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BMC Microbiology

Open Access

Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function

BMC Microbiology20066:75

https://doi.org/10.1186/1471-2180-6-75

Received: 25 July 2006

Accepted: 31 August 2006

Published: 31 August 2006

Abstract

Background

Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome.

Results

Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.

Conclusion

This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.

Background

Comparative genomics is based on the conservation of the molecular function of genes in different organisms throughout evolution. Orthologous genes can be studied in simple, genetically tractable model systems, as a first step to address their function in higher organisms, including humans and evaluate their possible roles in diseases. The completion of Dictyostelium genome now offers the opportunity to study the function of conserved genes present in the social amoeba and other organisms in a systematic way [1].

Dictyostelium is a primitive eukaryote, living as a single cell organism while bacteria, its source of nutrients, are present in the soil. When bacteria are consumed, starvation triggers a complex response allowing the cells to aggregate by chemotaxis and form a multicellular structure. Many different aspects of its biology including motility, chemotaxis, cytokinesis, cell-differentiation and morphogenesis among others, are more closely related to those in higher organisms than to unicellular models, such as yeasts [24]. We have performed a systematic knock-out approach to begin to address the function of genes of unknown function present in Dictyostelium and human but absent from S. cerevisiae and S. pombe [5].

The classical approach of gene inactivation in Dictyostelium is performed by the insertion of a resistant cassette in the target gene by homologous recombination. Obtaining the disruption construct is time consuming since large flanking sequences are necessary to assure a high efficiency of the process. A PCR-based method and an in vitro transposition strategy have been developed facilitating the technique [6, 7]. In the last method, the cloned gene is subjected to random insertion of a transposon containing a blasticidine-resistance cassette. The construct in which the transposon has interrupted the cloned gene is identified, expanded, digested and used for transformation of Dictyostelium cells. Since transposon insertion is random, it is sometimes necessary to screen a large number of clones, mainly when the gene is small. Besides, after transformation, the variable efficiency of homologous recombination in Dictyostelium makes the isolation of the disruptant strain tedious, since many different transformants must be screened to distinguish between random insertion and homologous recombination. Although all these difficulties are not a threat when few knock-outs are being performed, they can be overwhelming when we try to scale up the number of genes to study. We have therefore optimized all the steps from cloning of the gene, construction of the disruption vector, preparation of DNA for transformation and the screening of Dictyostelium transformants. We describe in detail the methods and illustrate its usefulness to disrupt a subset of genes of unknown function highly conserved between Dictyostelium and human.

Results and discussion

Optimizing the construction of disruption vectors in Dictyostelium

We have essentially followed the protocol described by [7] designed to insert a transposable cassette (EZTN:tetr-bsr), containing blasticidin- and tetracycline-resistance cassettes, into a cloned gene. The gene loci were previously amplified by PCR and cloned into pGEM-t vector as described in the methods section. Ideally, insertion events of the transposon must be in the middle of the clone, leaving large flanking regions to allow for an efficient homologous recombination. Besides, it might be important to interrupt the gene as much 5' as possible to disrupt the coded protein near the N-terminus. Since in vitro transposition is random, many different clones must be analyzed to find a correct location of the cassette. Consequently, the selection of the right insertion might be time-consuming, mainly when the targeted gene is small in comparison with the cloned insert. We have therefore designed a simple PCR strategy for a rapid assessment of the insertion point of the cassette after an in vitro transposition reaction. A single PCR reaction will provide information about the point of insertion and the same reaction can be sequenced directly to obtain the precise location of the insertion.

