ADAR1 is required for hematopoietic progenitor cell survival via RNA editing

Abstract

Adenosine Deaminase Acting on RNA 1 (ADAR1) is an RNA-editing enzyme that converts adenosine to inosine, following RNA transcription. ADAR1's essential role in embryonic development, especially within the hematopoietic lineage, has been demonstrated in knock-out mice. However, a specific role for ADAR1 in adult hematopoietic progenitor cells (HPCs) remains elusive. In this report, we show that ADAR1 is required for survival of differentiating HPCs as opposed to more primitive cells in adult mice by multiple strategies targeting floxed ADAR1 for deletion by Cre recombinase. As a consequence, ADAR1-deficient hematopoietic stem cells (HSCs) were incapable of reconstituting irradiated recipients although being phenotypically present in the recipient bone marrow. While an effect on HSCs cannot be completely ruled out, the preferential effect of ADAR1 absence on HPCs over more primitive hematopoietic cells was consistent with the increased expression of ADAR1 within HPCs, as well as the inability of ADAR1-deficient HPCs to form differentiated colonies and increased apoptotic fraction during ex vivo culture. Moreover, we have obtained direct evidence that ADAR1 functions in HPCs via an RNA-editing dependent mechanism. Therefore, ADAR1 plays an essential role in adult hematopoiesis through its RNA editing activity in HPCs.

Hierarchical hematopoiesis is a highly regulated process in which hematopoietic stem cells (HSCs) symmetrically or asymmetrically differentiate into hematopoietic progenitor cells (HPCs) that are directly responsible for the production of all lineages within the blood (1). The balance between HSCs and HPCs depends on the developmental stage, age, as well as physiological and pathological conditions of the organism. Distinct molecular programs in HSCs as opposed to HPCs are the key to maintenance of homeostasis within the hematopoietic cascade and their disruption may contribute to pathological states, such as leukemogenesis. However, few molecules have been clearly demonstrated to have distinct roles in HSCs versus HPCs.

RNA editing recodes RNA molecules thereby posttranscriptionally regulating gene function in eukaryotic cells (2, 3). As the enzymes responsible for one type of RNA editing, the adenosine deaminases acting on RNA (ADAR) convert adenosine (A) residues to inosine (I) specifically on double-stranded RNAs thereby generating RNA molecules not encoded in the genome (4). Of the three members of the ADAR family in mammals (5), disruption of ADAR1 in mice leads to embryonic lethality, likely due to defective hematopoiesis in the fetal liver (6–8). Because ADAR1^−/− embryos die at 11–12 days post coitus, it was not possible to define the role of ADAR1 during and after definitive hematopoiesis in these mice. A recent study confirmed the previous finding of dysregulated hematopoiesis within the fetal liver, as well as adult bone marrow by ADAR1-deficient cells (9). However, the cellular mechanism underlying this phenomenon remains elusive, in particular the role of ADAR1 in HPCs as opposed to HSCs. By conditional deletion of floxed ADAR1 alleles we have demonstrated the inability of ADAR1-deficient HSCs to reconstitute irradiated recipient bone marrow despite their engraftment in the bone marrow. ADAR1-deficient HPCs failed to form differentiated colonies ex vivo, most likely due to increased apoptosis within these cells and a lack of ADAR1's RNA editing activity. HPCs are therefore dependent on ADAR1 for their survival and function during adult hematopoiesis.

Results

Conditional Deletion of ADAR1 in Hematopoietic Cells.

To determine the efficiency and functional consequence of ADAR1 deletion by Cre recombinase, hematopoietic cells isolated from 16-week old mice harboring floxed ADAR1 alleles (ADAR1^lox/lox) (8) were transduced with a Murine Stem Cell Virus (MSCV) (10) carrying Cre and GFP (MSCV-Cre) or GFP alone (MSCV) (Fig. 1 B and C). PCR confirmed efficient deletion of ADAR1^lox/lox alleles in hematopoietic cells transduced with MSCV-Cre (ADAR1^Δ/Δ) (Fig. 1D). Moreover, 82% of single sorted HSC-enriched GFP⁺Lineage⁻c-Kit⁺Sca-1⁺ (LKS⁺) cells isolated by micromanipulation were found to be ADAR1^Δ/Δ as early as 24 to 30 h after MSCV-Cre transduction (Fig. 1E). To functionally validate ADAR1 deletion in hematopoietic cells, in vitro proliferation of transduced Lin⁻ bone marrow cells was measured. While the total cell number in the ADAR1^lox/lox group increased 5-fold over 1 week of culture, ADAR1^Δ/Δ cells decreased in number by 75% within the first 4 days of culture (Fig. 1F) similar to the results observed in previous studies (6, 9). Thus retroviral delivery of Cre recombinase efficiently deleted ADAR1 in hematopoietic cells.

