©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth and Differentiation Proceeds Normally in Cells Deficient in the Immediate Early Gene NGFI-A (*)

Stephen L. Lee (2), LeAnne C. Tourtellotte (2), Robin L. Wesselschmidt (1), Jeffrey Milbrandt (2)(§)

From the (1) Division of Bone Marrow Transplantation and Stem Cell Biology, Departments of Medicine and Genetics at Jewish Hospital, and (2) Departments of Pathology and Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

NGFI-A (also known as EGR-1, zif/268, and Krox-24) is a zinc finger transcription factor induced in many cell types by a variety of growth and differentiation stimuli. To determine if NGFI-A plays a requisite role in these processes, we used homologous recombination to mutate both alleles of NGFI-A in embryonic stem (ES) cells and examined its effect on growth and differentiation. We find that ES cells lacking NGFI-A exhibit similar growth rates and serum-induced gene expression profiles compared to wild-type parental cells. They are capable of differentiating into neurons, cardiac myocytes, chondrocytes, and squamous epithelium. Chimeric mice were generated from targeted ES cells, and their progeny were crossed to produce homozygous mutant mice. Growth and histological analyses of mice lacking NGFI-A confirm the finding in ES cells that NGFI-A is not required for many of the processes associated with its expression and suggest that the function of NGFI-A is either more subtle in vivo or masked by redundant expression provided by other gene family members such as NGFI-C, Krox-20, or EGR3.


INTRODUCTION

The cellular immediate early genes (IEGs)() are thought to mediate processes of cell growth and differentiation (1, 2) . Upon stimulation by growth factors, cytokines, or membrane depolarization, these basally quiescent genes become rapidly expressed without the requirement for de novo protein synthesis. Many of these genes encode transcription factors such as c -fos and c -jun, as well as NGFI-A and NGFI-B (also known as nur/77) which are thought to regulate genetic programs that ultimately result in long term phenotypic changes (3, 4) .

The NGFI-A gene (also termed EGR-1, Krox-24, and zif/268) (5, 6, 7, 8) was originally identified by virtue of its induction by nerve growth factor in the rat pheochromocytoma cell line PC12 and by mitogenic stimuli in fibroblasts and in lymphocytes (5, 6, 7, 8, 9) . The NGFI-A gene encodes a Cys-Hiszinc finger protein which shares strong sequence homology to a family of genes that includes NGFI-C (10) , Krox-20 (11) , EGR3 (12) , and more distantly, the Wilms' tumor suppressor gene WT1 (13, 14, 15) . The expression of NGFI-A has been detected in a variety of differentiation paradigms such as embryonal carcinoma cells into myocytes or neurons, pre-osteoblasts into osteoblasts, and myelomonocytic precursors into macrophages (16, 17, 18, 19) . While most of the evidence for the involvement of NGFI-A in these processes has been correlative, antisense strategies directed against NGFI-A have shown that NGFI-A is necessary for macrophage differentiation as well as for T lymphocyte proliferation (20, 21) .

To assess the importance of NGFI-A in the variety of cellular processes associated with its induction, we used homologous recombination followed by culture in elevated G418 to inactivate both copies of NGFI-A in embryonic stem (ES) cells. In this report, we demonstrate that NGFI-A is not required for the growth and differentiation of ES cells. We confirm and extend these findings by examining homozygous mutant mice derived from the targeted ES cells and find that mice lacking NGFI-A exhibit no overt defects in growth or differentiation.


MATERIALS AND METHODS

NGFI-A Gene Targeting

To construct the targeting vectors, a 4.5-kb EcoRI/ XbaI fragment encompassing 1.0 kb relative to the transcription start site to the 3`-UTR of the NGFI-A gene was isolated from a Dash II Balb/c mouse genomic library (Stratagene) and cloned into the corresponding sites in pGEM-7z(+) (Promega). The neomycin resistance gene cassette MC1neopAAct (22) , PGKneo, or PGKneopA (23) was cloned into a unique NdeI site located upstream of the DNA binding domain using XhoI linkers. The thymidine kinase gene cassette MC1TK was cloned 3` of the short arm of homology as an XhoI/ SalI fragment into a SalI site carried over from the phage polylinker to create either pA(MC1neopA)TK, pA(PGKneopA)TK, or pA(PGKneo)TK.