Oligonucleotides A, B and EZTN-R (whose location is depicted in Figure 1) were used in a PCR reaction with DNA isolated from the bacterial colonies obtained after in vitro transposition as described in detail in the methods section. In those plasmids where the transposon was inserted in the vector, as illustrated in Fig 1A, a band corresponding to the size of the insert is expected (Fig 1C, lanes 1,3,5). However, if the insertion took place in the insert (see Fig 1B), EZTN-R oligo would be located in the right position for amplification with oligo A or B (depending on the orientation of the transposon). In this case, a band smaller than the size of the insert is expected, and its own size will provide an estimate of the position of the transposon (Fig 1C, lanes 2,4,6,7). In the case shown in Figure 1C, lanes 6 and 7 suggested good candidates. The specific insertion point can then be obtained by direct sequencing of the PCR product with oligo EZTN-R. The use of DNA extracted directly from the bacterial colonies and the procedure to obtained quality DNA for sequencing are optimized steps carefully described in the methods section.
Figure 1

Optimizing the selection of the transposable cassette insertion into the target gene. A model of a cloned gene into the vector pGEM-t is shown in panels A and B. Two possible insertion events of the EZTN transposon are depicted: one in the vector (A) and the other in the cloned gene (B). The oligonucleotides used for cloning and analysis are shown as arrows. Panel C shows a PCR analysis of bacterial isolates after in vitro transposition. Oligonucleotides A, B and EZTN-R were used to estimate the approximate location of the transposon insertion. Lanes 1,3 and 5 correspond to insertion events probably located in the vector, as shown in the diagram of panel A. In this case, a 2.5 Kb insert corresponding to the size of the cloned gene is expected. Lanes 2,4,6 and 7 indicate insertion events in different points along the cloned gene, a situation illustrated in panel B. Panel C (right side) also shows the expected sequence obtained from the PCR products of lanes 2,4,6 and 7 by using the EZTN-R primer. The specific gene sequence located after the end of the transposon will indicate the precise location of the insertion as well as the orientation of the transposon.

Analysis of gene disruption by PCR

The region containing the flanking sequences and the inserted transposon was amplified by PCR using the universal oligonucleotides A and B (see figure 2A,B for a schematic representation). Transformation of the PCR product is a faster alternative to the isolation of DNA by maxi-prep and subsequent digestion. Moreover, we have observed a higher transformation efficiency with PCR-derived DNA than that obtained with maxi-preps.
Figure 2

Rapid analysis of gene disruption by PCR. Panel A shows a diagram of a hypothetical disruption construct in which the cloned gene has been interrupted by the transposon. This construct was used as template for PCR with oligos A and B. The resulting fragment (depicted in panel B) was transfected into Dictyostelium cells. Panel C shows an example of the analysis of clonal isolates after transformation and selection with blasticidin. PCR with primers g3 and g4 generates a 0.5 Kb band, which is absent when the gene has been disrupted (lanes 1,4,5,6). The upper band confirms the insertion of the 3 Kb transposon in the locus.

After selection, transformants were plated in association with Klebsiella aerogenes for clonal isolation. As soon as the lysis plaques are visible, cells from the growing zone were picked up and DNA extracted for PCR analysis. As depicted in Fig 2A,B, two close oligonucleotides surrounding the transposon insertion were previously synthethized for each targeted gene (g3, g4) to allow efficient amplification of the genomic region. A typical example of the analysis of Dictyostelium colonies by PCR is shown in figure 2C. Lanes 1,4,5 and 6 show the absence of the lower band corresponding to the amplification of the unaffected gene with oligos g3 and g4. Concomitantly, an upper band indicates the insertion of the transposon in the locus. An internal control corresponding to the amplification of an unrelated locus is included. The selected strains were grown and saved frozen for future analysis.