Fig. 1.

Conditional gene deletion of ADAR1 in adult hematopoietic cells. (A) Relative positions of the Lox/P elements within the ADAR1 catalytic domains are shown before and after Cre recombination. (B) Diagram of the MSCV and MSCV-Cre vectors. LTR: long terminal repeat; ψ: packaging signal; PGK: phosphoglycerate kinase promoter; IRES: internal ribosome entry site; GFP: green fluorescent protein. (C) Retroviral transduction procedure and experimental design. (D) Confirmation of ADAR1 gene deletion by Cre in whole cell populations by PCR; floxed and deleted ADAR1 alleles (8) are indicated. (E) A representative PCR result for ADAR1 deletion within single GFP⁺LKS⁺ cells 24–30 h following Cre transduction. One of three independent experiments with similar results is shown here. (F) Impaired growth of hematopoietic cells in vitro in the absence of ADAR1. The GFP⁺ cell number in liquid culture (IMDM containing 10% FBS, SCF, Flt3 ligand and TPO) was monitored, starting at 48 h after transduction and lasting for 7 days. Data shown are mean + SD in triplicate wells from one of four experiments. Cell morphology at the seventh day of culture is shown under the curve.

Lack of Hematopoietic Reconstitution but Preservation of Phenotypic HSCs in ADAR1^Δ/Δ Transplant Recipients.

To study the specific role of ADAR1 in HSCs versus HPCs, the in vivo reconstituting ability of ADAR1^Δ/Δ cells was determined by transplantation into irradiated NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NOD/SCID-γc^null) (11) mice due to the mixed background of the ADAR1^lox/lox mice and unavailability of a congenic strain. One hundred thousand to 1.5 × 10⁵ GFP⁺ ADAR1^Δ/Δ or ADAR1^lox/lox bone marrow cells were transplanted into sublethally irradiated (3.5 Gy) NOD/SCID-γc^null recipients and multilineage engraftment in the peripheral blood was monitored for up to 3 months. NOD/SCID-γc^null mice transplanted with ADAR1^lox/lox cells transduced with MSCV or wild-type cells transduced with MSCV-Cre were used as controls. While control cells expressing ADAR1 were able to reconstitute up to 90% of the peripheral blood in transplant recipients (Fig. 2 A and B), engraftment of ADAR1^Δ/Δ cells was less than 1% in all host mice (Fig. 2A). Engraftment levels in the thymus and spleen were consistent with that of the peripheral blood (Table S1). Flow cytometric analysis of the blood (Fig. 2C) and flow and histological studies of the spleen (Fig. S1) showed an absence of ADAR1^Δ/Δ cells in the periphery. Bone marrow engraftment was significantly higher in control recipients compared to ADAR1^Δ/Δ recipients, in which the donor-derived cells were less than 0.1% (Fig. 2 D and E and Table S1). A defect in HSC homing was not observed as CFSE-labeled ADAR1^Δ/Δ cells were present in the bone marrow 1 day following transplantation (Fig. S2).

Fig. 2.

