double mutant jak2 rl tet2  (MedChemExpress)

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Jak2 V617F deletion abolishes JAK/STAT signaling and abrogates the MPN phenotype. A, Schematic representation of the dual-recombinase Jak2 V617F conditional knock-in/knock-out allele ( Jak2 RL ), the Jak2 RL knock-in allele following Dre recombination, and the null recombined allele following Cre-mediated deletion. Semicircles indicate Rox sequences; triangles indicate lox P sequences. B, Representative Western blot depicting phospho-STAT5 abundance of Dre-mediated Jak2 V617F knock-in (+Dre) vs. Jak2 V617F -deleted (+Dre +Cre) states from isolated splenocytes 7 days following tamoxifen (TAM) administration in comparison with unrecombined (Unrec.) Jak2 RL cells ( n = 2 biological replicates each; representative of n = 2 independent experiments). C, Peripheral blood count trends (weeks 0–24) of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice: WBCs (left), Hct (right; n ≥ 10 per arm; mean ± SEM). Gray bar represents duration of tamoxifen pulse/chow administration. Representative of n = 2 independent transplants. **, P ≤ 0.01; ****, P ≤ 0.0001. D, Kaplan–Meier survival analysis of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice ( n ≥ 12 per arm; log-rank test). Gray bar represents duration of tamoxifen pulse/chow administration. ****, P ≤ 0.0001. E, Spleen weights of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice at timed sacrifice (24 weeks) in comparison with WT control mice (mean ± SEM). Representative of n = 2 independent transplants. ****, P ≤ 0.0001. F, Heat map scaled using Z-scores of serum cytokine/chemokine concentrations of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice harvested at time of sacrifice 18–24 weeks posttransplant in comparison with WT control mice ( n = 4–7 biological replicates per arm pooled from n = 3 transplants). Asterisks denote cytokines with FDR ≤ 0.05. Kruskal–Wallis test with FDR correction. G, Representative hematoxylin and eosin (H&E) and reticulin stains of bone marrow of MPN (Control) vs. tamoxifen ( Jak2 V617F -deleted) treated mice from timed sacrifice at 24 weeks. Representative micrographs of n = 6 individual mouse replicates per arm. All images represented at 400× magnification. Scale bar, 20 μm.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Knock-In, Knock-Out, Western Blot, Isolation, Comparison, Control

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Jak2 V617F reversal impairs the fitness of MPN cells, including MPN stem cells. A, Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (weeks 0–24) of early (3 weeks posttransplant) tamoxifen (TAM; Jak2 V617F -deleted) treated (gold bar) and late (12 weeks posttransplant) tamoxifen-treated (maroon bar) mice ( n = 8 each) in comparison with MPN (dark gray bar; n = 6) mice (mean ± SEM). Gray bars represent duration of tamoxifen pulse/chow administration. Representative of n = 2 independent transplants. **, P ≤ 0.01; ***, P ≤ 0.001. B, Bone marrow–mutant cell fraction within LSK (Lineage − Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage − cKit + Sca1 − Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage − cKit + Sca1 − Cd34 − Fcg − ) compartments of early (3 weeks posttransplant) tamoxifen ( Jak2 V617F -deleted) treated and late (12 weeks posttransplant) tamoxifen-treated mice in comparison with MPN mice at timed sacrifice of 24 weeks ( n = 6–8 individual biological replicates per arm; mean ± SEM). Representative of n = 2 independent transplants. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. C, Gene-set enrichment analysis (GSEA) of significant Hallmark gene sets of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated LSKs isolated 7 days after initiation of tamoxifen ( n = 3–4 biological replicates per arm). D, Volcano plot demonstrating differential gene expression of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated LSKs 7 days following initiation of tamoxifen ( n = 3–4 biological replicates per arm). E, GMP and MEP stem cell frequencies of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated mice 7 days following initiation of tamoxifen ( n = 8 biological replicates per arm across two independent transplants; mean ± SEM). F, Row normalized heat map of RNA-seq data of key erythroid differentiation factor genes from harvested MEPs at baseline (MPN), day 3 (D3), and day 7 (D7) following initiation of tamoxifen ( Jak2 V617F deletion). G, HOMER motif analysis from ATAC-seq data demonstrating decreased accessibility of Gata motif signatures with concomitant increased accessibility of Cebp motif signatures of tamoxifen-treated ( Jak2 V617F -deleted) cKit + bone marrow cells isolated 7 days following initiation of treatment in comparison with MPN cells ( n = 3 biological replicates per arm). Non-Sig., nonsignificant.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Mutagenesis, Comparison, Isolation, Gene Expression, RNA Sequencing