Targeting genes of unknown function

In order to prove the value of the method, we have constructed 41 disruption constructs as described above for genes of unknown function present in Dictyostelium and human but absent from the genome of the yeasts S. cerevisiae and S. pombe. The level of homology of the putative proteins between Dictyostelium and human, as determined by E-value, was equal or lower than E-20. The disruption constructs were amplified by PCR and transformed in Dictyostelium by electroporation. 28 genes were successfully disrupted and for the remaining 13 we did not detect any homologous recombination events even after checking more than 100 independent transformants. Either the efficiency of homologous recombination in those loci was very low or alternatively, disruption of the gene leads to lethality. The data has been compiled in table 1 including those genes previously described (marked with an asterisk) [5]. Curated models of these predicted genes can be found at DictyBase [8]. The possible phenotype of the disruptants is now under investigation and will open the possibility to use Dictyostelium as a suitable model to address the function of these genes as previously described for MidA, a new mitochondrial protein involved in bioenergetics [5]. As expected, most of the genes are also represented in other model systems such as Drosophila, Caenorhabditis and Arabidopsis. However, a group of them are absent in some of the models. Remarkably, 6 of them (DDB0232143, DDB0217693, DDB0232153, DDB0217633, DDB0201847, DDB0187448) are present in human and Dictyostelium and no homologues can be recognized in the genomes of the mentioned model systems. In these particular cases, Dictyostelium is probably the only non-vertebrate model to study their function. The absence of these genes in the yeast models suggests that their function might be related with those aspects that are closer in Dictyostelium and higher eukaryotes than to unicellular protists. According to this, our preliminary phenotype analysis of the KO strains has revealed the involvement of some of these proteins in processes such as chemotaxis, motility, cytokinesis, phagocytosis and development (unpublished data).
Table 1

Conserved genes of unkown function in Dictyostelium

Dicty gene (Dictybase ID)

Human gene (Genebank #)

Disruption vector (total aa/point of insertion)

Disrupted gene

DDB0232140*

NP_653337

425/77

Yes

 

DDB0232141*

CAH72141

164/135

Yes

 

DDB0232143*

CAI14128

366/207

Yes

 

DDB0232144*

AAH40291

715/233

Yes

 

DDB0232146*

CAI12688

794/291

Yes

 

DDB0232147*

BAA76798

1133/794

Yes

 

DDB0232148*

AAY15088

260/92

Yes

 

DDB0232149*

CAB64339

660/249

Yes

 

DDB0232150*

BAB85063

930/375

Yes

 

DDB0232151*

BAD96633

527/153

Yes

 

DDB0232152*

BAA20779

2117/501

Yes

 

DDB0232153*

BAA25518

1479/87

Yes

 

DDB0232154*

BAD96633

567/183

Yes

 

DDB0232155*

CAI22059

128/23

Yes

 

DDB0232156*

NP_579877

688/327

Yes

 

DDB0229859*

AAH04865

619/214

Yes

 

DDB0187500

AAR27292

514/301

Yes

 

DDB0218401

BAA13240

3080/2602

Yes

 

DDB0187448

BAA92650

1099/407

Yes

 

DDB0201847

BAA92650

1032/377

Yes

 

DDB0202130

AAH94704

3779/388

Yes

 

DDB0185464

NP_056090

1135/423

Yes

 

DDB0217693

BAA25482

2322/1973

Yes

 

DDB0188660

BAA20807

1550/614

Yes

 

DDB0205660

AAH01079

183/61

Yes

 

DDB0168698

CAH18422

315/64

Yes

 

DDB0186376

CAB66646

403/35

Yes

 

DDB0186924

BAB71412

675/430

Yes

 

DDB0185679

AAH22898

195/47

 

No

DDB0185616

NP_056138

799/340

 

No

DDB0187996

BAD97348

1164/718

 

No

DDB0187107

BAB71377

978/344

 

No

DDB0185973

NP_115675

238/107

 

No

DDB0219747

BAA90931

676/274?

 

No

DDB0216747

CAG38656

1297/813

 

No

DDB0218704

Q8N201

2479/444

 

No

DDB0189962

AAI05095

1519/352

 

No

DDB0205483

AAH16707

508/301

 

No

DDB0186187

BAD96352

320/27

 

No

DDB0217633

NP_006484

322/150?

