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ANIMALBIOTECHNOLOGY

ANIMAL BIOTECHNOLOGY

ModelsinDiscoveryandTranslation

SECONDEDITION

Editedby

PROF.ASHISHS.VERMA,PH.D.(ZOOLOGY)

JadavpurUniversity,Kolkata,700032,W.B.,India

DR.ANCHALSINGH,PH.D.(BIOCHEMISTRY)

DepartmentofBiochemistry,InstituteofScience,BanarasHinduUniversity,Varanasi,221005,U.P.India

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ListofContributorsxvii

Prefacexxi Acknowledgmentsxxiii

SectionI

Humandiseases:invivoandinvitro

models

1.Drosophila:amodelforbiotechnologist

K.RAVIRAMANDD.KARCHOWDHURI

Summary3 Whatyoucanexpecttoknow3 Introduction3 History7 Principle8 Methodology8 Protocols9

Ethicalissues11

Translationalsignificance11 Clinicalsignificance11

Turningpoint14

WorldWideWebresources15

Acknowledgments16 References16

Furtherreading17

Glossary17 Abbreviations18

Longanswerquestions18

Shortanswerquestions18

Answerstoshortanswerquestions18 Yes/notypequestions18 Answerstoyes/notypequestions18

2.Animalmodelsoftuberculosis

DEVYANIDUBE,RAJEEVSHARMA,NISHIMODY,MADHUGUPTA, UDITAAGRAWALANDSURESHP.VYAS

Summary21 Introduction21

Comparativepathologyoftuberculosisinhumansandanimals22

Pathogendiversity:crossingspeciesbarriers23

Hostdiversity:fundamentalprocessesandfine-tuning23

Animalmodelsoftuberculosis:limitsandlessons24

Animalmodels:contributionsintuberculosisvaccinetesting24 Variousanimalmodels25

Protocols28 Ethicalissues32 Translationalsignificance36 Worldwidewebresources37 Safetyconsiderations39 References39 Furtherreading40 Glossary40 Abbreviations40 Long-answerquestions40 Short-answerquestions40 Answerstoshort-answerquestions40 Yes/no-typequestions41 Answersforyes/notypequestions41

3.Animalmodelsforneurodegenerative disorders

KOJIYAMANAKA

Summary43 Whatyoucanexpecttoknow43 Historyandmethods43 Principles46 Methodology49 Examplesandtheirapplications53 Clinicalcorrelations57 Protocols57 Ethicalissues58 Translationalsignificance58 WorldWideWebresources58 Acknowledgment59 References59 Furtherreading60 Glossary60 Abbreviations60 Longanswerquestions60 Shortanswerquestions61 Answerstoshortanswerquestions61 Yes/notypequestions61 Answersforyes/notypequestions61

4.Epigeneticsandanimalmodels: applicationsincancercontrolandtreatment MUKESHVERMA,NEELESHAGARWAL,MUDITVERMAAND VINEETKUMAR

Summary63 Whatyouexpecttoknow63 Introduction63 History64

Principle65

Methodology69 Protocols69

Ethicalissues70

Translationsignificance71

WorldWideWebresources73

Somethinginterestingaboutthischapter73 References73

Glossary(termsusedintextwithexamples)74

Abbreviations74

Longanswerquestions74

Answerstolonganswerquestions74

Shortanswerquestions75

Answerstoshortanswerquestions75 Yes/notypequestions75

Answerstoyes/notypequestions75

5.Developmentofmousemodelsforcancerresearch

PARTHAK.CHANDRA,AMRITADATTAANDDEBASISMONDAL

Summary77

Whatyoucanexpecttoknow77

Introduction77

History78 Principle79

Methodology80

Exampleswithapplications81

Checklistforasuccessfulinvivoexperiment88 Protocols90

Ethicalissues95

Translationalsignificance95

WorldWideWebresources96 References98

Glossary99

Abbreviations99

Longanswerquestions100

Shortanswerquestions100

Answerstoshortanswerquestions100 Yes/notypequestions101

Answerstoyes/notypequestions102

6.Theclinico-molecularapproachesfor detectionofhumanpapillomavirus

MAUSUMIBHARADWAJ,SHOWKETHUSSAIN,RICHATRIPATHI, NEHASINGHANDRAVIMEHROTRA

Summary103

Whatyoucanexpecttoknow103

Introduction104

Historicaloverview104

Mistakentheoriesofcervicalcancercausation104

Thefirstbreakthrough105

Prevalenceandepidemiologyofcervicalcancer106

Screeninganddiagnosticmethodologiesofcervicalcancer109

Methodsusedforscreening/diagnosisofcervicalcancer110 Applications115 Treatment115 Stage-wisemanagementofcervicalcancer116

Ethicalissues118 Translationalsignificance120 Conclusion122 WorldWideWebresources122 Furtherinformation123 Ablativetechniques124 Excisionaltechniques125 References125 Glossary126 Abbreviations126 Longanswerquestions127 Answerstolonganswerquestions127 Shortanswerquestions128 Answerstoshortanswerquestions128 Yes/notypequestions129 Answerstoyes/notypequestions129

7.HumanDNAtumorvirusesandoncogenesis

PRAVINKUMARPURUSHOTHAMAN,TIMSYUPPALANDSUBHASH CHANDRAVERMA

Summary131 Whatyoucanexpecttoknow131 Historyandmethods131 Transformationandoncogenesis132 HistoryofhumanDNAtumorvirusesandcancer134 Principle140 Clinicalcorrelation144 Turningpoint:modelingEpstein Barrvirusinfectionand pathogenesis146 Currentresearchperspectives147 Ethicalissues148 Translationalsignificance148 WorldWideWebresources149 References149 Furtherreading150 Glossary150 Abbreviations150 Longanswerquestions150 Shortanswerquestions151 Answerstoshortanswerquestions151 Yes/notypequestions151 Answerstoyes/notypequestions151

8.Animalmodelsforhumandisease

M.REZAKHORRAMIZADEHANDFARSHIDSAADAT Summary153 Outline153

Whatyouexpecttoknow153 Introduction153 Rheumatoidarthritis154 Multiplesclerosis159 Ethicalissues165 Translationalsignificance165 Clinicalcorrelations166 Conclusion166 WorldWideWebresources167

References167

Furtherreading168

Glossary168

Abbreviations170

Long-answerquestions170

Shortanswerquestions170

Answerstoshortanswerquestions170 Yes/notypequestions171

Answerstoyes/notypequestions171

9.HIVandantiretroviraldrugs

ANCHALSINGH,ASHISHSWARUPVERMAANDVIPINKUMAR

Summary173

Whatyoucanexpecttoknow173

Historyandmethods173

DiscoveryandoriginofHIV174

HistoryofHIVandAIDS174

Globaldiseaseburden175

ClinicalstagesofHIV176

MolecularbiologyofHIV178

Replication:stepsanddrugtargets180 Antiretroviraldrugs182 HIVresistanceandantiretroviraltreatment184

Newtypesofantiretrovirals184

Methodologyandprinciples186 UnderstandingHIVreservoir190 NeuroAIDS:anemerginghealthconcern192

Bonemarrowtransplantation:aprobablecureforHIV193 Ethicalissues194

Translationalsignificance194

Clinicalcorrelation195

Acknowledgments195

WorldWideWebresources195 References195 Furtherreading196

Glossary196

Abbreviations197

Longanswerquestions197

Shortanswerquestions197

Answerstoshortanswerquestions197 Yes/notypequestions198

Answerstoyes/notypequestions198

10.Animalmodelsinadvancementof researchinentericdiseases

DEBASISPORE,KAZIMIRAJULHOQUEANDMANOJK.CHAKRABARTI

Summary199

Whatyoucanexpecttoknow199 Introduction199

Animalmodelsforenterotoxigenic

Animalmodelsfor

Animalmodelsfor

Animalmodelsfor

Animalmodelsfor

Animalmodelsfornontyphoidal

WorldWideWebresources216 References216 Furtherreading217 Glossary217 Longanswerquestions218 Shortanswerquestions218 Yes/notypesquestions218 Answerstoyes/noquestions218

11.Chickchorioallantoicmembraneassay:a3D animalmodelforcancerinvasionandmetastasis

NOORALOKMAN,CARMELARICCIARDELLIANDMARTINKOEHLER

Summary221

Whatyoucanexpecttoknow221 Introduction221 History221 Principle222 Conclusions228 EthicalIssues228 Translationalsignificance228 References228 Furtherreading230 Glossary230 Abbreviations230 Longanswerquestions230 Shortanswerquestions231 Answerstoshortanswerquestions231 Yes/notypequestions231