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Differential efficacy of Jak2 V617F deletion compared with JAK inhibitor therapy. A, Scatter plot depicting −log 10 ( P adj )*sign(log 2 Fold Change) of ruxolitinib (RUX) treated vs. tamoxifen (TAM; Jak2 V617F -deleted) treated LSKs (Lineage − Sca1 + cKit + ) in comparison with MPN control LSKs isolated after 7 days of treatment ( n = 2–3 biological replicates per arm); differentially expressed genes as indicated by color (see Supplementary Tables S1 and S3). B, Gene-set enrichment analysis (GSEA) depicting a positive enrichment in heme metabolism in ruxolitinib-treated ( n = 3) vs. negative enrichment in tamoxifen ( Jak2 V617F -deleted) treated ( n = 3) LSKs isolated after 7 days of treatment. C, Box plot of the top leading edge genes in the Hallmark heme metabolism gene set of ruxolitinib-treated (blue) or tamoxifen ( Jak2 V617F -deleted) treated (red) megakaryocytic-erythroid progenitor (MEP; Lineage − cKit + Sca1 − Cd34 − Fcg − ) cells as compared with untreated MPN cohorts. D, Box plots of scATAC-seq motif accessibility for either NFKB1 or REL transcription factors for untreated human JAK2 WT ( n = 188 cells from 4 patients; gray), untreated JAK2 V617F -mutant ( n = 105 cells from 4 patients; gray), and ruxolitinib-treated JAK2 V617F -mutant ( n = 87 cells from 3 patients; blue) HSPCs . P values indicated are from linear mixture model explicitly modeling patient identity as random effect to account for patient-specific effects, followed by likelihood ratio test. ****, P ≤ 0.0001. E, Peripheral blood counts of vehicle (VEH), ruxolitinib (RUX), the type II JAK2 inhibitor CHZ868 (CHZ), or tamoxifen ( Jak2 V617F -deleted) treated mice at the conclusion of a 6-week in vivo trial: WBCs (left), Hct (right; n ≥ 4 each; mean ± SEM). **, P ≤ 0.01; ***, P ≤ 0.001; ****, p ≤ 0.0001. F, Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (0–6 weeks) of vehicle, ruxolitinib, CHZ868, or tamoxifen ( Jak2 V617F -deleted) treated mice ( n ≥ 4 each; mean ± SEM). *, P ≤ 0.05. G, Bone marrow–mutant cell fraction of LSK (Lineage − Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage − cKit + Sca1 − Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage − cKit + Sca1 − Cd34 − Fcg − ) compartments of vehicle, ruxolitinib, CHZ868, or tamoxifen ( Jak2 V617F -deleted) treated mice at the conclusion of the 6-week in vivo trial ( n ≥ 4 each; mean ± SEM). *, P ≤ 0.05; ****, P ≤ 0.0001. H, GSEA depicting a negative enrichment in downregulation of KRAS signaling targets in ruxolitinib-treated ( n = 3) vs. positive enrichment in tamoxifen ( Jak2 V617F -deleted) treated ( n = 3) MEPs isolated after 7 days of respective treatment. I, IHC of phospho-ERK on sectioned bone marrow of vehicle, ruxolitinib, or tamoxifen ( Jak2 V617F -deleted) treated mice following 7 days of treatment ( n = 3 individual biological replicates per arm). All images represented at 400× magnification. Scale bar, 20 μm. J, Quantitative PCR demonstrating relative Ybx1 expression levels from isolated cKit + bone marrow of vehicle vs. ruxolitinib vs. tamoxifen ( Jak2 V617F -deleted) treated mice after 7 days of treatment ( n = 2–4 individual biological replicates per arm; mean ± SEM). *, P ≤ 0.05; **, P ≤ 0.01. E–G, Representative of n = 3 independent experiments.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Comparison, Control, Isolation, Mutagenesis, In Vivo, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer Discovery

Article Title: Jak2 V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms

doi: 10.1158/2159-8290.CD-22-0952

Figure Lengend Snippet: Jak2 V617F dependency with cooperative Tet2 loss. A, Schematic of the experimental setup for the double-mutant Jak2 RL / Tet2 f/f competitive transplants. Downward arrows represent initial pulse tamoxifen (TAM) administration to genetically inactivate Tet2 . B, WBC counts of primary Jak2 RL vs. Tet2 −/− vs. Jak2 RL / Tet2 −/− transplanted mice at 16 weeks posttransplant ( n = 5–6 each; mean ± SEM). Representative of n = 2 independent transplants. *, P ≤ 0.05; ***, P ≤ 0.001. C, Spleen weights of primary Jak2 RL vs. Tet2 −/− vs. Jak2 RL /Tet2 −/− transplanted mice at time of sacrifice ( n = 5–6 each; mean ± SEM). Representative of n = 2 independent transplants. *, P ≤ 0.05; **, P ≤ 0.01. D, Peripheral blood Cd45.2-mutant percent chimerism of Jak2 RL vs. Tet2 −/− vs. Jak2 RL / Tet2 −/− secondary competitive transplant mice at 9 weeks posttransplant ( n ≥ 10 per arm; mean ± SEM). Representative of n = 2 independent transplants. *, P ≤ 0.05. E, Peripheral blood count trends (weeks 0–21) of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− competitive transplant mice: WBCs (left), hematocrit (Hct; right; n = 3–4 per arm; mean ± SEM). Gray bars represent duration of tamoxifen pulse/chow administration. Representative of n = 2 independent transplants. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. F, Fold change from baseline (pretreatment) to posttreatment of Cd45.2-mutant peripheral blood chimerism of Jak2 RL vs. Tet2 −/− vs. Jak2 RL / Tet2 −/− transplanted mice treated for 6 weeks with either vehicle, ruxolitinib (RUX; 60 mg/kg twice daily), or tamoxifen ( Jak2 VF deletion; n = 4–5 per arm; mean ± SEM). *, P ≤ 0.05. G, Reticulin stains of bone marrow from MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− mice at timed sacrifice (21 weeks). Representative micrographs of n = 3 individual mouse replicates per arm. All images represented at 400× magnification. Scale bar, 20 μm. H, Bone marrow–mutant Cd45.2 percent chimerism within the LSK (Lineage − Sca1 + cKit + ) compartment of MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− mice at timed sacrifice (21 weeks; n ≥ 7 biological replicates per arm across two independent transplants; mean ± SEM). *, P ≤ 0.05; ***, P ≤ 0.001. I, Serial replating assay of plated MPN vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL / Tet2 −/− bone marrow cells harvested at timed sacrifice 21 weeks and scored at day 8 after each plating (each sample plated in triplicate, representative of n = 2 independent experiments, mean ± SD). cGy, centigray; KI, knock-in; KO, knock-out; Lin-neg BM, lineage-negative bone marrow; trx, transplant.

