "Western Blot Analysis"
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"name": "JAK Inhibitor Assays"
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"name": "Murine Bone Marrow Transplant Assay"
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"@type": "ScholarlyArticle",
"headline": "MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia",
"datePublished": "2006",
"author": [
{
"@type": "Person",
"name": "Yana Pikman"
},
{
"@type": "Person",
"name": "Benjamin H Lee"
},
{
"@type": "Person",
"name": "Thomas Mercher"
},
{
"@type": "Person",
"name": "Elizabeth McDowell"
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{
"@type": "Person",
"name": "Benjamin L Ebert"
},
{
"@type": "Person",
"name": "Maricel Gozo"
},
{
"@type": "Person",
"name": "Adam Cuker"
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{
"@type": "Person",
"name": "Gerlinde Wernig"
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"@type": "Person",
"name": "Sandra Moore"
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"@type": "Person",
"name": "Ilene Galinsky"
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"@type": "Person",
"name": "Daniel J DeAngelo"
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"name": "Jennifer J Clark"
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"@type": "Person",
"name": "Stephanie J Lee"
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"@type": "Person",
"name": "Todd R Golub"
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"@type": "Person",
"name": "Martha Wadleigh"
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{
"@type": "Person",
"name": "D. Gary Gilliland"
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"@type": "Person",
"name": "Ross L Levine"
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"identifier": "10.1371/journal.pmed.0030270"
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