AB1 and D3 ES cells were passaged every 2-3 days, maintained with daily changes of ES medium (DMEM containing 15% fetal calf serum (HyClone or Sigma), 10 µM 2-mercaptoethanol, 10-20 µg of LIF (Life Technologies, Inc.) antibiotics), and cultured on -irradiated neomycin-resistant primary murine embryonic fibroblasts (MEFs). ES cells were shown in preliminary experiments to contribute extensively to chimeric mice (30-75% as determined by coat color) when injected into day 3.5 C57Bl/6 host blastocysts (24) . NsiI-linearized targeting vector (25 µg) was electroporated into approximately 10trypsinized ES cells suspended in 0.9 ml of HEPES-buffered saline (pH 7.2) using a BTX electroporator at 260 V, 500 µF. After plating onto 10-cm MEF feeder plates for 24 h, drug selection was applied using G418 (330 µg/ml) (Life Technologies, Inc.) with or without 2 µM ganciclovir (Syntex). Approximately 10colonies survived selection in G418 alone with 2-5-fold reduction in numbers with ganciclovir, while no reduction was seen in cells transfected with a vector lacking the TK expression cassette. Colony halves were picked and pooled in groups of five for PCR analysis, while the corresponding half was plated onto individual 96 wells containing MEF feeders.

DNA for PCR was prepared from cells by lysis in 50 µL of 1 PCR buffer, digested for 3 h to overnight in 10 µg/ml proteinase K, which was then heat-inactivated at 95-100 °C for 15 min (25) . Half of the lysate was used for PCR analysis using primers specific for the neomycin resistance gene and the 3`-untranslated region of NGFI-A external to the targeting vector, with conditions previously determined from cells transfected with a positive control template. Individual clones were identified from positive pools and expanded for Southern analysis. Blots were exposed to x-ray film (Amersham Corp.) or to PhosphorImager screens (Molecular Dynamics).

Serum Response and Proliferation Assays

ES cells conditioned to grow in the absence of MEF feeders were plated in triplicate onto gelatinized six-well plates to yield approximately 1 10colonies. At various times, cells were trypsinized, and cell counts were determined in duplicate for each well. Error bars represent standard deviation from the mean.

For serum response analysis, ES cells deprived of serum for 12-24 h in DMEM containing 0.5% fetal calf serum were induced with fetal calf serum at a final concentration of 20% and harvested at various times after induction. Total RNA was prepared by the guanidinium isothiocyanate method (26) . 15 µg of total RNA were electrophoresed in 2.2 M formaldehyde gels, transferred onto Sureblot nylon membranes (Oncor), and probed with either a 400-bp BglII/ XbaI fragment of the 3`-UTR of NGFI-A, full-length c- fos cDNA (ATCC), a 410-bp BglII fragment of the 3`-UTR of NGFI-B, or full-length cyclophilin cDNA, and exposed for 12-24 h to PhosphorImager screens. Immunoblots were prepared (27) from 5 10uninduced or serum-stimulated ES cells or NGF-induced PC12 cells and probed by ECL chemiluminescence (Amersham Corp.) using the anti-NGFI-A mAb 6H10.

For lymphocyte proliferation assays, single cell suspensions of spleens from adult mice were prepared by mincing between frosted slides. Cells were washed three times in Hanks' balanced salt solution and plated into flat bottom 96-well plates at 5 10cells/well in 200 µl of RPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, 50 µg/ml gentamycin, 10 mM HEPES, and 2 10 M 2-mercaptoethanol. Following exposure to either concanavalin A (Sigma) or anti-CD3 antibody, cells were incubated at 37 °C at 5% COfor 72 h, followed by an additional incubation for 20 h in the presence of added [H]thymidine (0.4 µCi/well). For stimulation by anti-CD3 antibody, cells were plated onto anti-CD3 antibody-coated plates which were prepared by incubating 2C11 hybridoma cell culture supernatant at various dilutions for 12 h at 37 °C, after which the medium was aspirated and the plates were allowed to dry.