Elimination of hematopoietic reconstitution and preservation of phenotypic hematopoietic stem cells in transplant recipients upon ADAR1 deletion. (A) Total percentage engraftment of GFP+ cells in the peripheral blood 9–12 weeks following transplantation. There is a significant difference between ADAR1^Δ/Δ and control groups (P < 0.001, by Mann-Whitney, n = 9–13). The data were pooled from three independent experiments. (B) Multilineage distribution of donor-derived cells that was only detectable in the control group due to low overall engraftment of ADAR1^Δ/^Δ cells. (C) Representative flow cytometric analysis of peripheral blood engraftment in ADAR1^lox/lox and ADAR1^Δ/^Δ recipients. The donor-derived cells are CD45.1⁺GFP⁺ positive, as indicated in the upper square. (D) Percent bone marrow engraftment in mice killed 13 weeks after transplantation on a log scale. n = 9 and 11 for ADAR1^Δ/^Δ and control groups respectively (P < 0.001 by Mann-Whitney test). (E) Multilineage analysis of donor derived cells in the bone marrow for the control recipients. (F) Increased percentage of primitive cells in ADAR1^Δ/^Δ transplant recipients. Flow cytometric plots of Lin⁻ gated donor-derived LKS⁺ cells, indicated in the overlaid square, from control and ADAR1^Δ/^Δ recipients 13 weeks after transplantation. The number shown is the percentage of LKS⁺ cells in the total donor-derived cell population. (G) Average percentage of LKS⁺ cells within the donor-derived cells (P < 0.01, n = 6–7). (H) The absolute yield of donor derived LKS⁺ cells in transplant recipients. There is no statistical difference between the two groups by Student's t test. (I) Independent assessment for the preservation of HSCs following transplantation by SLAM markers. Representative flow cytometric plots show long-term (6 month) engraftment of ADAR1^Δ/wt and ADAR1^Δ/Δ HSCs defined by the SLAM markers (Lin⁻CD48⁻CD150⁺). The number in the square indicates the percentage of HSCs within the donor derived Lin⁻ cells. (J) The frequency of SLAM HSCs within the donor-derived cell population (P < 0.01, n = 3–4). (K) Absolute yield of SLAM HSCs in recipient mice (P > 0.05, n = 3–4).

Interestingly, within this small population of donor-derived cells in the bone marrow of ADAR1^Δ/Δ recipients, the percentage of HSC-enriched LKS⁺ cells was more than 40-fold higher than that in control recipients (Fig. 2 F and G). However, the absolute number of LKS⁺ cells was not statistically different between the two groups (Fig. 2H). A similar lack of hematopoietic reconstitution despite the presence of phenotypically-defined HSCs in the bone marrow was observed when HSC-enriched ADAR1^Δ/Δ LKS⁺ cells were transplanted into irradiated recipients (Fig. S3).

Given that the LKS phenotype becomes less reliable for HSC identification in transplant recipients (12) and retroviral transduction can have a negative impact on HSCs (13), SLAM markers (14–16) were used to quantify long term repopulating HSCs in mice transplanted with cells carrying floxed ADAR1 alleles and Cre driven by an estrogen responsive promoter (ER-Cre-ADAR^lox/lox). Fifteen hundred LKS⁺ cells heterogenous for CD45 isoform expression (CD45.1⁺/CD45.2⁺) from ER-Cre-ADAR1^lox/lox or ER-Cre-ADAR1^lox/wild mice as a control were sorted and transplanted into 10-Gy irradiated C57/BL6 recipients together with 100,000 competitor cells (CD45.2). Three months after transplantation, floxed ADAR1 alleles were deleted by oral administration of tamoxifen. One month later, peripheral blood engraftment in ER-Cre-ADAR1^lox/lox recipients was significantly decreased compared to mice transplanted with ER-Cre-ADAR1^lox/wild cells (Fig. S4) similar to the engraftment observed with ADAR1^lox/lox hematopoietic cells transduced with MSCV-Cre (Fig. 2A). Six months after ADAR1 deletion, the recipients were killed and a 22 to 240-fold increase in the frequency of SLAM (Lin⁻CD48⁻CD150⁺) donor-derived HSCs in the bone marrow was observed even though the overall bone marrow engraftment was <1% in ER-Cre-ADAR1^lox/lox recipients (Fig. 2 I and J). Consistent with ADAR1^Δ/Δ transplant recipients (Fig. 2H), an almost identical absolute number of donor-derived SLAM HSCs in the bone marrow of ER-Cre-ADAR1^lox/lox and ER-Cre-ADAR1^lox/wt recipient groups was found (Fig. 2K). Together, this in vivo data from distinct gene deletion strategies and HSC phenotypes strongly suggest that ADAR1-deficient HSCs engrafted and were sustained in the bone marrow although the production or maintenance of mature blood cells in the recipients was disrupted.

A Selective Effect of ADAR1 Absence on HPCs Versus More Primitive Cells.