SectionII

Animalbiotechnology:toolsand techniques

12.Animalbiotechnologyasatooltounderstand andfightaging

PAWANKUMARMAURYA

Summary235

Whatyouexpecttoknow235 Introduction235 Principle238 Methodology:measurementoffreeradicalsandmethodsto monitoraging241 Commonlaboratoryanimalexperimentalmodelsforaging research242

Polyphenolsasanagenttofightaging243

Animalbiotechnologyasatooltounderstandagingandfight aging246

Humanaging:atranslationalperspectiveandsignificance247 Ethicalissuesrelatedwithagingresearch247 Worldwidewebresources247 Clinicalcorrelation248 References248 Furtherreading248

Glossary248

Abbreviations249

Long-answerquestions249

Short-answerquestions249

Answerstoshort-answerquestions249

Yes/no-typequestions250

Answerstoyes/no-typequestions250

13.Multicellulartumorspheroidsasinvitromodels forstudyingtumorresponsestoanticancertherapies

SUCHITKHANNA,ANKITCHAUHAN,ANANTNARAYANBHATTAND BILIKERESRINIVASARAODWARAKANATH

Summary251

Whatyoucanexpecttoknow251

Historyandmethods252

Multicellulartumorspheroids252

Historicalfactstowardthedevelopmentoftissueculture technologyfrom2Dand3Dcultures256

Techniquesforthegenerationofspheroids258

Protocolfortumorspheroidgeneration260

Drugtreatmentprotocol260

Parameterstomonitordrugefficacyin3Dcultures261

Radiationresponseoftumorcellsanditsmodifications261

Responsetoanticancerdrugs262

Responsetophotodynamictherapy262

Responsetoantiangiogenesistherapeutics263

Evaluationofresponsetoimmunotherapy263

Applicationof3Dculturesinotherdiseases264

Conclusions265

Ethicalissues265

Translationalsignificance265

WorldWideWebresources266

References266

Glossary267

Abbreviations267

Longanswerquestions268 Shortanswerquestions268 Answerstoshortanswerquestions268

14.Animaltissuecultureprinciplesand applications

ANJUVERMA,MEGHAVERMAANDANCHALSINGH

Summary269

Whatyoucanexpecttoknow269

Historyandmethods269

Developmentofanimalcellculture270

Basicconceptofcellculture270

Typesofcellculture273

Cellline273

Growthcycle274

Monitoringcellgrowth275

Cellviability276

Culturemedia277

Characterizationofcelllines280

Advantagesofanimalcellculture280

Disadvantagesofanimalcellculture281 Ethicalissues281 Useoffetalbovineseruminanimalcultureofmedia281 Translationalsignificance281 Antiviralvaccines282 Recombinanttherapeuticproteins283 Genetherapy285 Biopesticides286 Monoclonalantibodies287 Stemcells287 WorldWideWebresources289 References290 Furtherreading291 Glossary291 Abbreviations292 Longanswerquestions292 Shortanswerquestions292 Answerstoshortanswerquestions292 Yes/notypequestions292 Answerstoyes/notypequestions293

15.Conceptsoftissueengineering

POONAMVERMAANDVIPINVERMA

Summary295 Whatcanyouexpecttoknow?295 Introduction295 History296 Basicapproachtotissueengineering:principlesand methodology296 Scaffolddesign298 Materialsforscaffolds298 Scaffoldfabricationmethods299 Examplesoftissue-engineeredorgans302 Tissueengineeringusingstemcells303 Issuesandchallenges303 Ethicalissues304 Translationalsignificance304 Worldwidewebresources304 References304 Furtherreading305 Glossary306 Abbreviations306 Long-answerquestions306 Short-answerquestions307 Answerstoshort-answerquestions307 Kindlystateyesornoagainstthefollowingstatements307 Answerstoyes/nostatements307

16.Nanotechnologyanditsapplicationsto animalbiotechnology

ASHOKK.ADYAANDELISABETTACANETTA

Summary309

Whatyoucanexpecttoknow309 Historyandmethods309 Methodologies311

Examplesofnanotechnologyapplicationstoanimal biotechnology318 Ethicalissues321 Translationalsignificance321 Acknowledgments321 Worldwidewebresources321 References322 Furtherreading323 Glossary324 Abbreviations324 LongAnswerQuestions324 Shortanswerquestions324 Answerstoshortanswerquestions325 Yes/notypequestions326 Answersto“Yes/no”questions326

17.Antibodies:monoclonalandpolyclonal ANCHALSINGH,AYUSHIMISHRAANDANJUVERMA

Summary327

Whatyoucanexpecttoknow327 Historyandmethods327 TiseliusandKabat’sexperiment328 History328 Elucidationofimmunoglobulinstructure329 ImmunoglobulinG:aprototypeforimmunoglobulin330 Polyclonalantibodyversusmonoclonalantibody331 Antibodiesastherapeutics:adverseeffects334 Applicationsofantibodies336 Methodology,principles,andprotocols337 Biochemicalpathway:hybridomaselection344 Ethicalissues345 Camelidnanobodies/single-domainantibodies/variable domainofcamelidheavychainonlyantibody346

Translationalsignificance347 Clinicalcorrelations347 WorldWideWebresources348 Acknowledgments349 References349 Furtherreading350 Glossary350 Abbreviations350 Longanswerquestions350 Shortanswerquestions351 Answerstoshortanswerquestions351 Yes/notypequestion351 Answerstoyes/notypequestions351

18.Molecularmarkers:toolforgeneticanalysis AVINASHMARWALANDRAJARSHIKUMARGAUR Summary353 Whatcanyouexpecttoknow353

Turningpoint367 Worldwidewebresources367 References368 Furtherreading369 Glossary369 Abbreviations370 Long-answerquestions370 Short-answerquestions370 Answerstoshortquestions371 Yes/no-typequestions371 Answerstoyes/No-typequestions372

19.Ribotyping:atoolformoleculartaxonomy SUDHIRKUMARKASHYAP,SUNILMAHERCHANDANIAND NAVEENKUMAR

Summary373

Whatyoucanexpecttoknow373 Historyandmethods373 Historicaldevelopmentsinbacterialtaxonomy374 Typingmethodsusedforbacterialsystematics375 BasisofusingrRNAandrRNAgenesastaxonomictools377 Differenttechniquesofribotyping379 Limitationsofribotyping387 Futureperspectives390 Ethicalissues390 Translationalsignificance390 WorldWideWebresources391 References391 Abbreviations392 Longanswerquestions392 Shortanswerquestions392 Answerstoshortanswerquestions393 Yes/notypequestions393 Answerstoyes/notypequestions393

20.Nextgenerationsequencingandits applications

ANUJKUMARGUPTAANDUDGUPTA

Summary395

Whatyoucanexpecttoknow395 Introduction395 HistoryofDNAsequencing395 Generationofsequencingtechnologies396 PrincipleofSangersequencingversusNGS397 NGStechnologies:Initialphase397 NGStechnologies:recentphase399 Othernewersequencingtechnologies401 Downstreambioinformatics403 GeneralprinciplesofNGSmethodsinvariousapplications405 Animalbiotechnologyandthecattlegenome409 ApplicationsofNGSinanimalbiotechnology409 ApplicationsofNGSinhumanhealth413 Clinicalcorrelation414 Translationalsignificance416 Ethicalissues416 Futureperspectives416

Challenges417

Worldwidewebresources417 References417

Glossary418

Abbreviations419

Longanswerquestions419 Shortanswerquestions419

Answerstoshortanswerquestions419 Yes/Notypequestions420 AnswerstoYes/Notypequestions420

21.Biomoleculardisplaytechnology:anew toolfordrugdiscovery

MANISHBIYANI,MADHUBIYANIANDKOICHINISHIGAKI

Summary423

Whatyoucanexpecttoknow423 Introduction423 Principle424

Necessity:smallmoleculeversusbiomolecular(biologics) drugs425

Methodology:biomoleculardisplaytechnologies426

Translationalsignificance435

WorldWideWebresources435

Conclusionandfutureperspective436 References436

Furtherreading437

Glossary437

Abbreviations438

Longanswerquestions438

Shortanswerquestions438

Answerstoshortanswerquestions438 Yes/notypequestions439

Answerstoyes/notypequestions439

22.Insilicodiseasemodel:fromsimple networkstocomplexdiseases

DEBMALYABARH,EUGENIACH.YIANNAKOPOULOU, EMMANUELO.SALAWU,ATANUBHATTACHARJEE, SUDHIRCHOWBINA,JOSEPHJ.NALLURI,PREETAMGHOSH ANDVASCOAZEVEDO