Article Snippet: Single-mutant Tet2 −/− or double-mutant Jak2 RL / Tet2 −/− transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily ×4; purchased from MedChemExpress) 6–8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Mutagenesis, Knock-In, Knock-Out

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Schematic representation of the dual-recombinase Jak2 V617F conditional knock- in/knock-out allele ( Jak2 RL ), the Jak2 RL knock-in allele following Dre recombination, and the null recombined allele following Cre-mediated deletion. Semi-circles indicate Rox sequences; triangles indicate lox P sequences. b. Representative western blot depicting phospho-STAT5 abundance of Dre-mediated Jak2 V617F knock-in (+Dre) vs. Jak2 V617F - deleted (+Dre +Cre) states from isolated splenocytes 7 days following tamoxifen administration in comparison to unrecombined (Unrec.) Jak2 RL cells ( n = 2 biological replicates each; representative of n = 2 independent experiments). c. Peripheral blood count trends (weeks 0-24) of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F - deleted) treated mice: white blood cells (WBC; left panel), hematocrit (Hct; right panel) ( n ≥ 10 per arm; mean ± s.e.m). Gray bar represents duration of TAM pulse/chow administration. Representative of n = 2 independent transplants. ** p < 0.01, **** p < 0.0001. d. Kaplan–Meier survival analysis of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice ( n ≥ 12 per arm; Log-rank test). Gray bar represents duration of TAM pulse/chow administration. **** p < 0.0001. e. Spleen weights of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice at timed sacrifice (24 weeks) in comparison to wild-type control mice (mean ± s.e.m.). Representative of n = 2 independent transplants. **** p < 0.0001. f. Heatmap demonstrating cytokine/chemokine concentrations (log10 concentration; pg/mL) in peripheral blood serum of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice in comparison to wild-type control mice ( n = 5 biological replicates per arm). g. Representative hematoxylin and eosin (H&E) and reticulin stains of bone marrow of MPN (Control) vs. tamoxifen ( Jak2 V617F -deleted) treated mice from timed sacrifice 24 weeks. Representative micrographs of n = 6 individual mouse replicates per arm. All images represented at 400X magnification. Scale bar: 20μm.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Knock-In, Knock-Out, Western Blot, Isolation, Comparison, Control, Concentration Assay

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Sanger sequencing of the Jak2 RL locus from genomic DNA of sorted Cre TdTomato+ reporter bone marrow cells following tamoxifen administration ( Jak2 RL + Cre) compared to the non-recombined Jak2 RL locus ( Jak2 RL ) demonstrating retainment of lox / rox sites despite prior Cre exposure. Representative of n = 3 individual biological replicates. b. Schematic of the Dre mRNA Jak2 RL bone marrow electroporation transplant protocol. Lin-neg BM: lineage-negative bone marrow; cGy: centigray. c. Representative polymerase chain reaction (PCR) of genomic DNA isolated from peripheral blood mononuclear cells in primary transplant recipients following Dre mRNA electroporation (+Dre) confirming the presence of Jak2 V617F knock-in bands (KI) in relation to the Jak2 wild-type (WT) allele in comparison to unrecombined (-Dre) Jak2 RL cells. d. Peripheral blood counts of wild-type (WT) vs. Jak2 RL knock-in mice in comparison to the previously published Cre-lox conditional Jak2 V617F knock-in mouse model ( Jak2 Crelox ) at timed sacrifice of 16 weeks: white blood cells (WBC; left panel), hematocrit (Hct; middle panel), platelets (Plt; right panel) ( n = 5 per arm; mean ± s.d.). * p < 0.05, ** p < 0.01, **** p < 0.0001. e. Ter119 + Cd71 + erythroid precursor fractions (left panel) and representative flow cytometry plots (right panel) from harvested spleen cells of Jak2 RL knock-in mice in comparison to wild-type (WT) and the Jak2 Crelox knock-in model ( n = 5 per arm; mean ± s.e.m). Black boxes on flow plots denote Ter119 + erythroid progenitor stages I-IV. ** p < 0.01. f. Bone marrow myeloid progenitor (Lineage - cKit + Sca1 - ) frequencies of Jak2 RL knock-in mice in comparison to wild-type (WT) and the Jak2 Crelox knock-in model ( n = 5 per arm; mean ± s.e.m). ** p < 0.01. g. Spleen weights (left panel) and representative spleens (right panel) of Jak2 RL knock-in mice in comparison to wild-type (WT) and the Jak2 Crelox knock-in model at timed sacrifice 16 weeks post-transplant ( n = 5 per arm; mean ± s.d.). ** p < 0.01, *** p < 0.001. h. Hematoxylin and eosin (H&E) stains of bone marrow and spleen of Jak2 RL knock-in mice in comparison to wild-type controls at timed sacrifice 16 weeks post-transplant. Representative micrographs of n = 5 individual mouse replicates per arm. Bone marrow images represented at 400X magnification (scale bar 20μm); spleen images 100X (scale bar 100μm). d-h. Representative of an n = 3 independent transplants.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Sequencing, Electroporation, Polymerase Chain Reaction, Isolation, Knock-In, Comparison, Flow Cytometry

