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. 2023 Jan 30:851:147049.
doi: 10.1016/j.gene.2022.147049. Epub 2022 Nov 13.

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells

Affiliations

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells

Cai Ping Koh et al. Gene. .

Abstract

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Keywords: AML1; Enhancer; Hematopoietic stem cell; Leukemia stem cell; RUNX1.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.. Highly efficient HSC marking in adult and child eR1-CreERT2 mice.
(A) Schematic diagram of the mouse Runx1 locus showing eR1 location (red arrow). Black bars represent exons while grey boxes indicate UTR. (B) Transgene construct used to generate the eR1-CreERT2 Tg mouse. (C) Schematic diagram demonstrates analysis schedule. 3-week-old or 6-week-old eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mice were analyzed at 48 h (initial target cells), 1 month (lineage tracing) and 1 year (long-term label retaining cells) after TMX injection. (D) Expected dynamics of tdTomato+ cells in eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mice. (E) Flow cytometry analysis of BM cells from eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mouse at 48 h after TMX injection. Note the highest percentage of tdTomato+ cells in CD34Flt3KSL fraction (LT-HSC). (F) Flow cytometry analysis of BM cells from eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mouse at 1 month after TMX injection. All lineage marker-positive cells exhibit tdTomato signals. (G) Long-term kinetics of tdTomato+ cells in PB of eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mice with single 0.2 mg/g TMX injection into 6-week-old mice (black line) or six times 0.2 mg/g TMX into 3-week-old mice (grey line). 5-FU challenge was conducted at one month after TMX injection (dotted lines). (H) Labeling efficiency in BM at 1 month after TMX injection with indicated dosages to 3-week-old mice. Abbreviations: TMX, tamoxifen; HSC, hematopoietic stem cell; HPC, hematopoietic cell; BM, bone marrow; PB, peripheral blood; 5-FU, 5-fluorouracil; UTR, untranslated region; LT-HSC, long-term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte and macrophage progenitor; MEP, megakaryocyte and erythroid progenitor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2.
Fig. 2.. eR1-CreERT2 system allows for further HSC enrichment in LT-HSCs.
(A) Schematic diagram depicts BMT procedure to examine eR1 activity-based enrichment of HSCs. (B) Gating strategy to isolate KSL T+, KSL T, CD34Flt3KSL T+ and CD34Flt3KSL T populations. (C) & (D) Chimerism in PB of recipient mice transplanted with tdTomato+ (red) or tdTomato (black) cells in KSL or CD34Flt3KSL fractions at different time points. Each line indicates percentage of chimerism in an individual recipient mouse. (E) Limiting dilution analysis to calculate HSC frequencies in tdTomato+ population (red diamonds) or tdTomato population (black squares) within CD34Flt3KSL (LT-HSC) fraction at six months after BMT. Mice were considered negative when chimerism in PB was<0.5% in any examined lineage. (F) HSC frequencies were calculated using Poisson statistic with extreme limiting dilution analysis (ELDA) (Hu and Smyth 2009). (G) Long-term marking in different stem cell and progenitor fractions in the bone marrow or peripheral blood from 6-week-old cohort of eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mice. Abbreviations: T, tdTomato; PB, peripheral blood; BM, bone marrow; BMT, bone marrow transplantation; HSC, hematopoietic stem cells; LT-HSC, long term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
Fig. 3.. in situ visualization of HSCs in child and adult mouse bone marrow.
(A-D) Bones from 3-week-old (A) or 3-month-old (C) eR1-EGFP Tg mice were obtained for immunostaining. Bones were stained with anti-GFP (green) and anti-c-Kit (red) antibodies. Results shown in (B) and (D) were obtained from quantification of cells in (A) and (C), respectively. P-value was obtained from unpaired t test. At least five fields from each region were counted. EGFP+c-Kit+ HSCs were distributed equally throughout the whole BM in 3-week-old mouse while EGFP+ c-Kit+ HSCs were localized at metaphysis (trabecular region) with greater abundance compared to diaphysis in 3-month-old mouse. (E-I) Single dose 0.2 mg/g of TMX were administered into 4-month-old eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mice. Bones were harvested 48 h after TMX injection and subjected to immunostaining. Bones were stained with anti-tdTomato (green) and anti-c-Kit (red) antibodies. Images were taken from (E) trabecular region and (F) diaphysis. (G), (H), and (I) are enlarged images taken from metaphysis (trabecular region). Abbreviations: TMX, tamoxifen; HSCs, hematopoietic stem cells; GFP, green fluorescence protein; EGFP, enhanced green fluorescence protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4.
Fig. 4.. Fetal HSCs and hemogenic ECs are marked in eR1-driven CreERT2 Tg mice.