Summary441

Whatyoucanexpecttoknow441

Bioinformaticsinanimalbiotechnology442

Bioinformaticsandsystemsbiology443

Commoncomputationalmethodsinsystemsbiology444

Experimentalmethodsinsystemsbiology444

Protein proteininteractions444

Transcriptionalcontrolnetworks444

Signaltransductionnetworks444

Mathematicalmodelingtechniques445

Conceptofmodeling446

Insilicomodelsofcells447

Applicationsofinsilicodiseasemodeling451 Conclusion457 References457

Longanswerquestions459 Shortanswerquestions459 Answerstoshortanswerquestions459 Yes/notypequestions460 Answerstoyes/notypequestions460

SectionIII

Animalbiotechnology:applications andconcerns

23.Transgenicanimalsinresearchandindustry

NISHUNISHU,SHETMASIH,SHIVALIKAMAL,POOJAJAINAND ZAFARK.KHAN

Summary463

Whatyouexpecttoknow463 Introduction463 Creatingtransgenicanimals465 Transgenicanimalsasdiseasemodels469 Transgenicanimalsasbiologicalmodels471 Transgenicanimalsasxenotransplanters472 Transgenicanimalsasfoodsource473 Transgenicanimalsfordrugandindustrialproduction473 Transgenicanimals’impactontheenvironment474 Patentingtransgenicanimals475 Ethicsintransgenesis476 FDAguidelinesongeneticallyengineeredanimals477 Translationalsignificance477 Clinicalcorrelations477 Turningpoint477 WorldWideWebresources478 References478 Furtherreading479 Glossary479 Longanswerquestions479 Shortanswerquestions479 Answerstoshortanswerquestions479 Yes/notypeofquestions480 Answerstoyes/notypequestions480

24.Roleofcytogeneticsandmolecular geneticsinhumanhealthandmedicine

MADHUMITAROYCHOWDHURY,ANCHALSINGHAND SUDHISHADUBEY

Summary481 Whatyoucanexpecttoknow481 Introduction481 Cytogenetics:anoverview482 Chromosomemorphologyandclassification482 Chromosomaldisorders484 Methodology:applicationofdifferentcytogenetic techniquesinthediagnosisofgeneticdisorders487

Moleculargenetics:anoverview488

Methodology:applicationofdifferentmoleculartechniques fordiagnosisofgeneticdisorders494 Ethicalissues498

Translationalsignificance498 WorldWideWebresources499 References499 FurtherReading500

Glossary500 Longanswerquestions500

Shortanswerquestions500

Answerstoshortanswerquestions501 Yes/notypequestions501 Answerstoyes/notypequestions501

25.Antibodiesandtheirapplications

FAHIMHALIMKHAN

Summary503

Whatyoucanexpecttoknow503 Historyandmethods503 Ethicalissues517

Translationalsignificance518 WorldWideWebresources519

Furtherreading520 Glossary521 Abbreviations521

Longanswerquestions521

Shortanswerquestions521

Answerstoshortanswerquestions522 Yes/Notypequestions522 AnswerstoYes/Notypequestions522

26.Vaccines:presentstatusandapplications

DINESHK.YADAV,NEELAMYADAVANDSATYENDRAMOHANPAUL KHURANA

Summary523

Whatyoucanexpecttoknow523 Introduction523 Typesofvaccines525 Molecularfarmingusingplantsasbioreactor533 Advancementinvaccineadjuvants534 Immunostimulatingcomplexes536 Futurechallengesinvaccinedevelopment536 Ethicalissues537 Mandates538 Vaccineresearchandtesting538 Informedconsent538 Accessissues538 Translationalsignificance538 WorldWideWebresources539 Protocols539 References540 Furtherreading540 Glossary540 Longanswerquestions541 Shortanswerquestions541

Answerstoshortanswerquestions541 Yes/notypequestions541 Answerstoyes/notypequestions541

27.Perspectivesonthehumangenome

KAILASHC.UPADHYAYAANDARUNAKUMAR

Summary543 Whatyoucanexpecttoknow543 Historyandmethods543 Humangenomesequencingproject544 Ethicalissues558 Translationalsignificance558 WorldWideWebresources560 References560 Furtherreading561 Glossary561 Abbreviations562 Longanswerquestions562 Shortanswerquestions562 Answerstoshortanswerquestions562 Yes/notypequestions564 Answerstoyes/notypequestions565

28.Marineresourcesandanimalsinmodern biotechnology

SURAJITDASANDBHAKTIPATEL

Summary567

Whatcanyouexpecttoknow567 Introduction567 Marinebiologicaldiversity567 Advancesinmariculture571 Geneticengineeringtechnology575 Marinemetagenomics577 Marineanimalsandnanotechnology578 Pharmaceuticalsandtherapeuticsfrommarineanimals579 Ethicalissues585 Translationalsignificance586 Futuredirections586 Worldwidewebresources587 References587 FurtherReading589 Glossary589 Long-answerquestions590 Short-answerquestions590 Answerstoshortanswerquestions590 Yes/no-typequestions590 Answerstoyes/no-typequestions590

29.Nanotechnologyanddetectionofmicrobial pathogens

RISHISHANKER,GULSHANSINGH,ANURAGJYOTI, PREMENDRAD.DWIVEDIANDSURINDERPALSINGH

Summary593

Whatyoucanexpecttoknow593 Introduction593

Indicatorsofmicrobialwaterquality594

Needfordetectionofwaterborneandfoodborne pathogens595

Conventionalmethodstodetectfecalindicatororganismand otherpathogenicbacteria596

Molecularmethodsbasedongeneticsignatureoftarget pathogen598

Nanotechnologyandpromises601

History603

Detectionprinciple603

Methodology604

Examplesofapplicationofgoldnanoparticlesandfew examplesofsilvernanoparticleandquantumdotsfor detectionofbacteria605

Ethicalissues607

Translationalsignificance607

Futuristicapproach607

WorldWideWebresources608

Clinicalcorrelations608

Turningpoint608

Awards/recognitions609

Acknowledgment609

References609

Furtherreading610

Glossary610

Abbreviations610

Longanswerquestions610

Shortanswerquestions610

Answerstoshortanswerquestions610

Yes/notypequestions611

Answerstoyes/notypequestions611

30.Herbalmedicineandbiotechnologyforthe benefitofhumanhealth

PRIYANKASRIVASTAVA,MITHILESHSINGHANDRAKHICHATURVEDI

Summary613

Whatyoucanexpecttoknow613

Introduction613

Methodology615

Identification,quantification,andcharacterizationofbioactive compounds618

Biotechnologicalapproachesforherbaldrugproduction621

Opportunitiesandchallenges622

Conclusionsandoutlook624

Ethicalissues625

Translationalsignificance625 WorldWideWebresources626

References626

Furtherreading627

Glossary628

Abbreviations628

Longanswerquestions628

Shortanswerquestions628

31.Enzymeinhibitionassayformetabolic disorders—exploringleadsfrommedicinal plants

PULOKK.MUKHERJEE,RANJITK.HARWANSH,SHIVBAHADUR, JOYDEBCHANDA,SAYANBISWASANDSUBHADIPBANERJEE

Summary631

Whatyoucanexpecttoknow631 Introduction631 Methodology637 Exampleswithapplications640 Invitroenzymeinhibitionassaysforscreeningof medicinalplantsinmetabolicdisorders641 Medicinalplantsusedinmetabolicdisorders646 Ethicalissues646 Translationalsignificance646 WorldWideWebresources648 Acknowledgment649 References649 Glossary650 Abbreviations650 Longanswerquestions650 Answerstolonganswerquestions650 Shortanswerquestions652 Answerstoshortanswerquestions652 Yes/notypequestions652 Answerstoyes/notypequestions652