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Schematic of the bone marrow endothelial cell (BMEC) co-culture assay. Lin-neg BM: lineage-negative BM; EtOH: ethanol vehicle; 4-OHT: 4-hydroxy-tamoxifen. b. Representative Jak2 V617F knock-in and knock-out genotyping by polymerase chain reaction (PCR) of genomic DNA isolated from cultured bone marrow cells following Dre-mediated knock-in (+Dre) and/or subsequent Cre- mediated deletion (+Dre+Cre) in comparison to the unrecombined Jak2 RL allele (CTRL). Representative bands of n = 4 individual replicates per arm. c. Total cell output of cultured wild- type (gray) vs. Jak2 RL knock-in (maroon) cells in comparison to Cre-inducible Jak2 V617F knock-in ( Jak2 Crelox ; gold) cells following 7 days of culture over BMECs in the presence of increasing doses (0 nM - 1000 nM) of 4-hydroxy-tamoxifen (4-OHT) ( n = 3 technical replicates each; mean ± s.d.). ns: not significant, ** p < 0.01, **** p < 0.0001. d. Total cell, e. Mac1 + Gr1 high mature neutrophil, f. Myeloid progenitor (Lineage - cKit + Sca1 - ), and g. Megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ) cell output of Jak2 RL knock-in cells in comparison to wild-type vs. Cre-inducible Jak2 Crelox MPN cells following 7 days of culture over BMECs in the presence of vehicle (VEH) vs. 400 nM 4-hydroxy-tamoxifen (4-OHT) ( n = 3 technical replicates each; mean ± s.d.). *** p < 0.001, **** p < 0.0001. h. Total erythroid precursor cell numbers by Ter119/Cd71 erythroid progenitor flow cytometry (I-IV; left panel) and representative Ter119/Cd71 flow plots (right panel) of cultured Jak2 RL MPN cells following 7 days of culture over BMECs in the presence of vehicle (VEH) vs. 400 nM 4-hydroxy-tamoxifen (4-OHT) vs. 250 nM ruxolitinib (RUX) ( n = 3 technical replicates each; mean ± s.d.). ** p < 0.01, *** p < 0.001, **** p < 0.0001. i. Percentages of Mac1 + Gr1 high Annexin V+ apoptotic cells of Jak2 RL cells in comparison to wild-type vs. Jak2 Crelox cells following 7 days of culture over BMECs in the presence of vehicle (VEH) vs. 200 nM 4-hydroxy-tamoxifen (4-OHT) ( n = 3 technical replicates each; mean ± s.d.). * p < 0.05, ** p < 0.01. c-i. Representative of n = 2 independent experiments.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Co-culture Assay, Knock-In, Knock-Out, Polymerase Chain Reaction, Isolation, Cell Culture, Comparison, Flow Cytometry

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Schematic of the experimental set up for the Jak2 RL knock-in/knock-out transplants. BM: bone marrow; TAM: tamoxifen; cGy: centigray. b. Representative polymerase chain reaction (PCR) demonstrating presence or loss of Jak2 RL knock-in (KI) bands in relation to the Jak2 wild-type (WT) allele from reporter-sorted whole bone marrow mononuclear cells based on different recombined states: Pre-Dre: unrecombined Jak2 RL cells; double-neg: reporter-negative cells following Dre recombination; TdTomato+: Jak2 V617F knock-in population following Dre recombination; GFP+: green fluorescent protein (GFP) population: Jak2 V617F -deleted population (i.e. Dre followed by Cre). c. Jak2 V617F mutant RNA transcript variant allele frequency (VAF) from isolated megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ) cells 7 days following tamoxifen (TAM; Jak2 V617F deletion) treatment in comparison to MPN (CTRL) mice ( n = 3-5 each; mean ± s.d.). **** p < 0.0001. d. Platelet (Plt) counts of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice at timed sacrifice 24 weeks ( n ≥ 10 each; mean ± s.e.m). Representative of n = 2 independent transplants. *** p < 0.001. e. Representative polymerase chain reaction (PCR) demonstrating presence of Jak2 V617F knock-in (KI) bands in relation to the Jak2 wild-type (WT) allele from isolated bone marrow mononuclear cells of transplant recipients previously treated with tamoxifen ( Jak2 V617F deletion) and exhibiting phenotypic features of recurrent MPN ( n = 2) in comparison to those with no recurrent MPN (representative of n = 8 biological replicates). f. Representative hematoxylin and eosin (H&E) stains and Ter119 immunohistochemistry of sectioned spleen of MPN control vs. tamoxifen ( Jak2 V617F -deleted) treated mice at timed sacrifice 24 weeks. Representative micrographs of n = 6 individual mouse replicates per arm. All images represented at 100X magnification. Scale bar: 100μm.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Knock-In, Knock-Out, Polymerase Chain Reaction, Mutagenesis, Variant Assay, Isolation, Comparison, Immunohistochemistry, Control

