. 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

Cai Ping Koh¹, Avinash Govind Bahirvani², Chelsia Qiuxia Wang³, Tomomasa Yokomizo⁴, Cherry Ee Lin Ng², Linsen Du², Vinay Tergaonkar⁵, Dominic Chih-Cheng Voon⁶, Hiroaki Kitamura², Hiroki Hosoi⁷, Takashi Sonoki⁸, Mok Meng Huang Michelle², Lii Jye Tan⁹, Akiko Niibori-Nambu¹⁰, Yi Zhang¹¹, Archibald S Perkins¹¹, Zakir Hossain², Daniel G Tenen², Yoshiaki Ito², Byrappa Venkatesh¹², Motomi Osato¹³

Affiliations

¹ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Department of Biochemistry, Faculty of Medicine, Quest International University, Perak 30250, Malaysia.
² Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore.
³ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore.
⁴ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; International Research Center for Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan.
⁵ Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore.
⁶ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Cancer Research Institute, Kanazawa University, Ishikawa 920-1192, Japan; Institute for Frontier Science Initiative, Kanazawa University, Ishikawa 920-1192, Japan.
⁷ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Department of Hematology/Oncology, Wakayama Medical University, Wakayama, Japan.
⁸ Department of Hematology/Oncology, Wakayama Medical University, Wakayama, Japan.
⁹ Department of Forensic Medicine, Hospital Raja Permaisuri Bainun, Ipoh, Perak Daruk Ridzuan, Malaysia.
¹⁰ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Institute of Life Sciences, Kumamoto University, Kumamoto, Japan.
¹¹ Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, United States of America.
¹² Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore. Electronic address: mcbbv@imcb.a-star.edu.sg.
¹³ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; International Research Center for Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan; Department of Pediatrics, National University of Singapore, Singapore 119228, Singapore. Electronic address: csimo@nus.edu.sg.

PMID: 36384171
PMCID: PMC10492510
DOI: 10.1016/j.gene.2022.147049

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

Cai Ping Koh et al. Gene. 2023.

. 2023 Jan 30:851:147049.

doi: 10.1016/j.gene.2022.147049. Epub 2022 Nov 13.

Authors

Affiliations

¹ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Department of Biochemistry, Faculty of Medicine, Quest International University, Perak 30250, Malaysia.
² Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore.
³ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore.
⁴ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; International Research Center for Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan.
⁵ Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore.
⁶ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Cancer Research Institute, Kanazawa University, Ishikawa 920-1192, Japan; Institute for Frontier Science Initiative, Kanazawa University, Ishikawa 920-1192, Japan.
⁷ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Department of Hematology/Oncology, Wakayama Medical University, Wakayama, Japan.
⁸ Department of Hematology/Oncology, Wakayama Medical University, Wakayama, Japan.
⁹ Department of Forensic Medicine, Hospital Raja Permaisuri Bainun, Ipoh, Perak Daruk Ridzuan, Malaysia.
¹⁰ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Institute of Life Sciences, Kumamoto University, Kumamoto, Japan.
¹¹ Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, United States of America.
¹² Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore. Electronic address: mcbbv@imcb.a-star.edu.sg.
¹³ Cancer Science Institute of Singapore, National University of Singapore, Singapore 117456, Singapore; International Research Center for Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan; Department of Pediatrics, National University of Singapore, Singapore 119228, Singapore. Electronic address: csimo@nus.edu.sg.

PMID: 36384171
PMCID: PMC10492510
DOI: 10.1016/j.gene.2022.147049

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 CreER^T2 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 CreER^T2 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-CreER^T2 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.

PubMed Disclaimer

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.. Highly efficient HSC marking in adult and child eR1-CreER^T2 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-CreER^T2 Tg mouse. (C) Schematic diagram demonstrates analysis schedule. 3-week-old or 6-week-old eR1-CreER^T2 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-CreER^T2 Tg; Rosa26-LSL-tdTomato mice. (E) Flow cytometry analysis of BM cells from eR1-CreER^T2 Tg; Rosa26-LSL-tdTomato mouse at 48 h after TMX injection. Note the highest percentage of tdTomato⁺ cells in CD34⁻Flt3⁻KSL fraction (LT-HSC). (F) Flow cytometry analysis of BM cells from eR1-CreER^T2 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-CreER^T2 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.. eR1-CreER^T2 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⁻, CD34⁻Flt3⁻KSL T⁺ and CD34⁻Flt3⁻KSL T⁻ populations. (C) & (D) Chimerism in PB of recipient mice transplanted with tdTomato⁺ (red) or tdTomato⁻ (black) cells in KSL or CD34⁻Flt3⁻KSL 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 CD34⁻Flt3⁻KSL (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-CreER^T2 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.. *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-CreER^T2 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.. Fetal HSCs and hemogenic ECs are marked in eR1-driven CreER^T2 Tg mice.**
(A) Schematic diagram demonstrates activation of eR1-driven CreER^T2 at embryonic stage. (B) Long-term HPCs marking in PB of eR1-CreER^T2 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-CreER^T2 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-CreER^T2 Tg; Rosa26-LSL-tdTomato mouse embryo (lateral view). tdTomato (green) signals represent CreER^T2 activity. Within DA, the rostral (R) and caudal (C) regions are indicated. (D) Image enlarged from boxed area in (C). eR1-driven CreER^T2 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-CreER^T2 Tg; Rosa26-LSL-tdTomato E10.5 embryos. Control mice were eR1-CreER^T2 Tg embryo (littermate of test cohort) and TMX non-injected eR1-CreER^T2 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.. eR1 is active in leukemia stem cells.**
(A) Kaplan-Meier survival curve for eR1-CreER^T2 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-CreER^T2 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-CreER^T2 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⁺TG^high and c-Kit⁺TG^low population. (E) Survival curve for mice transplanted with T and TG population. (F) Survival curve for mice transplanted with different amount of c-Kit⁺TG^high and c-Kit⁺TG^low 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-CreER^T2; T, tdTomato⁺; TG, tdTomato⁺EGFP⁺; TG high, c-Kit⁺tdTomato⁺EGFP^high; TG low, c-Kit⁺tdTomato⁺EGFP^low.

**Fig. 6.. Activation of Kras^G12D at distinct ages demonstrates different tumor spectra.**
(A) Survival curve for eR1-CreER^T2 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.. Ablation of the Evi1 gene in fetal, child and adult HSCs leads to distinct phenotypes.**
eR1-CreER^T2 Tg; Evi1^fl3/fl3mice were sacrificed one month after TMX injection. Control mice were the TMX injected littermates of eR1-CreER^T2 Tg; Evi1^fl3/fl3 mice without eR1-CreER^T2 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, Evi1^fl3/fl3; eR1-CreER^T2 Tg negantive; lanes 4-6, Evi1^fl3/fl3; R1-CreER^T2 Tg positive mice. (B) Flow cytometric profiles of BM cells from WT and eR1-CreER^T2 Tg; Evi1^fl3/fl3 mice at one month after TMX injection. (C) Percentages of LT-HSC, ST-HSC and MPP in eR1-CreER^T2 Tg; Evi1^Δ/Δ adult cohort mice 1 month after TMX injection. (D) Percentages of LT-HSCs in the embryo, child and adult eR1-CreER^T2 Tg; Evi1^Δ/Δ mouse cohorts at 1 year after TMX injection. (E) Platelet counts in the embryo, child and adult eR1-CreER^T2 Tg; Evi1^Δ/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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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

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases

Miscellaneous