 

No

DDB0168130

CAB66565

385/183

 

No

41

  

28

13

Conclusion

A complete protocol from gene cloning to isolation of disruptant strains in Dictyostelium is presented in this methodological report. Every step was optimized from gene cloning to selection of homologous recombination, with the aim to allow large-scale gene disruption strategies, such as the one described for the analysis of genes of unknown function. Once optimized, we have been able to complete the described protocol for a group of 10 genes in approximately 5 weeks of one person's work. It is conceivable for a well-trained technician to process 100 genes in a year. Extrapolating our results, we might expect to fail obtaining knock-out strains in around 30% of any group of genes, in part due to lethality. Other simple approaches must be designed to cope with these difficulties. Meanwhile, the existing methods, such as the one described here, will help to the rapid development of functional genomics in Dictyostelium.

Methods

Dictyostelium cell culture

Cells were grown axenically in HL5 medium (14 g/l tryptone, 7 g/l yeast extract, 0.35 g/l Na2HPO4, 1.2 g/l KH2PO4, 14 g/l glucose, pH 6.5) or in association with Klebsiella aerogenes in SM plates (10 g/l glucose, 1 g/l yeast extract, 10 g/l peptone, 1 g/l MgSO4·6H20, 1.9 g/l KH2PO4, 0.6 g/l K2HPO4, 20 g/l agar, pH 6.5) as described by [9].

PCR-amplification and cloning of the genes of interest

Specific oligonucleotides were designed to amplify a genomic region of 2–3 Kb corresponding to the genes of interest. 1–2 μg of genomic DNA from AX4 was used as template in a standard 50 μl PCR reaction containing dNTPs at 0.5 mM each, oligonucleotides at 1 pmol/μl each, 1.5 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. Different cycle programs were used and the best results were obtained in the following ranges: initial melting: 5 min at 95°C; melting: 1 min at 95°C, annealing: 1 min at 45–50°C, elongation: 5–6 min at 62–65°C, 30 cycles; final elongation: 10 min at 62–65°C. The low extending temperature (62–65°C) allowed the amplification of highly A+T-rich templates as described previously [10]. 5 μl were used for checking the size and purity of the PCR product by agarose gel, the remaining of the reaction was purified with QIAquick PCR purification Kit from Qiagen and eluted in 30 μl. 3 μl were used for cloning into pGEM-T easy vector following manufacture's instructions. Ligation reactions were transformed into E. coli DH5α and the plasmids containing the inserts were recognized by restriction with NotI, which releases the inserted fragment. Plasmid DNA from alkaline mini-preps were purified with QIAquick PCR purification kit from Qiagen and eluted in 30 μl as described by manufacture's instructions. This purification step is essential for the efficiency of the in vitro transposition reaction described below, avoiding the necessity to perform maxi-preparation of the DNA.

Insertion of the transposable cassette into the target gene

10 μg of the EZTN plasmid described by [7] were digested with PvuII and the reaction was directly purified with QIAquick PCR purification kit from Qiagen and eluted in 30 μl. We found that gel purification of the transposon was not necessary and increased the yield of the procedure. We used 1.5 μl of the pGEMT-cloned insert and 1 μl of the digested transposon in a reaction with 1 μl of transposase from Epicentre in a 5 μl final volume, following the manufacturer's instructions. After performing the transposition reaction and precipitation as described [7], E. coli DH5α were transformed and plated in LB-agar containing 15 μg/ml tetracycline, 50 μg/ml ampicillin. After 36 hours of incubation at 37°C, 10–20 bacterial colonies from each transposition were analyzed by PCR as described in figure 1. Bacterial colonies were picked up with a pipette tip, resuspended in 20 μl of distilled water and boiled for 5 minutes. 5 μl were used in a 50 μl PCR reaction with universal primers A and B, from pGEMt and EZTN-R oligo (5'-GCCAATATGCGAGAACACCCGAG-3'), derived from the sequence of the transposon [11]. The conditions for the PCR were as follows: 50 μl PCR reaction containing dNTPs at 0.25 mM each, oligonucleotides A, B and EZTN-R at 0.5 pmol/μl each, 1.5 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. The cycling program was as follows: initial melting: 5 min at 95°C; melting, 1 min at 95°C, annealing: 1 min at 50°C, elongation: 2 min at 65°C, 30 cycles; final elongation: 10 min at 65°C. 5 μl of the PCR reactions were used for analysis in agarose gels. The specific insertion point was obtained by direct sequencing of the PCR product with oligo EZTN-R. For this purpose, the remaining 45 μl of the PCR reactions were purified with QIAquick PCR purification kit from Qiagen and eluted in 30 μl. 3 μl were used for sequencing in an Applied Biosystems 377 sequencer. The expected sequence from oligo EZTN-R contains the transposase recognition sequence (which is indicated in Fig 1C), followed by the specific sequence of the targeted gene.