In Vitro Differentiation of ES Cells and Teratoma Production

For differentiation assays, ES cells were lightly trypsinized, aggregated to form simple embryoid bodies (24) , and plated 4-5 days later onto gelatinized plates. Neurons, beating cardiac myocytes, and other cell types were distinguishable morphologically between 4 and 12 days after plating. Indirect immunofluorescence staining of cardiac myosin was performed on cells fixed in 1% paraformaldehyde in PBS by incubating mAb 3A6.8 made against cardiac myosin() followed with rhodamine-coupled goat anti-mouse secondary antibody and visualized on a Zeiss Axiophot fluorescence microscope.

Teratomas were produced by intraperitoneal or subcutaneous injection of 10ES cells into SCID mice. Tumors were evident by 3 weeks after injection, at which time the mice were sacrificed. Tumors were fixed in 4% paraformaldehyde in PBS overnight, incubated in 30% sucrose in PBS, embedded in OCT (TissueTek), cut into 7-µm sections, and stained with hematoxylin and eosin.

Production and Analysis of Mice

Blastocyst injection (24) of two D3 ES cell-derived clones A4 and Z, singly targeted by the vectors pA(MC1neopA)TK and pA(PGKneo)TK, respectively, resulted in chimeras which transmitted the agouti allele as well as the targeted allele to their offspring. The resulting heterozygotes were mated to produce homozygous mutants lacking the wild type allele. All subsequent analyses were performed on mice derived from either of these clones.

For histological analysis, mice were perfused transcardially, and their tissues fixed overnight in 4% paraformaldehyde in PBS. Bones were decalcified following fixation by overnight incubation in 10% formic acid in PBS. Tissues were processed for paraffin embedding, sectioned into 6-7-µm sections, and stained as described in figure legends.


RESULTS

Production of Homozygous Mutant NGFI-A ES Cells

To disrupt NGFI-A, we used a 4.5-kb genomic fragment of the NGFI-A gene to construct a replacement-type targeting vector (28) , which upon homologous recombination, introduces several in-frame stop codons that truncate the NGFI-A coding sequence upstream of the DNA binding domain. The negative selectable marker thymidine kinase (MC1TK) was attached at the end of the genomic fragment to select against nonhomologous recombination (Fig. 1 a) (29) . Linearized targeting vector was electroporated into AB1 or D3 ES cells and transfectants were selected by culture in G418 and ganciclovir. From 580 doubly resistant colonies, four had incorporated the targeting vector by homologous recombination as assessed by PCR and verified by Southern blot (Fig. 1 b). No other integration sites were detected when blots were probed specifically for the neomycin cassette (data not shown). From these heterozygous clones, homozygous mutant ES cells were generated by culturing in elevated concentrations of G418 (3-4 mg/ml) (30) . Surviving colonies were screened by Southern blot to identify clones homozygous for the targeted allele. (Fig. 1 c).


Figure 1: Targeted inactivation of both alleles of the NGFI-A gene. a, schematic representation of the targeting vector pA(MC1neopA)TK, wild type, and mutated NGFI-A gene, indicating predicted restriction fragment lengths when probed with probe A3`. Arrowheads denote the location of primers used for PCR screens. Similar results were obtained with the targeting vectors pA(PGKneopA)TK and pA(PGKneo)TK (data not shown). b, Southern blot of PstI-digested DNA from two positive clones ( lanes 1 and 2) and a non-targeted clone ( lane 3) demonstrating the presence of the 1.9-kb band corresponding to the mutated NGFI-A allele. c, Southern blot of PstI-digested DNA from 12 colonies derived from the parent clone A1 ( lane C) surviving selection in high G418 (3 mg/ml) demonstrating the loss of the 2.5-kb band corresponding to the wild type allele ( lanes 1-4, 6, 10, and 11).