32.Safetyassessmentoffoodderivedfrom geneticallymodifiedcrops

PREMENDRAD.DWIVEDI,MUKULDAS,SANDEEPKUMARAND ALOKKUMARVERMA

Summary655

Whatyoucanexpecttoknow655 Historyandmethods655 Rationalefortheallergenicityassessmentofgenetically modifiedFoods657 Mechanismoffoodprotein-inducedallergenicity657 Simulatedgastricfluidassay658 Thermaltreatmentassay665 Ethicalissues669 Translationalsignificance669 Acknowledgment669 WorldWideWebresources670 References670 Furtherreading671 Glossary671 Abbreviations671 Longanswerquestions672 Shortanswerquestions672 Answerstoshortanswerquestions672

Answerstoshortanswerquestions628 Yes/notypequestions628 Answerstoyes/notypequestions629

Yes/notypequestions672

Answerstoyes/notypequestions673

33.CorrelatingAyurvedaandbiotechnology: approachesforthe21stcenturyandbeyond

CHANDRAKANTKATIYAR,RAJATHAZRAANDSATYAJYOTIKANJILAL

Summary675

Whatyoucanexpecttoknow675 Introduction675

Ageingprocess683

Prakriti andgenomics684 WorldWideWebresources685 Wayforward685

Acknowledgment685 References685 Furtherreading686

Glossary686

Longanswerquestions686

Shortanswerquestions686 Yes/notypequestions686

Answerstoyes/noquestions686

34.Nanoparticlesynthesisharnessingbenigngreen routes

SOUMENMUKHERJEE,SHANTADUTTA,ASHISHSWARUPVERMAAND MALAYKUMARSAHA

Summary689

Whatyoucanexpecttoknow689 Introduction689 History690 Principle692 Methodology692 Examples694 Protocol697 Ethicalissues700

Translationalsignificance701 WorldWideWebresources703 Turningpoint703 Acknowledgement705 References705 Furtherreading706 Glossary706

Abbreviations707

Longanswerquestions707

Shortanswerquestions707

Answerstoshortanswerquestions707 Yes/Notypequestions708

AnswerstoYes/Notypequestions708

35.Ethicalissuesinanimalbiotechnology

ABHIKGUPTA

Summary709

Whatyoucanexpecttoknow709 Historyandmethods710 Abriefoverviewofethicalthoughtsandprinciples711 Principles712 Methodology713 Applicationofethicsinanimalbiotechnology714 Ethicalconcernsinanimalbiotechnology714 Somechallengingethicalissuesinanimalbiotechnology720 Translationalsignificance725 Conclusions725 WorldWideWebresources725 References726 Glossary727 Abbreviations728 Long-answerquestions728 Short-answerquestions728 Answerstoshort-answerquestions728 Yes/no-typequestions729 Answerstoyes/no-typequestions729

36.Approachestothehumaneeuthanasiaof researchanimals

MARKA.SUCKOWANDJESSICAL.GIMPEL

Summary731 Whatyoucanexpecttolearn731 Introductionandbackground731 Propertrainingofpersonnel731 Methodology,equipment,andprinciples732 Chemicalmethods733 Physicalmethods735 Specialconsiderations:fetusesandneonateanimals736 Ethicalissues736 Translationalsignificance738 WorldWideWebresources738 References738 Furtherreading739 Glossary739 Abbreviations739 Longanswerquestions739 Shortanswerquestions739 Answerstoshortanswerquestions739 Yes/Notypequestions740 AnswerstoYes/Notypequestions740 Index741

ListofContributors

AshokK.Adya BIONTHE(Bio-andNano-technologiesfor Health&Environment)Centre,DivisionofBiotechnology andForensicSciences,SchoolofContemporarySciences, UniversityofAbertay,Dundee,Scotland,United Kingdom

NeeleshAgarwal EpidemiologyandGenomicsResearch Program,DivisionofCancerControlandPopulation Sciences,NationalCancerInstitute,NationalInstitutesof Health(NIH),MedicalCenterDrive,Rockville,MD, UnitedStates

UditaAgrawal DepartmentofPharmaceuticalSciences,Dr. HariSinghGourUniversity,Sagar,India

VascoAzevedo DepartmentofGeneralBiology,Federal UniversityofMinasGerais,BeloHorizonte,Brazil

ShivBahadur SchoolofNaturalProductStudies, DepartmentofPharmaceuticalTechnology,Jadavpur University,Kolkata,India

SubhadipBanerjee SchoolofNaturalProductStudies, DepartmentofPharmaceuticalTechnology,Jadavpur University,Kolkata,India

DebmalyaBarh CentreforGenomicsandAppliedGene Technology,InstituteofIntegrativeOmicsandApplied Biotechnology(IIOAB),Nonakuri,PurbaMedinipur,West Bengal,India

MausumiBharadwaj ICMR-NationalInstituteofCancer PreventionandResearch,Noida,India

AnantNarayanBhatt DivisionofRadiatonBiosciences, InstituteofNuclearMedicineandAlliedSciences,Delhi, India

AtanuBhattacharjee BioinformaticsLaboratory,Department ofBiotechnologyandBioinformatics,NorthEasternHill University,Shillong,India

SayanBiswas SchoolofNaturalProductStudies, DepartmentofPharmaceuticalTechnology,Jadavpur University,Kolkata,India

MadhuBiyani DepartmentofFunctionalMaterialsScience, SaitamaUniversity,Saitama,Japan

ManishBiyani BioSeedsCorporation,IshikawaCreateLab, Ishikawa,Japan;DepartmentofBioscienceand Biotechnology,JapanAdvancedInstituteofScienceand Technology,Ishikawa,Japan;DepartmentofFunctional MaterialsScience,SaitamaUniversity,Saitama,Japan

ElisabettaCanetta FacultyofSport,HealthandApplied Science,StMary’sUniversity—Twickenham,London, UnitedKingdom

ManojK.Chakrabarti DivisionofPathophysiology, NationalInstituteofCholeraandEntericDiseases, Kolkata,India

JoydebChanda SchoolofNaturalProductStudies, DepartmentofPharmaceuticalTechnology,Jadavpur University,Kolkata,India

ParthaK.Chandra TulaneUniversity,NewOrleans,LA, UnitedStates

RakhiChaturvedi DepartmentofBiosciencesand Bioengineering,IndianInstituteofTechnologyGuwahati, Guwahati,India

AnkitChauhan DivisionofRadiatonBiosciences,Institute ofNuclearMedicineandAlliedSciences,Delhi,India

SudhirChowbina AdvancedBiomedicalComputing Center,SAIC-Frederick,Inc.,FrederickNational LaboratoryforCancerResearch,NationalCancer Institute,Frederick,MD,UnitedStates

D.KarChowdhuri EmbryotoxicologyLaboratory,CSIRIndianInstituteofToxicologyResearch,Lucknow,India

MadhumitaRoyChowdhury GeneticUnit,Departmentof Pediatrics,AllIndiaInstituteofMedicalSciences, NewDelhi,India

MukulDas Food,DrugandChemicalToxicologyGroup, CSIR—IndianInstituteofToxicologyResearch,Lucknow, India

AmritaDatta TulaneUniversity,NewOrleans,LA, UnitedStates

DevyaniDube ISFCollegeofPharmacy,Moga,India

SudhishaDubey InstituteofMedicalGeneticsand Genomics,SirGangaRamHospital,NewDelhi,India

ShantaDutta DivisionofBacteriology,ICMR-National InstituteofCholeraandEntericDiseases,Kolkata,India

BilikereSrinivasaRaoDwarakanath ShanghaiProtonand HeavyIonCenter,Pudong,Shanghai,China

PremendraD.Dwivedi Food,DrugandChemical ToxicologyGroup,CSIR—IndianInstituteofToxicology Research,Lucknow,India

RajarshiKumarGaur DepartmentofBiotechnology,Deen DayalUpadhyayaGorakhpurUniversity,Gorakhpur, UttarPradesh,India

PreetamGhosh DepartmentofComputerScience,Virginia CommonwealthUniversity,Richmond,VA,UnitedStates

JessicaL.Gimpel FacultyofMedicine,PontificalCatholic UniversityofChile,Santiago,Chile

AbhikGupta DepartmentofEcologyandEnvironmental Science,AssamUniversity,Silchar,India

AnujKumarGupta C-11/Y-1,C-Block,DilshadGarden, Delhi,India

MadhuGupta DepartmentofPharmaceuticalSciences,Dr. HariSinghGourUniversity,Sagar,India

UDGupta NationalJALMAInstituteforLeprosyand OtherMycobacterialDiseases(ICMR)Tajganj,Agra,Uttar pradesh,India

RanjitK.Harwansh SchoolofNaturalProductStudies, DepartmentofPharmaceuticalTechnology,Jadavpur University,Kolkata,India