To understand the observed preferential dependence of HSC progeny rather than HSCs themselves on ADAR1 following transplantation, the expression of ADAR1 in hematopoietic cells at different stages of maturation was determined by real-time RT-PCR. ADAR1 expression was relatively low in more primitive HSCs (CD34⁻LKS⁺) and mature bone marrow populations as compared to progenitor cell types (Fig. 3A). Increased expression of ADAR1 at the protein level in HPCs by intracellular staining analyzed by flow cytometry (Fig. S5) further suggested a preferential role of ADAR1 within HPCs.

Fig. 3.

Preferential impact of ADAR1 deletion on differentiating hematopoietic progenitor cells. (A)The gene expression pattern of ADAR1 in hematopoietic cells at different stages. The mRNA levels of ADAR1 were examined using real-time RT-PCR in different HSC/HPC subsets and mature hematopoietic cell populations. LT-HSCs, long-term hematopoietic stem cells; ST-HSCs, short-term hematopoietic stem cells; CMPs, common myeloid progenitors; CLPs, common lymphoid progenitors; GMPs, granulocyte-monocyte progenitors; MEPs, megakaryocyte-erythrocyte progenitor; CD11b, peripheral blood myeloid cells; B220, peripheral blood B cells; CD3, peripheral blood T cells. Data shown are summarized from 6 experiments. The expression level of ADAR1 in LT-HSC is significantly lower than that in ST-HSC and different HPC populations (*, P < 0.05; **, P < 0.01). (B) The proliferation of LKS⁻ HPCs in liquid culture within 3.5 days (three pooled independent experiments; ***, P < 0.001). (C) The colony forming ability of LKS⁻ HPCs following 7 days of culture (three pooled independent experiments, ***, P < 0.001). (D) The morphology of colonies examined in panel C. (E) May-Grunwald/Giemsa stained cells from the individual colonies. (F) The expansion of Lin⁻ScaI⁺ and Lin⁺ cell populations in the presence or absence of ADAR1 during 4 days of in vitro culture. The data shown are from one of two independent experiments with similar results. (G) The percentage of GFP⁺Lin⁻ScaI⁺ cells within ADAR1^lox/lox and ADAR1^Δ/Δ groups. (H) TUNEL staining of sorted Lin⁻ and Lin⁺ cells at 48 h after transduction. Apoptotic cells are labeled by red fluorescence and the DAPI-stained nucleus is shown in blue. The numbers in the parentheses to the left of the images indicate the positive cell number vs. total cell number counted in four fields. (I) Percentage of TUNEL⁺ cells shown in panel H. More than 200 cells in four different fields were counted on each slide.

To define this association functionally, sorted Lin⁻c-Kit⁺ bone marrow cells were transduced with MSCV-Cre or MSCV and single HSC enriched GFP⁺LKS⁺ cells were sorted and deposited into Terasaki plates by micromanipulation. Similar proliferative rates were observed in ADAR1^Δ/Δ and ADAR1^lox/lox LKS⁺ cells within the first 72 h of culture (Table 1). In contrast, there was limited growth of the HSC-depleted and HPC-enriched Lin⁻c-Kit⁺Sca-1⁻ (LKS⁻) cells (17) upon ADAR1 deletion during 3 days of liquid culture (Fig. 3B), a result similar to that found with the previous data derived from liquid culture of total GFP⁺ cells following transduction (Fig. 1F). Assessment of the in vitro proliferation of ER-Cre-ADAR1^lox/lox cells following tamoxifen induction of Cre expression confirmed the selective adverse effect of ADAR1 absence on LKS⁻ cells over more primitive LKS⁺ cells (Fig. S6).

Table 1.

Cell survival and division rates in single stem cell culture

	≥1 cell			≥1 division			≥3 divisions
	24 h	48 h	72 h	24 h	48 h	72 h	in 72 h
Experiment one
ADARΔ/Δ	57	27	22	15	17	18	4
ADARlox/lox	40	28	20	15	19	19	7
ADARwt/wt	55	23	16	16	18	18	5
Experiment two
ADARΔ/Δ	80	27	26	32	32	34	6
ADARlox/lox	81	37	25	41	44	45	10

Single sorted GFP⁺Lin^-Sca-1⁺ cells were picked and deposited into Terasaki plates by micromanipulation, and cell division was monitored daily for up to 1 week. Initially, 144 cells were input at specific time point for each group. The number in the table indicates the wells containing live cells (first column) or accumulative wells with cell divisions. There was no statistical significance among groups in terms of survival and division rates according to Chi-Square test (P > 0.05).