The Loss of NGFI-A Does Not Affect ES Cell Growth

Because NGFI-A is rapidly induced in quiescent fibroblasts upon treatment with serum (6, 8, 31) , we examined the possibility that serum also induced NGFI-A in quiescent ES cells. After treatment of ES cells with serum for 1 h, NGFI-A mRNA and protein were detected in wild type cells, but were absent in the homozygous mutant cells, confirming that the targeting vector disrupted the NGFI-A gene (Fig. 2, a and b). We next examined the effect of this mutation on ES cells in 1) their growth rate and 2) their serum induction profiles of other immediate-early genes. To compare growth rates, equivalent numbers of wild type and mutant cells were plated and cell counts were taken at various times afterward. No significant differences were observed in cell number during the exponential growth phase (Fig. 2 c), with cell doubling times ranging from 13-15 h in both wild type and mutant cells. The expression profiles of c- fos and NGFI-B mRNAs demonstrated characteristic serum induction patterns, as evidenced by an increase in mRNA levels within 1 h, but no differences were observed between wild type ( WT) and mutant ( KO) cells (Fig. 2 b). We also examined whether the loss of NGFI-A affected the expression of the closely related genes Krox-20, EGR-3, and NGFI-C, but no differences were observed (data not shown). Thus, NGFI-A is not essential for ES cell proliferation or the response to serum.


Figure 2: Analysis of RNA, protein, and growth rate of NGFI-A ES cells. a, homozygous mutant ES cells lack NGFI-A protein. 5 10wild type ( WT) and mutant ( KO) ES cells were serum starved for 24 h before stimulating 1 h with 20% fetal calf serum (+ serum) or DMEM alone ( serum). The cells were lysed in Laemmli buffer and protein blot analysis was performed using the NGFI-A-specific mouse mAb 6H10 (27). PC12 cells stimulated with NGF for 1 h were used for comparison ( lane 5). Molecular size markers (in kilodaltons) are indicated on the right. b, Northern analysis comparing the serum induction profiles of NGFI-A, c -fos and NGFI-B mRNA in wild type ( WT) and homozygous mutant ( KO) ES cells. Cells were serum stimulated as in a and ES cells harvested for RNA at the indicated time points ( h). 15 µg of total RNA were blotted and probed for NGFI-A (3.5 kb), NGFI-B (2.4 kb), c -fos (1.3 kb), and cyclophilin (0.8 kb). Blots were exposed to PhosphorImager screens for 24 h except for NGFI-B, which was exposed for 48 h. c, growth rates of wild type D3 (+/+) and homozygous mutant (/) ES cells. Approximately 1 10ES cells/well were plated onto gelatin-coated 6-well plates, and cell counts determined at indicated times. Each point represents mean (± S.D.) of triplicate determinations. Similar results were obtained with AB1 ES cells (not shown).



The Loss of NGFI-A Does Not Affect ES Cell Differentiation

NGFI-A is induced during the differentiation of a variety of cell types (5, 17, 18) . Additionally, NGFI-A has been implicated in the regulation of -cardiac myosin heavy chain gene expression (32) . To determine whether the loss of NGFI-A affected these processes, we assessed the ability of ES cells to differentiate into neurons and cardiac myocytes (24) . ES cells were aggregated to form embryoid bodies which were then allowed to attach and undergo differentiation. After several days, both wild type and NGFI-Acells formed neurons with extensive neurite outgrowth (Fig. 3 a). Rhythmically contracting myocytes were also observed, and these stained positive with a monoclonal antibody which recognizes cardiac myosin (Fig. 3 b).


Figure 3: Analysis of differentiation of ES cells in vitro. Embryoid bodies formed by aggregation of NGFI-AES cells were plated and cultured for 4-12 days. Various cell types were observed including ( a) neurons sending out extensive neurites (phase contrast, 200) or ( b) beating cardiac myocytes, stained here with monoclonal antibody made against cardiac myosin followed with rhodamine conjugated goat anti-mouse antibody. Fluorescence specifically localizes to regions of contractile myocytes ( 350). Similar results were obtained with +/ and +/+ ES cells (not shown).



To examine the role of NGFI-A in the formation of a greater variety of tissues, we examined the ability of NGFI-A deficient ES cells to form teratomas after injection into SCID mice (24) . Three weeks after injection, tumors from these mice were analyzed histologically. Hematoxylin and eosin-stained tumor sections revealed the presence of a variety of tissue types including cartilage, with their chondrocytes arranged in characteristic groups or cell ``nests'' (Fig. 4 a, arrow); squamous epithelium, forming a stratified layer of flattened cells (Fig. 4 b, arrowheads); and striated muscle, arranged in longitudinal myofibrils containing cross-striations (Fig. 4 c, arrows). The presence of these tissues indicate that, although NGFI-A is expessed in these tissues in embryonic and adult animals, it is not required for their formation (33) .() Taken together, the in vitro differentiation and histological analysis of teratomas demonstrate that the absence of NGFI-A does not abolish the ability of ES cells to differentiate into a variety of cell types.