RajatHazra EmamiLimited,Kolkata,India

KaziMirajulHoque DivisionofPathophysiology,National InstituteofCholeraandEntericDiseases,Kolkata,India

ShowketHussain ICMR-NationalInstituteofCancer PreventionandResearch,Noida,India

PoojaJain DepartmentofMicrobiologyandImmunology, DrexelUniversityCollegeofMedicine,Philadelphia,PA, UnitedStates

AnuragJyoti AmityInstituteofBiotechnology,Amity University,Gwalior,India

ShivaliKamal NationalAIDSControlOrganization, MinistryofHealth,NewDelhi,India

SatyajyotiKanjilal EmamiLimited,Kolkata,India

SudhirKumarKashyap DepartmentofVeterinary MicrobiologyandBiotechnology,RajasthanUniversityof VeterinaryandAnimalSciences,Bikaner,India

ChandraKantKatiyar EmamiLimited,Kolkata,India

FahimHalimKhan DepartmentofBiochemistry,Facultyof LifeSciences,AligarhMuslimUniversity,Aligarh,India

ZafarK.Khan DepartmentofMicrobiologyand Immunology,DrexelUniversityCollegeofMedicine, Philadelphia,PA,UnitedStates

SuchitKhanna DivisionofRadiatonBiosciences,Institute ofNuclearMedicineandAlliedSciences,Delhi,India

SatyendraMohanPaulKhurana AmityInstituteof Biotechnology,AmityUniversity,Manesar,India

ArunaKumar SchoolofLifeSciences,JawaharlalNehru University,NewDelhi,IndiaAmityInstituteof Biotechnology,AmityUniversity,Noida,India

NaveenKumar NationalCenterforVeterinaryType Cultures,ICAR-NRConEquines,Hisar,India

SandeepKumar Food,DrugandChemicalToxicology Group,CSIR—IndianInstituteofToxicologyResearch, Lucknow,India

VineetKumar DepartmentofPharmacology,YongLooLin SchoolofMedicine,NationalUniversityofSingapore, Singapore

VipinKumar DepartmentofBiochemistry,Instituteof Science,BanarasHinduUinversity,Varanasi, UttarPradesh,India

NoorALokman DisciplineofObstetricsandGynaecology, AdelaideMedicalSchool,RobinsonResearchInstitute, TheUniversityofAdelaide,Adelaide,SA,Australia

SunilMaherchandani DepartmentofVeterinaryMicrobiology andBiotechnology,RajasthanUniversityofVeterinaryand AnimalSciences,Bikaner,India

AvinashMarwal DepartmentofBiotechnology,Vigyan Bhawan BlockB,NewCampus,MohanlalSukhadia University,Udaipur,Rajasthan,India

ShetMasih MolecularDiagnostics&ResearchLaboratories (MDRL),Chandigarh,India

PawanKumarMaurya DepartmentofBiochemistry, CentralUniversityofHaryana,Mahendergarh(Haryana), India

RaviMehrotra ICMR-NationalInstituteofCancer PreventionandResearch,Noida,India

AyushiMishra DepartmentofBiochemistry,Instituteof Science,BanarasHinduUniversity,Varanasi,India

NishiMody DepartmentofPharmaceuticalSciences,Dr. HariSinghGourUniversity,Sagar,India

DebasisMondal LMU-DCOM,Knoxville,TN,United States

PulokK.Mukherjee SchoolofNaturalProductStudies, DepartmentofPharmaceuticalTechnology,Jadavpur University,Kolkata,India

SoumenMukherjee VirusResearchandDiagnostic Laboratory,ICMR-NationalInstituteofCholeraand EntericDiseases,Kolkata,India

JosephJ.Nalluri DepartmentofComputerScience, VirginiaCommonwealthUniversity,Richmond,VA, UnitedStates

KoichiNishigaki BioSeedsCorporation,IshikawaCreate Lab,Ishikawa,Japan;DepartmentofBioscienceand Biotechnology,JapanAdvancedInstituteofScienceand Technology,Ishikawa,Japan;DepartmentofFunctional MaterialsScience,SaitamaUniversity,Saitama,Japan

NishuNishu MolecularDiagnostics&Research Laboratories(MDRL),Chandigarh,India

MartinKOehler DisciplineofObstetricsandGynaecology, AdelaideMedicalSchool,RobinsonResearchInstitute, TheUniversityofAdelaide,Adelaide,SA,Australia; DepartmentofGynaecologicalOncology,RoyalAdelaide Hospital,Adelaide,SA,Australia;FutureIndustries Institute,UniversityofSouthAustralia,MawsonLakes, SA,Australia

BhaktiPatel LaboratoryofEnvironmentalMicrobiology andEcology(LEnME),DepartmentofLifeScience, NationalInstituteofTechnology,Rourkela,Odisha,India

DebasisPore DivisionofPharmaAnalytics,Excelra KnowledgeSolution,Hyderabad,India

PravinkumarPurushothaman DepartmentofMicrobiology andImmunology,UniversityofNevada,RenoSchoolof Medicine,CenterforMolecularMedicine,Reno,NV, UnitedStates

K.RaviRam EmbryotoxicologyLaboratory,CSIR-Indian InstituteofToxicologyResearch,Lucknow,India

M.RezaKhorramizadeh BiosensorResearchCenter, EndocrinologyandMetabolismMolecular-Cellular SciencesInstitute,TehranUniversityofMedicalSciences, Tehran,Iran;ZebrafishCoreFacility,Endocrinologyand MetabolismResearchInstitute,TehranUniversityof MedicalSciences,Tehran,Iran;Endocrinologyand MetabolismResearchCenter,Endocrinologyand MetabolismClinicalSciencesInstitute,TehranUniversity ofMedicalSciences,Tehran,Iran

CarmelaRicciardelli DisciplineofObstetricsand Gynaecology,AdelaideMedicalSchool,Robinson ResearchInstitute,TheUniversityofAdelaide,Adelaide, SA,Australia

FarshidSaadat DepartmentofImmunology,Schoolof Medicine,GuilanUniversityofMedicalSciences,Rasht, Iran

MalayKumarSaha DivisionofVirology,ICMR-National InstituteofCholeraandEntericDiseases,Kolkata,India

EmmanuelO.Salawu InstituteofBioinformaticsand StructuralBiology,NationalTsingHuaUniversity,Taipei, Taiwan;InstituteofInformationScience,AcademiaSinica, Taipei,Taiwan

RishiShanker ABCGenomics(India)PrivateLtd., BiotechnologyPark,Lucknow,India

RajeevSharma DepartmentofPharmaceuticalSciences,Dr. HariSinghGourUniversity,Sagar,India

AnchalSingh DepartmentofBiochemistry,Instituteof Science,BanarasHinduUniversity,Varanasi,Uttar Pradesh,India

GulshanSingh InstituteforWaterandWastewater Technology(IWWT),DurbanUniversityofTechnology, Durban,SouthAfrica

MithileshSingh G.B.PantNationalInstituteofHimalayan EnvironmentandSustainableDevelopment,SikkimUnit, Panthang,Gangtok,India

NehaSingh DepartmentofBiotechnology,Panjab University,Chandigarh,India

SurinderPalSingh CSIR-NationalPhysicalLaboratory, NewDelhi,India

PriyankaSrivastava SchoolofBioSciencesand Technology,VelloreInstituteofTechnology,Vellore,India

MarkA.Suckow DepartmentofBiomedicalEngineering, UniversityofKentucky,Lexington,KY,UnitedStates

SurajitDas LaboratoryofEnvironmentalMicrobiologyand Ecology(LEnME),DepartmentofLifeScience,National InstituteofTechnology,Rourkela,Odisha,India

RichaTripathi ICMR-NationalInstituteofCancer PreventionandResearch,Noida,India;Divisionof

PreventiveOncology,NationalInstituteofCancerPrevention andResearch(ICMR),Noida,India

KailashC.Upadhyaya SchoolofLifeSciences,Jawaharlal NehruUniversity,NewDelhi,India

TimsyUppal DepartmentofMicrobiologyand Immunology,UniversityofNevada,RenoSchoolof Medicine,CenterforMolecularMedicine,Reno,NV, UnitedStates

AlokKumarVerma Food,DrugandChemicalToxicology Group,CSIR—IndianInstituteofToxicologyResearch, Lucknow,India