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Peripheral blood count trend (weeks 0-15) of MPN (CTRL) vs. tamoxifen (TAM) treated mice transplanted with Cre-negative Jak2 RL knock-in cells in competition with Cd45.1 support: white blood cells (WBC; left panel), hematocrit (Hct; middle panel), platelets (PLT; right panel) ( n = 3- 4 each; mean ± s.e.m). Gray bars represent duration of TAM pulse/chow administration. ns = not significant, * p < 0.05. b. Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (weeks 0- 15) of MPN (CTRL) vs. tamoxifen (TAM) treated mice transplanted with Cre-negative Jak2 RL knock-in cells in competition with Cd45.1 support ( n = 3-4 each; mean ± s.e.m). Gray bar represents duration of TAM pulse/chow administration. c. Spleen weights (left panel) and representative isolated spleens (right panel) of MPN (CTRL) vs. tamoxifen (TAM) treated mice transplanted with Cre-negative Jak2 RL knock-in cells in competition with Cd45.1 support at timed sacrifice 15 weeks ( n = 3-4 each; mean ± s.e.m). d. Total bone marrow (BM) cellularity (femur) and e. Myeloid progenitor (Lineage - cKit + Sca1 - ) frequencies of MPN (CTRL) vs. tamoxifen (TAM) treated mice transplanted with Cre-negative Jak2 RL knock-in cells in competition with Cd45.1 support at timed sacrifice 15 weeks ( n = 3-4 each; mean ± s.e.m). f. Bone marrow mutant cell fraction within LSK (Lineage - Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage - cKit + Sca1 - Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ) compartments of MPN (CTRL) vs. tamoxifen (TAM) treated mice transplanted with Cre-negative Jak2 RL knock-in cells in competition with Cd45.1 support at timed sacrifice 15 weeks ( n = 3-4 each; mean ± s.e.m.). g. Hematoxylin and eosin (H&E) stains of bone marrow (BM) of MPN (CTRL) vs. tamoxifen (TAM) mice transplanted with Cre-negative Jak2 RL knock-in cells in competition with Cd45.1 support at timed sacrifice 15 weeks. Representative micrographs of n = 3-4 individual mouse replicates. Images represented at 400X magnification. Scale bar: 20μm. a-g. Representative of n = 3 independent experiments.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Knock-In, Mutagenesis, Isolation

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (weeks 0-24) of early (3 weeks post-transplant) tamoxifen (TAM; Jak2 V617F -deleted) treated (gold bar) and late (12 weeks post- transplant) tamoxifen treated (maroon bar) mice ( n = 8 each) in comparison to MPN (CTRL; dark gray bar; n = 6) mice (mean ± s.e.m.). Gray bars represent duration of TAM pulse/chow administration. Representative of n = 2 independent transplants. ** p < 0.01, *** p < 0.001. b. Bone marrow mutant cell fraction within LSK (Lineage - Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage - cKit + Sca1 - Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ) compartments of early (3 weeks post-transplant) tamoxifen (TAM; Jak2 V617F -deleted) treated and late (12 weeks post-transplant) tamoxifen treated mice in comparison to MPN (CTRL) mice at timed sacrifice 24 weeks ( n = 6-8 individual biological replicates per arm; mean ± s.e.m.). Representative of n = 2 independent transplants. ** p < 0.01, *** p < 0.001, **** p < 0.0001. c. Gene-set enrichment analysis (GSEA) of significant Hallmark gene sets of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated LSKs isolated 7 days following initiation of TAM ( n = 3-4 biological replicates per arm). d. Volcano plot demonstrating differential gene expression of MPN (CTRL) vs. TAM ( Jak2 V617F -deleted) treated LSKs 7 days following initiation of TAM ( n = 3-4 biological replicates per arm). e. GMP and MEP stem cell frequencies of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice 7 days following initiation of TAM ( n = 8 biological replicates per arm across 2 independent transplants; mean ± s.e.m.). f. Row normalized heatmap of RNA-sequencing data of key erythroid differentiation factor genes from harvested MEPs at baseline (CTRL), day 3 (D3) and day 7 (D7) following initiation of TAM ( Jak2 V617F deletion). g. HOMER motif analysis from ATAC-seq data demonstrating decreased accessibility of Gata motif signatures with concomitant increased accessibility of Cebp motif signatures of TAM treated ( Jak2 V617F -deleted) cKit + bone marrow cells isolated 7 days following initiation of treatment in comparison to MPN (CTRL) cells ( n = 3 biological replicates per arm).