(A) Schematic diagram demonstrates activation of eR1-driven CreERT2 at embryonic stage. (B) Long-term HPCs marking in PB of eR1-CreERT2 Tg; Rosa26-LSL-tdTomato progeny mice. The progeny mice were delivered from pregnant mothers injected with TMX on 9.5–10.5 or 14.5-d.p.c. (C) Whole-mount immunostaining of eR1-CreERT2 Tg; Rosa26-LSL-tdTomato E10.5 embryo with anti-CD31 (magenta) and anti-tdTomato (green) antibodies at 24 h after TMX injection. Three-dimensional reconstruction of the eR1-CreERT2 Tg; Rosa26-LSL-tdTomato mouse embryo (lateral view). tdTomato (green) signals represent CreERT2 activity. Within DA, the rostral (R) and caudal (C) regions are indicated. (D) Image enlarged from boxed area in (C). eR1-driven CreERT2 expression pattern in middle of DA within AGM region was observed. (E) Image shows ECs of DA. Some ECs were stained with anti-tdTomato antibody. Clustered HPCs emerge from ventral (F) and dorsal (G) artery wall in AGM region. Arrows depict newly generated HPC clusters from ventral and dorsal artery wall. (H) Flow cytometry plots display the percentage of tdTomato+ cells in eR1-CreERT2 Tg; Rosa26-LSL-tdTomato E10.5 embryos. Control mice were eR1-CreERT2 Tg embryo (littermate of test cohort) and TMX non-injected eR1-CreERT2 Tg; Rosa26-LSL-tdTomato embryo. Abbreviations: TMX, tamoxifen; PB, peripheral blood; UA, umbilical artery; UV, vitelline artery; AGM, aorta-gonad-mesonephros; d.p.c., days post coitum; DA, dorsal aorta; H, heart; FL, fetal liver, HPCs, hematopoietic cells; ECs, endothelial cells; FL, fetal liver. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5.
Fig. 5.. eR1 is active in leukemia stem cells.
(A) Kaplan-Meier survival curve for eR1-CreERT2 Tg; Kras-LSL-G12D mice after TMX injection. p-value obtained from Log-rank (Mantel-Cox) test is shown. (B) Comparison of Cre leakage and induction efficiency between Mx1(p)-Cre Tg and eR1-CreERT2 Tg. Induction was performed in adult mice (6-week-old). For Mx1(p)-Cre Tg mice, 250 μg of pIpC was administered inperitoneally every alternate day for a total of 3 doses. eR1-CreERT2 Tg mice were injected with a single dose of TMX (0.2 mg/g) Each line represents percentage of tdTomato+ cells in PB of individual mouse at 1 week after induction. Asterisks represent statistical significance (*** P < 0.0001, in unpaired Student’s t-test). (C) Schematic diagram depicts BMT procedure to evaluate eR1 activity in leukemia initiating cells. BM cells were harvested from diseased mice and sorted out for BMT. (D) Gating strategy to isolate T, TG, c-Kit+TGhigh and c-Kit+TGlow population. (E) Survival curve for mice transplanted with T and TG population. (F) Survival curve for mice transplanted with different amount of c-Kit+TGhigh and c-Kit+TGlow population. Abbreviations: HSC, hematopoietic stem cell; TMX, tamoxifen; PB, peripheral blood; BM, bone marrow; IP, intraperitoneally; BMT, bone marrow transplantation; K, Kras-LSL-G12D; C, eR1-CreERT2; T, tdTomato+; TG, tdTomato+EGFP+; TG high, c-Kit+tdTomato+EGFPhigh; TG low, c-Kit+tdTomato+EGFPlow.
Fig. 6.
Fig. 6.. Activation of KrasG12D at distinct ages demonstrates different tumor spectra.
(A) Survival curve for eR1-CreERT2 Tg; Kras-LSL-G12D upon TMX injection at different ages. Log-rank (Mantel-Cox) test was used for calculating statistical significance.(B) Comparison of tumor spectra caused by activation of oncogenic Kras at different ages.(C) Representative flow cytometry results of diseased mice in fetal, child and adult cohorts.(D) Differences in percentage of cell surface lineage markers amongst fetal, child and adult cohorts. Asterisks or hashes represent statistical significance (*, P < 0.01; **, P < 0.001; *** P < 0.0001, represents unpaired Student’s t-test; #, P < 0.01; # #, P < 0.001; # # #, P < 0.0001, # # # #, P < 0.00001, represents F test)Abbreviations: TMX, tamoxifen; w/o, without.
Fig. 7.
Fig. 7.. Ablation of the Evi1 gene in fetal, child and adult HSCs leads to distinct phenotypes.
eR1-CreERT2 Tg; Evi1fl3/fl3mice were sacrificed one month after TMX injection. Control mice were the TMX injected littermates of eR1-CreERT2 Tg; Evi1fl3/fl3 mice without eR1-CreERT2 transgene. (A) Genotype PCR for Evi1 gene to detect the presence of floxed or excised exon 3. Genomic DNA from BM cells were amplified by PCR using primers (Supplemental Table 3). Lanes 1-3, Evi1fl3/fl3; eR1-CreERT2 Tg negantive; lanes 4-6, Evi1fl3/fl3; R1-CreERT2 Tg positive mice. (B) Flow cytometric profiles of BM cells from WT and eR1-CreERT2 Tg; Evi1fl3/fl3 mice at one month after TMX injection. (C) Percentages of LT-HSC, ST-HSC and MPP in eR1-CreERT2 Tg; Evi1Δ/Δ adult cohort mice 1 month after TMX injection. (D) Percentages of LT-HSCs in the embryo, child and adult eR1-CreERT2 Tg; Evi1Δ/Δ mouse cohorts at 1 year after TMX injection. (E) Platelet counts in the embryo, child and adult eR1-CreERT2 Tg; Evi1Δ/Δ mouse cohorts at 1 year after TMX injection. (*, P < 0.01; **, P < 0.001). Abbreviations: TMX, tamoxifen; BM, bone marrow; PB, peripheral blood; WT, wild-type; Evi1, Ecotropic virus integration site 1; LT-HSC, long term hematopoietic stem cell; ST-HSC, short term hematopoietic stem cell; MPP, multipotent progenitors.

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