Furthermore, an equal number of sorted ADAR1^Δ/Δ or ADAR1^lox/lox LKS⁻ HPCs were seeded into semisolid methylcellulose medium, and the number of colonies examined after 7 days of culture. ADAR1^Δ/Δ LKS⁻ cells yielded a strikingly lower number of colonies, as compared to ADAR1^lox/lox LKS⁻ cells (Fig. 3C). Most colonies formed in the ADAR1^Δ/Δ group were tiny and of an atypical morphology (Fig. 3D). Those atypical colonies were picked, cytospun, and stained with May-Grunward/Giemsa. The cells compromising the colony were also small and atypical in morphology, as compared to the multilineage differentiated cells in the ADAR1^lox/lox group (Fig. 3E). The cells appeared to be dead, which suggests that the progenitor cells were able to proliferate at the beginning and then underwent apoptosis during culture.

Increased Apoptosis of Differentiating HPCs Following ADAR1 Deletion.

Our previous studies demonstrated that apoptosis is the primary cellular mechanism underlying the embryonic lethality of ADAR1^−/− mice (6, 8). To determine whether apoptosis could explain the decrease in colony formation upon ADAR1 deletion (Fig. 3C) and absence of peripheral engraftment in ADAR1^Δ/Δ reconstituted mice, ADAR1^lox/lox bone marrow cells were transduced with MSCV or MSCV-Cre and cultured for 4 days. The number of Lin⁺ cells diminished rapidly in the absence of ADAR1, whereas it continuously expanded in the ADAR1^lox/lox group during culture (Fig. 3F). In contrast, the absolute number of more primitive Lin⁻ScaI⁺ ADAR1^Δ/Δ cells was relatively steady during the culture period (Fig. 3F) and the percentage of Lin⁻ScaI⁺ cells was not significantly different between the ADAR^lox/lox and ADAR1^Δ/Δ groups (Fig. 3G). Apoptosis was measured at the end of 4 days of culture by a Terminal uridine deoxynucleotidyl transferase Nick End Labeling (TUNEL) assay. As compared to ADAR1^lox/lox cells, ADAR1^Δ/Δ Lin⁺ cells exhibited a 10-fold increase in apoptosis, while more primitive Lin⁻ cells only showed a slight increase in the percentage of apoptotic cells (Fig. 3 H and I). An increased rate of apoptosis in the differentiated cells was confirmed by Annexin V staining (Fig. S7). Furthermore, senescence was unlikely involved due to equivalent β-galactosidase staining in both Lin- and Lin+ populations of cells (Fig. S8). Therefore, an increase in apoptosis rather than senescence was largely responsible for the decrease of differentiating HPCs following ADAR1 deletion during culture and likely underlies the decreased colony formation and in vivo engraftment of ADAR1-deficient cells.

ADAR1 Functions in Hematopoietic Cells via Its RNA Editing Domain.

ADAR1 consists of a catalytic RNA editing domain, as well as RNA and Z-DNA binding domains, which are believed to mediate different functions of ADAR1 (8). To determine whether the RNA editing activity of ADAR1 is necessary for its function in hematopoietic cells, the ability of an MSCV-delivered wild type or mutant ADAR1 lacking RNA editing activity (Fig. 4A) to rescue the colony forming ability of ER-Cre-ADAR1^Δ/Δ HPCs was determined (Fig. 4B). Following the induction of Cre expression with 30 μM 4-hydroxytomoxifen, transduced wild-type ADAR1 (p150) rescued more than half of the colony forming ability of HPCs. There were significantly fewer colonies generated from cells transduced with ADAR1 (E/A) harboring a point mutation in its RNA editing catalytic site. The morphology of colonies generated from cells without ADAR1 or expressing E/A was similar to those generated by ADAR1^Δ/Δ (compare Figs. 3D and 4D). This demonstrates that the role of ADAR1 in HPCs is largely dependent on its RNA editing function.

Fig. 4.