AnjuVerma DivisionofPlantPathology,Centrefor AppliedGeneticTechnologies,UniversityofGeorgia, Athens,GA,UnitedStates;DepartmentofPlant Pathology,InstituteofPlantBreedingGenetics& Genomics,CenterforAppliedGeneticTechnologies, UniversityofGeorgia,Athens,GA,UnitedStates

AshishSwarupVerma JadavpurUniversity,RajaS.C.Mallick Road,Kolkatta,WestBengal,India

MeghaVerma CollegeofArtsandSciences,St.Louis,MO, UnitedStates

MuditVerma EpidemiologyandGenomicsResearch Program,DivisionofCancerControlandPopulation Sciences,NationalCancerInstitute,NationalInstitutesof Health(NIH),MedicalCenterDrive,Rockville,MD, UnitedStates

MukeshVerma EpidemiologyandGenomicsResearch Program,DivisionofCancerControlandPopulation Sciences,NationalCancerInstitute,NationalInstitutesof Health(NIH),MedicalCenterDrive,Rockville,MD, UnitedStates

PoonamVerma BioglobeResearchandSolutionsLLP, NewDelhi,India

SubhashChandraVerma DepartmentofMicrobiologyand Immunology,UniversityofNevada,RenoSchoolof Medicine,CenterforMolecularMedicine,Reno,NV, UnitedStates

VipinVerma BioglobeResearchandSolutionsLLP, NewDelhi,India

SureshP.Vyas DepartmentofPharmaceuticalSciences,Dr. HariSinghGourUniversity,Sagar,India

DineshK.Yadav DepartmentofBotany,Universityof Allahabad,Allahabad,India

NeelamYadav DepartmentofBotany,Universityof Allahabad,Allahabad,India

KojiYamanaka DepartmentofNeuroscienceand Pathobiology,ResearchInstituteofEnvironmental Medicine,NagoyaUniversity,Nagoya,Japan

EugeniaCh.Yiannakopoulou DepartmentofBasic MedicalLessonsFacultyofHealthandCaring Professions,TechnologicalEducationalInstituteof Athens,Athens,Greece

Preface

Intheyear2016ProfessorAshishS.Vermareceived acallfromAcademicpressofficewitharequestto comeupwiththesecondeditionofourbook Animal Biotechnology:ModelsinDiscoveryandTranslation.Soon hesharedthisinformationwithmeandbothofus werethrilledandsuperexcitedasithadbeenjust3 yearssincethefirsteditionwaslaunchedandwehad neverthoughtthatwewouldbeworkingonthesecondeditionsosoon.Thechallengewastoadd,update, andrevisethefirsteditionbecausebythattimeboth ofushadmovedtodifferentplaces,Ihadjoined BanarasHinduUniversity,Varanasi,India,andProf. VermabecametheactingVice-ChancellorofJadavpur University,Kolkata,India.Eventhoughhisunending administrativeresponsibilitieskepthimbusydayand nightstillhehadsolutionstoeveryproblemthatI couldfathomineditingthenexteditionatthattime. Ultimatelywestartedworkingtogetheronthisedition byreviewingalltheinputsandcriticismwehad receivedforthefirstedition,fromstudents,instructors,teachers,andscientists.

WealwaysfeltthatthefieldofAnimalBiotechnology assuchiswellexploredandiscontinuouslyevolvingto comeupasafulldisciplineverysoon.Nevertheless,itis stilldifficulttofinddedicatedbooksonthesubject,and atthesametimebooksthatcancatertheneedsofstudents,instructors,andteachersalike.Thefirsteditionof AnimalBiotechnology:ModelsinDiscoveryandTranslation wasdevelopedasaResourceBookasitwouldprovide sufficientinformationandliteratureforinstructorsto teachthesubjectswhilestudentswillfindampleinformationtogainabetterinsightofthetopic.Whileworkingonthesecondedition,wehavemaintainedthesame patternasbefore,infact,thesecondeditionincludes substantialrevisionandupdateofthecontentandinformationwhilecontinuingwiththeeasy-to-followlanguageanduniformityofthestyleandpresentations whichthefirsteditionhad.Consideringtherecent advancementsinnanotechnology,enzymetechnology, andayurvedawestronglyfelttohavechaptersthatwill helpinhavingaviewofAnimalBiotechnologyfrom theseperspectives.Asaresultthesecondeditionhas entirelynewchapterscomprisingofvariousaspectsof nanotechnology,ayurveda,enzymetechnology,euthanasia,CAMassays,andalsosomechaptersdealingwith newermodelstostudydifferenthumandiseases.

Almostallthechaptersfromthefirsteditionhave beenretainedbuttheyhavebeenrigorouslyupdated withthemostrecentinformationavailablewiththe

expertsofthefield.Moreovereachchaptercontains interestinginformationthatisaddedasaTurning point,it’ssomethingwhichwehopethatourreaders willcherishwhilereadingthetopicoftheirinterest. Wehavealsoaddedasub-topiconClinicalCo-relation ineachchaptersothatreaderswhohaveaninterestin theclinicalaspectofdifferenttopicscanfindtherelevantinformationinoneplacewhiledevelopingthe subject.Manysectionshavebeenrewrittenforbetter clarity,understanding,fluencyofstyle/presentation, andlanguage.Wehavetriedtodevelopeachchapter inanindividualmannersothatreaderswhopreferto purchaseindividualchapterscanfindallthedetails relatedtothetopicinthesamechapteritself.Wehave triedourbestforimprovementofthefiguresandillustrations,someofthesehavebeenrevised,fewnew figureshavebeenaddedineachchapterandthe figuresfromthelasteditionhavebeenupdated.Very similartothefirstedition,eachchapterhasasubsectionontheavailableinternetresourcesthatarerelated tothetopicandalistofreviewarticlesandtextbooks asfurtherreadingisalsoprovidedforthosereaders whosejet-speedcuriositiesneedstillmorejetfuel.

Whilewewereworkingonthesecondedition,Prof. Vermawastakenawaybythecruelhandsofdestiny onMay11,2019,andhissudden,untimelydemiseleft mecompletelyshocked.MeandProf.Vermahadbeen workingtogethersince2008andIwasdevastatedas thisentireprojectwasmid-wayandfromthenonI wouldbeeditingthebooksingle-handedly.Atthis pointImustappreciatethekindgestureoftheentire AcademicPressteamespeciallyMr.PeterLinsley (SeniorAcquisitionsEditor)andMr.TimothyBennett (EditorialProjectManager)whogavemesupportand showedtrustinmycapabilities.Theygenerously extendedthebooktimelinesduetowhichthisbook couldbecompletedinitspresentform.

Finally,Iwouldliketorequestthereadersofthis booktoextendtheirfullsupportastheyhadextended forthepreviousedition.Wearealwaysopentocriticism,suggestions,andrecommendationsthatcanbe helpfultoimprovethecontentsandpresentationof thebook.Yoursuggestions/criticismwillallowusto exploreotheraspectsofAnimalBiotechnologyandin ourfutureventuresandendeavors.

Acknowledgments

AsEditors,wewouldliketoexpressourgratitudeandthankstoallthecontributingauthors,whoseexpertise andexperienceisnowwithusintheformofbookchaptersintheirrespectivefields.Iunderstandthatourcontributorshaveworkedhardtoupdate,upgrade,andimprovetheirchapterssothatallchaptersofthebookcould beuniforminstyleandpresentation.Thisbookcouldneverhavebeenpossiblewithouttheirvaluableworkand timelysupport.

LateProf.AshishS.VermawouldhavesurelythankedhismotherMs.SushmaSaxena,forhergreateffortsto raiseandgroomhim.HisbrotherMr.SaumyaSwarup,sisterinlawMs.NimishaSwaruptheirkidsMr.Utkarsh andMs.Shreeparna,hissisters,andtheirfamiliesalsodeserveaspecialthanksfortheirloveandsupport.

I(Anchal)wouldliketothankmyPh.D.supervisorProf.SushmaRathaur,UGC-BSRfellow,Departmentof Biochemistry,BanarasHinduUniversity,India,whohasalwaysguidedandhelpedmeandhasstoodbesideme asthebiggestsupporttilldate.IwishtoexpressmygratitudetomydadMr.KanhaiyaJiSingh,MotherMs. MohiniSingh,brotherAbhisarandhiswifeMeenakshifortheirsupport,love,andhelp.MysonAviraland nephewAaravwerethe“Stressbreakers”andtheirfrequentrequeststoindulgeingamesandactivitieswere mostlyignoredforthesakeofmeetingthetimelinesofbookediting.