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Mutagenesis, Comparison, Isolation, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Schematic of the experimental set up for the Jak2 RL competitive transplant studies. TAM: tamoxifen; cGy: centigray. b. Peripheral blood count trends (weeks 0-24) of early (3 weeks post-transplant) tamoxifen (TAM; Jak2 V617F -deleted) treated (gold) and late (12 weeks post-transplant) tamoxifen treated (maroon) mice in comparison to MPN (CTRL; gray) mice: white blood cells (WBC; left panel), hematocrit (Hct; right panel) ( n = 6-8 per arm; mean ± s.e.m). Gray bars represent duration of TAM pulse/chow administration. Representative of n = 2 independent transplants. ** p < 0.01, *** p < 0.001, **** p < 0.0001. c. Peripheral blood Mac1 + Gr1 high mutant Cd45.2 percent chimerism trend (weeks 0-24) of early (3 weeks post- transplant) tamoxifen (TAM; Jak2 V617F -deleted) treated (gold) and late (12 weeks post-transplant) tamoxifen treated (maroon) mice in comparison to MPN (CTRL; gray) mice ( n = 6-8 each, mean ± s.e.m.). Gray bars represent duration of TAM pulse/chow administration. Representative of n = 2 independent transplants. ** p < 0.01, *** p < 0.001. d. Bone marrow mutant Cd45.2 percentage within the long-term hematopoietic stem cell (LT-HSC; Lineage - Sca1 + cKit + Cd150 + Cd48 - ) compartment of early (3 weeks post-transplant) tamoxifen (TAM; Jak2 V617F -deleted) treated and late (12 weeks post-transplant) tamoxifen treated mice in comparison to MPN (CTRL) mice at timed sacrifice 24 weeks ( n = 6-8 each; mean ± s.e.m.). Representative of n = 2 independent transplants. ** p < 0.01, **** p < 0.0001. e. Representative flow cytometry plots demonstrating mutant Cd45.2 to competitor Cd45.1 percentage of gated LSK (Lineage - Sca1 + cKit + ) cells of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice at timed sacrifice 24 weeks. Representative of n = 6-8 biological replicates per arm. f. Hematocrit (Hct) and g. Peripheral blood mutant Cd45.2 fraction of recipient mice transplanted with MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated unfractionated donor bone marrow cells at 12 weeks post-transplant ( n = 5 each; mean ± s.e.m). Representative of n = 2 independent experiments. * p < 0.05, ** p < 0.01. h. Polymerase chain reaction (PCR) demonstrating presence of a Jak2 V617F knock-in (KI) band in relation to the Jak2 wild-type (WT) allele in 1 of 5 recipients (red asterisk) transplanted with tamoxifen (TAM; Jak2 V617F -deleted) treated donor cells demonstrating recurrence of MPN phenotype in comparison to donor mice transplanted with MPN (CTRL) bone marrow donor cells.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Comparison, Mutagenesis, Flow Cytometry, Polymerase Chain Reaction, Knock-In

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Heatmap (left panel) and gene set enrichment analysis (GSEA; right panel) demonstrating negative enrichment of STAT5 gene targets, including negative regulators of JAK/STAT signaling, in LSK (Lineage - Sca1 + cKit + ) cells harvested 3 days (D3) +/- 7 days (D7) following initiation of tamoxifen ( Jak2 V617F deletion) treatment in comparison to MPN (CTRL) cells. b. GSEA demonstrating negative enrichment in KEGG:MAPK signaling targets from harvested LSKs 7 days following initiation of tamoxifen treatment in comparison to MPN control (CTRL) LSKs. c. Mac1 + immunohistochemistry (IHC) of sectioned bone marrow (BM; left panel) and bone marrow Mac1 + Gr1 high frequencies by flow cytometry (right panel) of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice at 7 days following treatment ( n = 4 each; mean ± s.e.m.). Representative micrographs of n = 4 individual mouse replicates each. Images represented at 400X magnification. Scale bar: 20μm. ** p < 0.01. d. Total erythroid progenitor bone marrow cell numbers, and e. Representative flow cytometry plots of Ter119/Cd71 erythroid progenitor populations of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice 7 days following treatment ( n = 4-7 per arm; mean ± s.e.m.). * p < 0.05, ** p < 0.01, *** p < 0.001. f. Burst-forming unit erythroid (BFU-E) colony output of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated marrow isolated 7 days following TAM administration and scored at day 8 after plating (each sample plated in triplicate, mean ± s.e.m.). g. Normalized ATAC-Seq signal at the EpoR locus (top panel) with associated quantified normalized peak counts (bottom panel) demonstrating decreased accessibility in tamoxifen (TAM; Jak2 V617F -deleted) treated cKit + bone marrow cells 7 days following initiation of TAM in comparison to MPN (CTRL) cells. d-f. Representative of n = 2 independent experiments.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Comparison, Control, Immunohistochemistry, Flow Cytometry, Isolation