Figure 4: Histological analysis of teratomas derived from NGFI-A ES cells. Three weeks after injection of NGFI-AES cells into SCID mice, the resulting teratomas were analyzed by histology and were shown to contain various tissues including: a, cartilage ( arrow); b, squamous epithelium ( arrowheads); and c, striated muscle tissue ( arrows). Magnification, 420; hematoxylin and eosin.



Mice Lacking NGFI-A Exhibit a Normal Growth Rate and Lack Any Obvious Defects in Cellular Differentiation

Injection of targeted ES cells into blastocysts resulted in chimeras which transmitted the agouti as well as the targeted allele to their offspring. Homozygous mutant mice were produced by intercross matings of the resulting heterozygotes. The NGFI-A mutant mice were born at expected Mendelian frequencies, appeared healthy, and were virtually indistinguishable from their littermates. The NGFI-A mutant mice were monitored for growth by weighing at various ages. In accord with the findings obtained from ES cells in vitro, NGFI-A does not appear to be required for growth in general as both mutant and wild type animals gained weight at the same rate (Fig. 5).


Figure 5: Growth assessment of NGFI-A mice. Weight comparison of NGFI-Amice and their wild type or heterozygous littermates at various ages. Values represent the mean of at least four animals/group ± S.D.



NGFI-A is highly expressed in the thymus (34) and in T-lymphocytes (6) which have been previously shown to require NGFI-A for proliferation (21) . Therefore, the T cell compartment of NGFI-Amice was further investigated. Histological analysis of the thymus indicated a normal architecture in NGFI-Amice, as evidenced by a well demarcated cortex ( C) and medulla ( M) (see Fig. 7 , c and d). To examine the proliferative response of peripheral lymphocytes, [H]thymidine incorporation was measured following activation by concanavalin A or anti-CD3 antibody. Surprisingly, both control and NGFI-Alymphocytes demonstrated an equivalent response (Fig. 6, A and B), indicating that NGFI-A is not required for T-lymphocyte proliferation in response to these stimuli.


Figure 7: Histological survey of NGFI-A and control mice. Histological analysis comparing littermate controls ( a, c, e, g) and NGFI-Amice ( b, d, f, h). a and b, coronal brain sections stained for neurons in the dentate gyrus ( dg) and pyramidal cell layer ( py) of the hippocampus (cresyl violet). c and d, sections of thymus showing the cortex ( c) and medulla ( m) (hematoxylin and eosin). e and f, longitudinal section of the humerus with epiphyseal plate (Alcian blue counterstained with hematoxylin and eosin). g and h, higher magnification of e and f depicting proliferative ( p), hypertrophic ( h), and calcifying ( c) zones of the epiphyseal plate. Magnification, a-f, 52; g and h, 420.




Figure 6: Proliferation assays of NGFI-A lymphocytes. Proliferative responses of peripheral lymphocytes to ( A) concanavalin A or ( B) anti-CD3 antibody as determined by incorporation of [H]thymidine. Values represent the mean of triplicate wells ± S.D.



To explore the possibility that NGFI-A is required for the formation of a variety of tissues and cell types, a histological survey was performed. Examination of coronal brain sections stained with cresyl violet revealed a normal neuronal organization and cellularity in the hippocampus (Fig. 7, a and b) and cortex (not shown), indicating that, despite the extensive expression of NGFI-A in the developing brain (34) , NGFI-A does not appear to be required for its basic organization. In situ hybridization analysis has also demonstrated coordinate regulation of NGFI-A and c- fos in the developing long ends of bones where they may regulate bone or cartilage formation (33) .Longitudinal sections of the humerus taken from mice at 9 months of age showed a bone marrow cavity with normal cellularity, contained within a spongy bone shaft and epiphysis (Fig. 7, e and f). Higher magnification of the epiphyseal plate demonstrated zones of proliferation ( p), hypertrophy ( h), and calcification ( c) of normal thickness compared to wild type samples (Fig. 7, g and h). Thus, NGFI-A does not appear to be required for bone or cartilage formation. These results confirm the findings in ES cells that differentiation into a variety of cell types remains intact despite the absence of NGFI-A.