Anchal’sresearchscholarsnamelyVipinKumarandAyushiMishragreatlycontributedtheirpartforthis bookandareacknowledgedfortheirhelp.Attimestheyofferedhelpandsupporttoorganizeusbetterandat othertimestheydidn’tfailtocriticizeus,butwhatevertheyhavedonetowardthisbookisadmiredbyus.We areindebtedtoMr.DineshKumarwhohasworkedwithusforthelast12yearsandhasbeeninstrumentalin providinghissecretarialassistance.

LastbutnotleastthisbookcouldneverhavebeencompletedwithoutElsevierpersonnelworkinginthisproject.Mr.PeterLinsley(SeniorAcquisitionsEditor),Mr.TimothyBennett(EditorialProjectManager),andMr. SreejithViswanathan(ProjectManager)whokeptsupporting,motivating,andpushingusthroughouttheproject duration.Weconveyourheartfeltthankstoeveryonewhohascontributeddirectlyorindirectlytothisbook.

Andallthiswouldneverhavebeenpossiblewithout“TheAlmighty”GODwhomweoweourexistenceto. Theonewhogiftedus(Humanbeings)abraintohypothesizeandanalyze,couragetodream,andmotivationto achieve,andhencewethankyouformakingourbookinstrumentalinspreadingknowledgeandfueling creativity.

LATE ASHISH S.VERMA ANCHAL SINGH

Drosophila:amodelforbiotechnologist

K.RaviRamandD.KarChowdhuri

EmbryotoxicologyLaboratory,CSIR-IndianInstituteofToxicologyResearch,Lucknow,India

Summary

Drosophilaisaminiatureyetversatileandmanipulable modeltoaddressbasicbiologicalquestionswithpotential implicationsplusapplicationstoothermetazoans.Inthis chapter,weemphasizethecontributionsofDrosophilato geneticsandbiotechnologyandtranslationalversatilityof thismodelalongwithassociatedethicalissuesandavailableresources.

NobellovestoryofDrosophila

Sofartheoutstandingcontributionsoffruitflyresearch havebeenrecognizedwithsixNobelprizesinphysiologyor medicine

1933 ThomasHuntMorgan—Chromosomesin heredity

1946 HermannJosephMuller—X-rayirradiationto increasemutationratesinfruitflies

1995 EdwardBLewis,ChristianeNusslein-Volhard, andEricFWieschaus—Geneticcontrolofembryonic development

2004 RichardAxel—Odorreceptorsandtheorganizationoftheolfactorysystem

2011 JulesAHoffmann—Activationofinnate immunity

2017 JeffreyCHall,MichaelRosbashandMichaelW Young—Molecularmechanismsregulatingcircadian rhythms

Whatyoucanexpecttoknow

Initially,thischapterfacilitateslearningofbasic conceptsofDrosophila,whichevolvedthisorganism asamodelforgeneticsandbiotechnology.Thehistoricalperspectivehelpsthereadertoassimilatethe contributionsofDrosophilaresearchfindingsto

biotechnology.Thelaterpartofthechapteracquaints thereaderwithaprotocoltogenerateDrosophila transgenicsandexemplifiesthetranslationalpotential ofthesametowardunderstandinghumandiseases.

Introduction

Innovativegenetictechnologieshaverevolutionized thewisdomofscience.Forexample,cloningand manipulationofgenesequenceshavehelpedinthe generationoftransgenicstowardasubstantialunderstandingofbiologicalconceptsandalsoforthebettermentoflife.However,thepivotalroleplayedby modelorganismsforbiotechnologiststoachievethese challengesisindeedphenomenal.Onesuchmodel organismthathashelpedbiotechnologiststorealize theirdreamsisDrosophila.Withpinnaclecontributionstogeneticsanddevelopmentover100years, Drosophilacontinuestoinspirethecreativityofbiotechnologists.Drosophilaissoamenabletogenetic manipulationthatitisconstrainedonlybytheimaginationofbiotechnologists.

Drosophilaisatinyflyalsoknownasvinegarlovingfly.Theterm Drosophila,meaning“dew-loving,” isamodernscientificLatinadaptationfromGreek words dro´sos,“dew,”and phı´los,“loving”withthe Latinfemininesuffix -a.ItbelongstothePhylum Arthropoda,classInsectaandorderDipteraandthe famousfamilyofDrosophilidae. Drosophila isasmall fly,typicallypaleyellowtoreddish-browntoblack, withredeyes.Theplumose(feathery)arista,bristling oftheheadandthorax,andwingvenationarethe charactersusedtodiagnosethefamily.Mostaresmall, about2 4mmlong,butsome,especiallymanyof theHawaiianspecies,arelargerthanahousefly.The genus Drosophila isfoundallaroundtheworldright fromdesertstotropicalrainforeststocitiestoalpine

zones.Mostspeciesbreedinvariouskindsofdecaying plantandfungalmaterialsincludingfruit,bark,slime fluxes,flowers,andmushrooms.

OfthevariousspeciesofDrosophila, Drosophilamelanogaster offersseveraladvantagesasamodelfor molecularstudies.Beingsmall,thesefliesare extremelysimpletohandle.Thesexualdimorphism (malesandfemalesaredifferent)permitseasydifferentiationofsexes.Further,thesefliesarenonpathogenic,haveashortergenerationtime(10 12days), andcanbeculturedatalowcostinalimitedspace.In addition,Drosophilaoffersvariousmolecularand genetictoolsthatabiotechnologistcandreamofand thefullysequencedgenomecoupledwithbioinformaticstoolsenhancethetranslationalutilityofthismodel.

Inthischapter,weinitiallydescribetheclassical aspectsofDrosophilasuchaslifecycle,cytology,and development.Subsequently,weprovideahistorical perspectiveofresearchhallmarksthatledtotheutility ofDrosophilaasamodelformolecularstudies.In addition,wediscussthetranslationalsignificanceof DrosophilabyemphasizingDrosophilamodelsavailableforhumandiseases.Finally,wediscusstheethical issuesandconcernsassociatedwiththismodel.

Classicalaspectsof Drosophilamelanogaster

Physicalappearance

ThebodyofDrosophila,likethatofanyother insect(andtypicalofArthropoda),issegmented.The bodyplantypicallyconsistsofhead,thorax,and abdomen.Whilemultiplesegmentsgiverisetothe head,threesegmentsconstitutethethoraxandeight segmentsformtheabdomen.Headconsistsofantennaewhereaslegsandwingsarisefromthoracicsegments. D.melanogaster hastransverseblackrings acrosstheirabdomen.Malesareeasilydistinguishablefromfemalesbythepresenceofadistinctblack patchattheabdomenthatisabsentinfemales.Males alsohavesexcombs,arowofdarkbristlesonthetarsusofthefirstleg,whichareabsentinfemales. Furthermore,maleshaveaclusterofspikyhairs calledclaspersthatsurroundtheanusandgenitals usedtoattachtothefemaleduringmating.

Lifecycle

D.melanogaster isapopularexperimentalanimal becauseitiseasilyculturedinmassoutofthewild,has ashortgenerationtime,andmutantanimalsarereadily obtainable.Typically,inalaboratory, D.melanogaster is grownoncornmeal yeast fruitjuicemixtureat25 C. Lifecycleofthisorganismconsistsofanumberof stages:embryogenesis,threelarvalstages,apupal stage,andtheadultstage.Thedevelopmentperiodfor

D.melanogaster varieswithtemperature.Thetime requiredforcompletedevelopmentat25 Cis8 9days. Femaleslaysome400eggs,aboutfiveatatimeon overripefruitorothersuitablematerials.Theeggs, whichareabout0.5mmlong,hatchafter20 22hours at25 C.Theresultinglarvaegrowforabout3days whilemoltingtwiceintosecondandthird-instarlarvae,atabout24and48hours,respectively,aftereclosion.Thelarvathenencapsulatesinthepuparium thatisimmobileandunder goesa4-day-longmetamorphosisat25 C.Aflyfinallyemergesfroma pupariumaftermetamorphosis,aprocessreferredto aseclosion.ThelifecycleofDrosophilaisschematicallydepictedin Fig.1.1.