ADAR1 functions in hematopoietic cells through its RNA editing activity. (A) Confirmation of the cDNA sequence of wild-type (MSCV-p150) or point-mutated (MSCV-E/A) ADAR1. The red arrow points to the A to C mutation within the catalytic domain. (B) Experimental design of ADAR1 rescue following induction of ADAR1 deletion by tamoxifen. (C) The number of colonies yielded from MSCV-p150 or MSCV-E/A transduced ER-Cre-ADAR1^Δ/Δ cells. MSCV: MSCV vector only transduced group; p150: MSCV-p150 transduced group; and E/A: MSCV-E/A transduced group; 4-HT: 4-hydroxytomoxifen (***, P < 0.001 by Student's t test). (D) The morphology of colonies counted in panel C.

Discussion

Our current study demonstrates a relatively selective effect of ADAR1 deletion in HPCs as compared to HSCs. Unlike some hematopoietic transcription factors such as MLL, SCL, and RUNX1 (18–20), ADAR1 is required for adult, but not primitive embryonic hematopoiesis and is more critical for differentiating HPCs, as opposed to more primitive HSCs or mature blood cells. This data, taken together with the fact that the growth of neither embryonic stem (ES) cells (7, 8) nor differentiated fibroblastic cells, (8, 21) appear to require ADAR1 (Fig. S9) suggests that the survival of differentiating progenitor cells such as HPCs may be more dependent on ADAR1 than other cell types. While we have evidence indicating that increased apoptosis may underlie the selective effect of ADAR1 deletion on differentiating HPCs, we could not formally exclude direct effects of ADAR1 on other functions of HSCs such as self renewal. However, given the equivalence of HSC numbers following transplantation of ADAR1^lox/lox and ADAR1^Δ/Δ cells (Fig. 2 H and K) and efficient homing of ADAR1^Δ/Δ cells (Fig. S2), it seems unlikely that ADAR1 plays an indispensable role in HSCs.

This work contrasts a recently published paper by Hartner et al. (9) claiming that ADAR1 is an essential regulator of HSC maintenance. In Hartner's work (9), there was no definitive evidence for the effect of ADAR1 on HSCs perhaps due to the fact that the authors failed to demonstrate successful deletion of ADAR1 in the analyzed cell populations in the conditional gene-deletion model, thereby jeopardizing their conclusions regarding the cellular mechanism of ADAR1. In fact, increased HSC abundance in ADAR1-null fetal liver shown in their study would support our current claim that ADAR1 is more essential in HPCs than HSCs (9). In contrast, we have direct evidence for efficient gene deletion in the cell population (Fig. 1D) as well as at the single cell level (Fig. 1E). Based on this important prerequisite, we have multiple lines of compelling evidence consistently demonstrating a selective effect of ADAR1 on differentiating progenitor cells over more primitive cells, that is dependent on its RNA editing function.

As one of the RNA processing mechanisms, adenosine to inosine RNA editing regulates gene activity through modification of mRNA (22, 23) noncoding RNA (24, 25), and regulatory small RNAs (e.g., microRNAs) (21, 26–28). The RNAs that code critical hematopoietic regulators, or the regulatory RNAs themselves, may require ADAR1-mediated editing before their physiological actions in these cells. Therefore, defining the targets of ADAR1 in differentiating hematopoietic cells will likely reveal a class of molecules that are critical for hematopoietic regeneration and perhaps also relevant for studies concerning leukemia or other hematopoietic disorders.

Materials and Methods

Mice.

ADAR1^lox/lox mice in a C57BL/6 and FVB mixed background were generated in our previous study (8). B6.Cg-Tg(CAG-cre/Esr1)5Amc/J (ER-Cre) mice were purchased from Jackson Laboratory and crossed to our ADAR1^lox/lox mice which were bred inhouse. Excision of floxed ADAR1 alleles in ER-Cre-ADAR1^lox/lox mice was accomplished by oral administration of 0.2 mg/g tamoxifen in 0.2 to 0.3 mL vegetable oil daily for 3 days. NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ (NOD/SCID-γc^null) mice were bred in the animal facility at University of Pittsburgh Cancer Institute. All mice were handled in accordance to institutional guidelines for animal care.

Retroviral Production.