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Scatter plot depicting log2FoldChange of ruxolitinib (RUX) treated vs. tamoxifen (TAM; Jak2 V617F -deleted) treated LSKs (Lineage - Sca1 + cKit + ) in comparison to MPN control LSKs isolated after 7 days of treatment ( n = 2-3 biological replicates per arm); differentially expressed genes as indicated by color (see Supplemental Tables 1 and 3). b. Gene set enrichment analysis (GSEA) depicting a positive enrichment in heme metabolism in ruxolitinib (RUX) treated ( n = 3) vs. negative enrichment in tamoxifen (TAM; Jak2 V617F -deleted) treated ( n = 3) LSKs isolated after 7 days of treatment. c. Row normalized heatmap of RNA-sequencing data of ruxolitinib (RUX) treated (blue) or tamoxifen (TAM; Jak2 V617F -deleted) treated (red) megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ) cells in comparison to MPN (CTRL) cohorts (gray). d. Box plots of single-cell ATAC-seq motif accessibility for either NFKB1 or REL transcription factors for untreated human JAK2 wild-type ( n = 188 cells from 4 patients; gray), untreated JAK2 V617F -mutant ( n = 105 cells from 4 patients; gray), and ruxolitinib- treated JAK2 V617F -mutant ( n = 87 cells from 3 patients; blue) HSPCs (from Myers, R.M. and Izzo, F. et al ., bioRxiv, 2022). P values indicated are from linear mixture model explicitly modeling patient identity as random effect to account for patient-specific effects, followed by likelihood ratio test. **** p < 0.0001. e. Peripheral blood counts of vehicle (VEH), ruxolitinib (RUX), or tamoxifen (TAM; Jak2 V617F -deleted) treated mice at the conclusion of a 6-week in vivo trial: white blood cells (WBC; left panel), hematocrit (Hct; right panel) ( n ≥ 4 each; mean ± s.e.m). ** p < 0.01, *** p < 0.001, **** p < 0.0001. f. Peripheral blood (PB) mutant Cd45.2 percent chimerism trend (0-6 weeks) of vehicle (VEH), ruxolitinib (RUX), or tamoxifen (TAM; Jak2 V617F -deleted) treated mice ( n ≥ 4 each; mean ± s.e.m.). * p < 0.05. g. Bone marrow mutant cell fraction of LSK (Lineage - Sca1 + cKit + ), granulocytic-monocytic progenitor (GMP; Lineage - cKit + Sca1 - Cd34 + Fcg + ), and megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ) compartments of vehicle (VEH), ruxolitinib (RUX), or tamoxifen (TAM; Jak2 V617F -deleted) treated mice at the conclusion of the 6-week in vivo trial ( n ≥ 4 each; mean ± s.e.m). * p < 0.05, **** p < 0.0001. h. GSEA depicting a negative enrichment in down-regulation of KRAS signaling targets in ruxolitinib (RUX) treated ( n = 3) vs. positive enrichment in tamoxifen (TAM; Jak2 V617F -deleted) treated ( n = 3) MEPs isolated following 7 days of respective treatment. i. Immunohistochemistry of phospho-ERK on sectioned bone marrow of vehicle, ruxolitinib, or tamoxifen ( Jak2 V617F - deleted) treated mice following 7 days of treatment ( n = 3 individual biological replicates per arm). All images represented at 400X magnification. Scale bar: 20μm. j. Quantitative polymerase-chain reaction demonstrating relative Ybx1 expression levels from isolated cKit + bone marrow of vehicle (VEH) vs. ruxolitinib (RUX) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated mice following 7 days of treatment ( n = 2-4 individual biological replicates per arm; mean ± s.e.m). * p < 0.05, ** p < 0.01. e-g. Representative of n = 3 independent experiments.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Comparison, Control, Isolation, RNA Sequencing, Mutagenesis, In Vivo, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Normalized read counts of representative JAK/STAT targets from LSK (Lineage - Sca1 + cKit + ) cells isolated 7 days following ruxolitinib (RUX) vs. tamoxifen (TAM; Jak2 V617F deletion) treatment compared to MPN controls (CTRL) ( n = 2-4 per arm; mean ± s.e.m.). LSK (Lineage - Sca1 + cKit + ; left panel), granulocytic-monocytic progenitor (GMP; Lineage - cKit + Sca1 - Cd34 + Fcg + ; middle panel), and megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ; right panel) bone marrow frequencies of vehicle (VEH), ruxolitinib (RUX), or tamoxifen (TAM; Jak2 V617F -deleted) treated mice 7 days following respective treatment ( n = 3- 5 per arm; mean ± s.e.m.). Data representative of n = 2 independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001. c. Heatmap of single-cell ATAC-seq motif accessibilities for NFκB transcription factors for untreated ( n = 105 cells from 4 patients) and ruxolitinib treated ( n = 87 cells from 3 patients) human JAK2 V617F mutant hematopoietic stem/progenitor cells (HSPCs) (from Myers, R. and Izzo, F. et al ., bioRxiv, 2022). d. Schematic of the competitive transplant set up for the Jak2 RL in vivo drug studies. cGy: centigray. e. Platelet (PLT) counts and f. Spleen weights of vehicle (VEH), ruxolitinib (RUX), or tamoxifen (TAM; Jak2 V617F -deleted) treated mice at the conclusion of the 6-week in vivo trial ( n ≥ 4 each; mean ± s.e.m). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Representative of n = 3 independent experiments. g. Hematoxylin and eosin (H&E) stains of bone marrow and spleen sections from vehicle, ruxolitinib, or tamoxifen ( Jak2 V617F -deleted) treated mice at the conclusion of the 6-week in vivo trial. Representative micrographs of n = 4 individual mouse replicates per arm. Bone marrow images represented at 400X magnification (scale bar 20μm); spleen images 100X (scale bar 100μm). h. Box plots of single-cell ATAC-seq motif accessibility for FOS (left panel) and JUN (right panel) transcription factors for untreated ( n = 188 cells from 4 patients; light gray) or ruxolitinib treated ( n = 55 cells from 3 patients; light blue) JAK2 wild-type human HSPCs in comparison to untreated ( n = 105 cells from 4 patients; dark gray) or ruxolitinib-treated ( n = 87 cells from 3 patients; dark blue) JAK2 V617F -mutant HSPCs (from Myers, R.M. and Izzo, F. et al ., bioRxiv, 2022). P values indicated are from linear mixture model explicitly modeling patient identity as random effect to account for patient-specific effects, followed by likelihood ratio test. ** p < 0.01, **** p < 0.0001.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Isolation, Mutagenesis, In Vivo, Comparison