PCR amplification of the whole construct and electroporation of Dictyostelium cells

Selected bacterial colonies containing the plasmid with the inserted transposon were grown overnight in LB supplemented with ampicilin and tetracycline. Alkaline mini-preps were performed and the plasmids digested with NotI for further confirmation. DNA from the mini-preps was purified with QIAquick PCR purification kit. We found this step essential for the quality of the DNA, necessary for PCR amplification in the subsequent steps. The region containing the flanking sequences and the inserted transposon was amplified by PCR using the universal oligonucleotides A and B (see figure 2A,B for a schematic representation). Four PCR reactions of 50 μl were set for each sample as follows: 1 μl of DNA from the purified mini-prep diluted to 1/50; dNTPs at 0.25 mM each, oligonucleotides A and B at 0.5 pmol/μl each, 2 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. Cycling program: initial melting: 5 min at 95°C, melting: 1 min at 95°C, annealing: 1 min at 55°C, elongation: 10 min at 62–65°C, 34 cycles; final elongation: 10 min at 62–65°C. The amplification product is expected to have 5.5–6 Kb (cloned insert plus transposon) and was checked by agarose electrophoresis. The four reactions were joined and purified with QIAquick PCR purification kit. DNA was eluted in 30 μl and 10 μl were electroporated in Dictyostelium cells as described by [12].

Rapid analysis of gene disruption by PCR

After selection, transformants were plated in association with Klebsiella aerogenes for clonal isolation. As soon as the lysis plaques are visible, cells from the growing zone were picked up with a pipette tip and resuspended in 10 μl of MasterAmp DNA extraction solution from Epicentre. Samples were incubated 10 min at 60°C followed by other incubation of 10 min at 95°C. The DNA prepared in this way will be suitable for PCR amplification to screen for those transformants in which the gene has been disrupted by homologous recombination. 1 μl of the DNA was used in 50 μl PCR reaction containing 0.25 mM dNTPs, oligonucleotides g3, g4 and control oligos for amplification of an unrelated locus, at 0.5 pmol/μl each, 1.25 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. Cycling program: initial melting, 5 min at 95°C; melting: 1 min at 95°C, annealing: 1 min at 45°C, elongation: 3 min at 62°C, 30 cycles; final elongation: 10 min at 62°C. If necessary, this analysis can be performed in pools of clones to allow the rapid assessment of thousands of independent transformants as previously described [13].

Declarations

Acknowledgements

This work was supported by grant BMC2003-02814 from the Spanish Ministerio de Educación y Ciencia. Sequence data for Dictyostelium were obtained from the Genome Sequencing Centers of the University of Cologne, Germany, the Institute of Molecular Biotechnology, Department of Genome Analysis, Jena, Germany, the Baylor Collage of Medicine in Houston, Texas, and the Sanger Center in Hinxton, Cambridge, UK. We thank Carlos Quijano for bioinformatics analysis, Dicty-base team for performing the curated gene models, and Juan J. Vicente and Rosa M. Calvo for critical reading of the manuscript.

Authors’ Affiliations

(1)
Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M.