The Murine Stem Cell Virus retroviral vector expressing both Cre recombinase and GFP (MSCV-Cre) was a gift from B. Lu (University of Pittsburgh). The control vector expressing GFP (MSCV) alone was generously provided by G. Sauvageau (Université de Montréal). The ADAR1-p150 and p150-E/A point mutation cDNAs were cloned into MSCV vector and the mutation confirmed by sequencing. The virus packaging cell line, 293T, was maintained in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO Life Technologies) supplemented with 10% heat-inactivated FBS (HyClone). To produce virus, 6 × 10⁶ 293T cells were transfected with 2.5 μg of each of the packaging vectors pKat and VSV-G as well as 10 μg of MSCV plasmid using Lipofectamine2000 (Invitrogen) per the manufacturer's instructions. The virus containing supernatant was collected and filtered with a 0.45 μm filter to remove the packaging cell debris at 48 h and 72 h after transfection. The retrovirus titer determined on NIH 3T3 cells was 5 × 10⁶ to 1 × 10⁷ IU/mL.

Retroviral Transduction of Murine Hematopoietic Cells.

Lineage⁻ (Lin⁻) bone marrow cells from 16-week-old ADAR1^lox/lox mice were enriched using the MACS™ Streptavidin Kit and separation column (Milteny Biotec) per the manufacturer's instructions. The cells were prestimulated with 100 ng/mL mSCF, 50 ng/mL Flt-3L, and 25 ng/mL TPO (Peprotech) in Iscove's Modified Dulbecco's Medium (IMDM, Invitrogen) with 10% FBS for 24 h at 37 °C, 5% CO₂. Cells were then plated in a 24-well plate precoated with Retronectin (takara Bio Inc.) at 1 to 5 × 10⁵ cells/well with 80% viral supernatant (MOI = 10 to 20) and 20% fresh medium supplemented with cytokines and 4 μg/mL polybrene (Sigma). The plate was then centrifuged at 1,700 rpm for 30 min, and cultured for 12–14 h at 32 °C, 5% CO₂. The cells were cultured in fresh medium at 37 °C for 8–10 h before a second round of transduction. The transduction of ER-Cre cells is described in Fig. 4B.

Flow Cytometry and Cell Sorting.

Cultured and freshly harvested bone marrow nucleated cells were stained with lineage (CD3, CD4, CD8, CD11b, CD45R, Gr-1, and Ter119), Sca-1, and/or CD45.1, CD45.2 (BD PharMingen) surface markers and examined by flow cytometry as previously described (17).

CFC Assay.

GFP⁺Lin⁻Sca-1⁻ cells were sorted 24 h after transduction, resuspended in semisolid methylcellulose medium M3434 (StemCell Technologies), and plated in triplicate in 24-well plates at 4,000 cells/well. The cells were cultured at 37 °C, 5% CO₂ for up to 14 days and colonies counted after 7–11 days in culture.

TUNEL Staining.

Forty-eight hours after transduction, GFP⁺Lin⁻ and GFP⁺Lin⁺ cell populations were sorted and 10,000—40,000 cells were immediately attached to glass slides by cytospin. Slides were stained with the In Situ Cell Death Detection Kit, TMR Red (Roche), followed by nuclear staining with 5 μg/μL DAPI (Sigma) per the manufacturer's instructions. Fluorescent images were taken immediately after staining and positive and total cell numbers were counted for more than 3 fields, or 200 to 300 cells, per slide.

Hematopoietic Cell and HSC Transplantation.

Six hours before transplantation, 6- to 8-week-old recipient NOD/SCID-γc^null mice were sublethally (3.5 Gy, 82 cGy/min, ¹³⁷Cs γ radiator) irradiated. 1.0 to 1.5 × 10⁵ transduced GFP⁺Lin⁻ or GFP⁺Lin⁻Sca-1⁺ cells were sorted and injected into the tail veins of recipients. Recipient mice were supplied with sterile food and acidified water. Multilineage engraftment in peripheral blood was monitored monthly for 3 months by flow cytometry.

Real-Time RT-PCR.

Five thousand hematopoietic cells from 6- to 8-week-old C57/Bl6 mice were directly sorted into cell lysis buffer and the RNA was extracted using Absolute RNA extraction kits (STRATAGENE) according to the manufacturer's instruction. cDNA was synthesized using SuperScript III (Invitrogen) and real-time RT-PCR was performed using Dynamo SYBR green qPCR kit (Finnzymes) on a PTC-200 thermo cycler (MJ Research) as previously described (6).

May-Grunwald/Giemsa Staining.

Cytospin slides were stained with May-Grunwald and Giemsa (Sigma) according to the manufacturer's instructions.