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Schematic of the experimental set up for the double-mutant Jak2 RL /Tet2 f/f competitive transplants. TAM: tamoxifen; KI: knock- in; KO: knock-out; Lin-neg BM: lineage-negative bone marrow; cGy: centigray. Downward arrows represent initial pulse TAM administration to genetically inactivate Tet2 . b. White blood cell (WBC) counts of primary Jak2 RL vs. Jak2 RL /Tet2 -/- transplanted mice at 16 weeks post- transplant ( n = 5 each; mean ± s.d.). Representative of an n = 2 independent transplants. * p < 0.05. c. Spleen weights of primary Jak2 RL vs. Jak2 RL /Tet2 -/- transplanted mice at time of sacrifice ( n = 5 each; mean ± s.d.). Representative of an n = 2 independent transplants. * p < 0.05. d. Peripheral blood Cd45.2 mutant percent chimerism of Jak2 RL vs. Jak2 RL /Tet2 -/- secondary competitive transplant mice at 16 weeks post-transplant ( n = 7-8 each; mean ± s.d.). Representative of an n = 2 independent transplants. ** p < 0.01. e. Peripheral blood count trends (weeks 0-21) of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL /Tet2 -/- competitive transplant mice: white blood cells (WBC; left panel), hematocrit (Hct; right panel) ( n = 3-4 per arm; mean ± s.e.m). Gray bars represent duration of TAM pulse/chow administration. Representative of n = 2 independent transplants. ** p < 0.01, *** p < 0.001, **** p < 0.0001. f. Percent change in Cd45.2 mutant peripheral blood chimerism pre- vs. post-tamoxifen ( Jak2 V617F - deletion) treatment of Jak2 RL vs. Jak2 RL /Tet2 -/- mice in relation to Cd45.1 competitor cells ( n = 3- 4 per arm; mean ± s.e.m). Representative of n = 2 independent transplants. g. Reticulin stains of bone marrow from MPN control vs. tamoxifen ( Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL /Tet2 - /- mice at timed sacrifice 21 weeks. Representative micrographs of n = 3 individual mouse replicates per arm. All images represented at 400X magnification. Scale bar: 20μm. h. Bone marrow mutant Cd45.2 percent chimerism within the LSK (Lineage - Sca1 + cKit + ) compartment of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL /Tet2 -/- mice at timed sacrifice 21 weeks ( n ≥ 7 biological replicates per arm across 2 independent transplants; mean ± s.e.m). * p < 0.05, *** p < 0.001. i. Serial replating assay of plated MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL /Tet2 -/- bone marrow cells harvested at timed sacrifice 21 weeks and scored at day 8 after each plating (each sample plated in triplicate, representative of n = 2 independent experiments, mean ± s.d.).

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Mutagenesis, Knock-In, Knock-Out, Control

Journal: bioRxiv

Article Title: Jak2 V617F Reversible Activation Shows an Essential Requirement for Jak2 V617F in Myeloproliferative Neoplasms

doi: 10.1101/2022.05.18.492332

Figure Lengend Snippet: a. Hematocrit (Hct; left panel) and platelet counts (Plt; right panel) of Jak2 RL vs. Jak2 RL /Tet2 -/- primary transplanted mice at 16 weeks post-transplant ( n = 5; mean ± s.d.). Representative of an n = 2 independent transplants. b. Serial replating assay of Jak2 RL vs. Jak2 RL /Tet2 -/- bone marrow cells in relation to wild-type bone marrow. Colonies were counted at day 8 after each plating (each sample plated in triplicate, n = 2 independent experiments, mean ± s.d.). c. Total Cd45.2 cell (left panel) and Mac1 + Gr1 high mature neutrophil cell (right panel) output of Jak2 RL vs. Jak2 RL /Tet2 -/- cells following 7 days of culture over bone marrow endothelial cells (BMECs) ( n = 3 replicates per arm; mean ± s.d.). Representative of n = 2 independent experiments. * p < 0.05, ** p < 0.01. d. Spleen weights (left panel) and representative isolated spleens (right panel) of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL /Tet2 -/- mice ( n ≥ 7 biological replicates per arm across 2 independent transplants; mean ± s.d.). * p < 0.05, **** p < 0.0001. e. Total bone marrow (BM) cellularity (femur), and f. Bone marrow mutant cell fraction within the granulocytic-monocytic progenitor (GMP; Lineage - cKit + Sca1 - Cd34 + Fcg + ; left panel) and megakaryocytic-erythroid progenitor (MEP; Lineage - cKit + Sca1 - Cd34 - Fcg - ; right panel) compartments of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F - deleted) treated Jak2 RL vs. Jak2 RL /Tet2 -/- mice ( n ≥ 7 biological replicates per arm across 2 independent transplants; mean ± s.e.m.). * p < 0.05, ** p < 0.01, *** p < 0.001. g. Representative Jak2 V617F knock-in (left panel) and Tet2 -/- excision genotyping (right panel) by polymerase chain reaction (PCR) of genomic DNA isolated from whole bone marrow of MPN (CTRL) vs. tamoxifen (TAM; Jak2 V617F -deleted) treated Jak2 RL vs. Jak2 RL /Tet2 -/- mice from time of sacrifice demonstrating presence or loss of Jak2 V617F knock-in (KI) and/or Tet2 -/- excised (knock-out; KO) bands in relation to respective wild-type (WT) bands. Representative bands of n = 4-7 individual replicates per arm across 2 independent transplants.

Article Snippet: Double-mutant Jak2 RL /Tet2 f/f transplants/electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily x4; purchased from MedChemExpress) 6-8 weeks prior to harvest and excision confirmed prior to Dre electroporation.

Techniques: Isolation, Mutagenesis, Knock-In, Polymerase Chain Reaction, Knock-Out