References

  1. Eichinger L, Pachebat JA, Glockner G, Rajandream MA, Sucgang R, Berriman M, Song J, Olsen R, Szafranski K, Xu Q, Tunggal B, Kummerfeld S, Madera M, Konfortov BA, Rivero F, Bankier AT, Lehmann R, Hamlin N, Davies R, Gaudet P, Fey P, Pilcher K, Chen G, Saunders D, Sodergren E, Davis P, Kerhornou A, Nie X, Hall N, Anjard C, Hemphill L, Bason N, Farbrother P, Desany B, Just E, Morio T, Rost R, Churcher C, Cooper J, Haydock S, van Driessche N, Cronin A, Goodhead I, Muzny D, Mourier T, Pain A, Lu M, Harper D, Lindsay R, Hauser H, James K, Quiles M, Madan Babu M, Saito T, Buchrieser C, Wardroper A, Felder M, Thangavelu M, Johnson D, Knights A, Loulseged H, Mungall K, Oliver K, Price C, Quail MA, Urushihara H, Hernandez J, Rabbinowitsch E, Steffen D, Sanders M, Ma J, Kohara Y, Sharp S, Simmonds M, Spiegler S, Tivey A, Sugano S, White B, Walker D, Woodward J, Winckler T, Tanaka Y, Shaulsky G, Schleicher M, Weinstock G, Rosenthal A, Cox EC, Chisholm RL, Gibbs R, Loomis WF, Platzer M, Kay RR, Williams J, Dear PH, Noegel AA, Barrell B, Kuspa A: The genome of the social amoeba Dictyostelium discoideum. Nature. 2005, 435: 43-57. 10.1038/nature03481.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Hereld D, Jin T: Moving toward understanding eukaryotic chemotaxis. Eur J Cell Biol. 2006,Google Scholar
  3. Robinson DN, Spudich JA: Mechanics and regulation of cytokinesis. Curr Opin Cell Biol. 2004, 16: 182-188. 10.1016/j.ceb.2004.02.002.View ArticlePubMedGoogle Scholar
  4. Escalante R, Vicente JJ: Dictyostelium discoideum: a model system for differentiation and patterning. Int J Dev Biol. 2000, 44: 819-835.PubMedGoogle Scholar
  5. Torija P, Vicente JJ, Rodrigues T, Robles A, Cerdan S, Sastre L, Calvo R, Escalante R: Functional genomics in Dictyostelium: MidA, a new conserved protein, is required for mitochondrial function and development. J Cell Sci. 2006, 119: 1154-1164. 10.1242/jcs.02819.View ArticlePubMedGoogle Scholar
  6. Kuwayama H, Obara S, Morio T, Katoh M, Urushihara H, Tanaka Y: PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucl Acids Res. 2002, 30: U14-U18. 10.1093/nar/30.2.e2.View ArticleGoogle Scholar
  7. Abe T, Langenick J, Williams JG: Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development. Nucl Acids Res. 2003, 31: E107-10.1093/nar/gng095. (10 pages)PubMed CentralView ArticlePubMedGoogle Scholar
  8. DictyBase.http://www.dictybase.org/
  9. Sussman M: Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Meth Cell Biol. 1987, 28: 9-29.View ArticleGoogle Scholar
  10. Su X, Wu Y, Sifri D, Wellems T: Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acid Research. 1996, 24: 1574-1575. 10.1093/nar/24.8.1574.View ArticleGoogle Scholar
  11. Epicentre Biotechnologies.http://www.epibio.com/sequences/pMod2_MCS.asp
  12. Pang KM, Lynes MA, Knecht DA: Variables controlling the expression level of exogenous genes in Dictyostelium. Plasmid. 1999, 41: 187-197. 10.1006/plas.1999.1391.View ArticlePubMedGoogle Scholar
  13. Charette SJ, Cornillon S, Cosson P: Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination. J Biotechnol. 2006, 122: 1-4. 10.1016/j.jbiotec.2005.08.023.View ArticlePubMedGoogle Scholar

Copyright

© Torija et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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