DISCUSSION

We have eliminated NGFI-A from ES cells by homologous recombination and have examined its effect on proliferative and differentiative processes associated with its expression. Our finding that ES cells proliferate normally without NGFI-A suggests either that NGFI-A has no role in ES cell proliferation, or that redundant mechanisms can compensate for NGFI-A function. We did not observe a compensatory increase, however, in the expression of other gene family members in the mutant cells. Similar observations have been reported for the targeted disruption of the immediate early genes c -fos and c -jun in which the lack of either gene did not significantly impair ES cell growth (35, 36) .

Several reports have described the induction of NGFI-A during differentiation into a variety of cell types, including neurons, myocytes, and osteoblasts. We have shown by in vitro differentiation and analysis of teratomas that NGFI-A is not essential for the differentiation of embryonic stem cells into multiple lineages. Additionally, NGFI-A is induced in cardiac tissue in response to hypertrophic signals and has been reported to regulate transcription of the -cardiac myosin heavy chain gene (CMHC) (32, 37) . Our observation that NGFI-AES cells can differentiate into beating embryoid bodies, which occurs only when both - and -cardiac myosin heavy chain genes are expressed (38) , suggests that NGFI-A is not essential for the expression of this gene.

As indicated by the weight analysis, growth in general of mice lacking NGFI-A appears to be normal. It is also surprising to find an intact proliferative response to mitogenic signals in NGFI-Alymphocytes, which is in direct contrast to previous studies using antisense oligonucleotides directed against NGFI-A (21) . Furthermore, histological analysis of NGFI-Amice indicate a variety of cell types including neurons, thymocytes, chondrocytes, and osteocytes. These results reinforce our findings in ES cells that NGFI-A is not required for many of the processes associated with its expression and suggest either that the expression of NGFI-A is superfluous to these processes, or that other factors or gene products provide functional redundancy. It is notable that NGFI-A is a member of a gene family that includes NGFI-C, EGR3, and Krox-20. Because these genes are often coordinately regulated, a likely explanation may be that these genes provide adequate functional compensation for the absence of NGFI-A without exhibiting a compensatory increase in expression. Significant overlap in function and expression has been observed in other gene families involved in signal transduction, most notably the src family of tyrosine kinases. Targeted mutation of these genes results in minimal or undetectable phenotypes despite their widespread pattern of expression. It may therefore be necessary to target other members of the NGFI-A gene family before the full range of function of these genes can be fully recognized.


FOOTNOTES

*
This research was supported by National Cancer Institute Grant P01 CA53514 (to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom reprint requests should be addressed: 314-362-4652; Fax: 314-362-8756.

The abbreviations used are: IEG, immediate early gene; ES, embryonic stem cells; DMEM, Dulbecco's modified Eagle's medium; MEF, murine embryonic fibroblast; PCR, polymerase chain reaction; mAb, monoclonal antibody; PBS, phosphate-buffered saline; SCID, severe combined immunodeficiency disease; kb, kilobase(s).

S. C. Smith, unpublished results.

M. A. Watson and J. Milbrandt, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Allan Bradley of Baylor University for the PGKneopA vector and the AB1 ES cells, Dr. Steven Potter of the University of Cincinnati for the D3 ES cells and advice on their culture, Dr. Andy McMahon of Harvard University for the MC1neopAact vector, Dr. Simon Halegua of the University of Pennsylvania for the cyclophilin probe, and Dr. Stacy Smith of Washington University for the gift of the 3A6.8 mAb. We especially thank Kathy Frederick and Dave Donermeyer of Dr. Paul Allen's laboratory at Washington University for help with the SCID mouse injections and for performing the lymphocyte proliferation assays, respectively. We also thank members of the Milbrandt laboratory for their suggestions and comments.


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