Drosophiladevelopment

Drosophilahascontributedtomostofourexisting knowledgeonthemechanismsofthedevelopmentof organisms.InDrosophila,thecomplexadultbody planisaccomplishedfromthefertilizedembryo throughdevelopmentalprocesses.Developmentin Drosophilaisholometabolous,whichinvolvesdevelopingstagesmorphologicallydistinctfromadults (Hartenstein,1993).Earlyprocessesofdevelopment occurinthefertilizedegglaidbythefemaletogive risetothelarva.Thelarvasubsequentlygivesriseto thepuparium,afterundergoingaseriesofmodificationsandtwomoltings,drivenbythehormonaltiters andmolecularsignals.Duringthepupalstage,many larvalstructuresarebrokendown,andadultstructuresundergorapiddevelopment.Inthissection,

III instarlarva

Pupa Egg

instar larva

instar larva

FIGURE1.1 Lifecyclestagesof Drosophilamelanogaster

wedescribethekeyaspectsofDrosophiladevelopment:embryogenesis,patternformation,andhomeotic genes.

EmbryogenesisinDrosophila

TheearlydevelopmentofDrosophilabeginswith theformationofoocytesthroughoogenesisinthe ovary(Hartenstein,1993).Theseoocytesarepacked withmaternalRNA,protein,ribosomes,andmitochondriathatassistintheeggtoembryotransition. Fertilization(unionoftheseoocytesandsperm)triggersmitosisintheembryo.Severalnucleardivisions withoutcytokinesis(divisionofcytoplasm)occurin theearlyembryo,resultinginacellwithmanynuclei inthecytoplasm.Atthe10th nucleardivision,these nucleimigratetowardthesurfaceoftheembryo, resultingintheformationofthesyncytialblastoderm. Atthe13th nucleardivision,membraneinvaginations enclosethenucleileadingtocellularizationandthe formationofthecellularblastoderm.Atthetimeofcellularization,themajorbodyaxesandsegmentboundariesaredetermined.Aftercellularization,theembryo proceedsthroughgastrulation:cellsfromtheventral surfaceinvaginatetocreatetheventralfurrow.Ventral furrowiscriticalfortheformationofthemesoderm. Subsequently,attheanteriorandposteriorendsofthe ventralfurrow,theinvaginationofprospectiveendodermoccurs.Gastrulationisfollowedbytheconvergenceofcertainectodermalcellsonthesurfacewith themesodermandtheirmigrationtowardtheventral midlinetoformthegermband,acollectionofcells thatwillformthetrunkoftheembryo.Thegermband extendsposteriorandwrapsaroundthedorsalsurface oftheembryo.Atthisextendedstate,severalmorphogeneticprocessesoccur:organogenesis,segmentation, andsegregationofimaginaldiscsthatwillunfoldduringmetamorphosistoformadultflystructuressuchas antennae,legs,andwings(Campos-Ortegaand Hartenstein,1985;MartinezArias,1993).Interestingly, atallthestagesofdevelopment,thegeneralbodyplan remainsthesame.Thegeneralizedbodyplanconsists ofasegmentedregionsandwichedbetweenadistinct headoranteriorregionandthetailorposteriorregion. Drosophila,withitsversatilegenetictools,hasledto theunderstandingofpatternformationanddifferentiationduringearlyanimaldevelopment.Threescientists,namely,EdLewis,ChristianeNusslein-Volhard, andEricWieschauspioneeredourunderstandingof patternformationanddifferentiation.Thesescientists notonlylaidtheplatformforourunderstandingof developmentbutalsodecipheredtheunderlying dynamics.Nusslein-VolhardandWieschausfocused theirstudiesonunderstandingearlyembryogenesis whileEdLewisconcentratedonlateembryogenesis.

PatternformationinDrosophila

Themetamorphosisofasimpleeggintoanadult withthecomplexbodyplansrequiresthreeclasses ofgenes.Theseclassescomprisethematernalgenes, thesegmentationgenes,andthehomeoticgenes. Nusslein-VolhardandWieschaus(1980) werethefirst toreportthekeycontributionsofeachgenethatregulatesaparticularpatternformationevent,thesegmentationofembryo.Theylookedforrecessiveembryonic lethalmutations,inasystematicgeneticscreenencompassingthewholegenometoidentifygenescriticalfor embryonicdevelopment.Subsequently,theyanalyzed thephenotypesofdeadembryosandclassifiedthese genesaccordingtotheirphenotypebeforedeath. Basedontheirphenotypicanalyses, Nusslein-Volhard andWieschaus(1980) identifiedthreecategoriesof mutations.Thefirstcategorycomprisedmutationsthat resultinthelossofmultipleadjacentsegments(called genesencodingthesameasgapgenes).Thesecond categoryincludedmutationsthatcausemissingof alternatesegmentsizeunits(accordingly,namedtheir genesaspair-rulegenes).Thethirdcategoryofmutationstriggeredthelossofpartofeachsegmentand duplicationoftheremainingpartofthesegment (namedassegmentpolaritygenes).Inviewoftheir findings, Nusslein-VolhardandWieschaus(1980) proposedthatgapgenes,pair-rulegenes,andsegment polaritygenes(togethercalledassegmentationgenes) arecriticalforsubdividingtheembryoandsegment formation.Anotherclassofgenes,includinghomeotic genes,definestheidentityofthesegment.However,to putthesegeneticcascadesintomotion,maternalcomponentsareessential.

Maternalcomponentsplayacriticalroleindeterminingtheembryonicpatterning(Hartenstein,1993). Asdiscussedabove,evenbeforefertilization, Drosophilaeggsareloadedwithregulatorymolecules thatdeterminetheanteroposterioraxisoftheeggand developmentoftheorganism.Eggsarepreloadedwith bicoidandnanosmRNA,andthesearetranslated uponfertilization(Hartenstein,1993).Ofthesetwo, bicoidisessentialfortheformationofthehead: femalescarryingmutantallelesofbicoidgiveriseto offspringwithdefectsinheaddevelopment.Priorto cellularization,theseproteinsformconcentrationgradientsintheembryo.Attheanteriorend,bicoidisata higherconcentration(Fig.1.2),whereasattheposterior end,nanosareabundant.Theseconcentrationgradientsarecriticalforregulatingsegmentationgenes(see above),whichdefinethesegmentationpattern. Temporalandspatialactivationofgenecascadesisthe hallmarkofdevelopment.Duringtheinitialphaseof development,bicoidandnanosdifferentiallyregulate thegapgenes.Attheanteriorend,bicoidtriggers thetranscriptionofagapgene, hunchback.However,at

FIGURE1.2 LocalizationofBicoidinDrosophilaembryo. ImmunostainingofDrosophilaembryoswithanti-Bicoidantibody revealstheaccumulationofBicoidprotein(green)intheanterior partoftheembryo.Bluecolorrepresentsthenuclearstainingby DAPI.

theposteriorend,nanosinhibit hunchback RNAfrom beingtranslated,therebyformingahunchbackprotein gradientintheembryo(Wredenetal.,1997). Thishunchbackproteintriggers,inaconcentrationdependentmanner,thetranscriptionofothergap genessuchas Kruppel,etc.(SchulzandTautz1994; Zuoetal.,1991),whichinturndefineslargeareassurroundingtheanteroposterioraxis(Gilbertetal.,2003). Thegapgenesencodetranscriptionfactorsthatregulatetheexpressionofcertainpair-rulegenes,whichin turnregulateotherpair-rulegenes.Thesepair-rule genes,whichareexpressedalongthestripesofthe embryo,dividetheembryointopairsofsegments. Pair-rulegenesencodetranscriptionfactorsthatregulatesegmentpolaritygenes.Thesesegmentpolarity genesdefinetheanteroposterioraxisofeachofthe segment(Fig.1.3).Oncethepatternofsegmentationis established,thesegmentsachieveuniqueidentities throughhomeoticgenes.

HomeoticgenesinDrosophila

Thetermhomeosisrepresentsthetransformationof onestructureofthebodyintothehomologous

FIGURE1.3 LocalizationofEngrailedinDrosophilaembryo. ImmunostainingofDrosophilaembryoswithanti-EngrailedantibodyrevealsEngrailedlocalization(red)withintheposteriorsection ofeverysegmentwithintheembryo.Bluecolorrepresentsthe nuclearstainingbyDAPI.

structureofanotherbodysegment.InDrosophila,as mentionedearlier,inadults,structuressuchaslegs, wingsdevelopfromthoracicsegmentswhereasantennaeappearonthehead.Thesegment-specificdevelopmentofthesestructuresrequirestheactionof homeoticgenes.Homeoticgenesareagroupofgenes thatregulatethepatternformation.Thesegenes, althoughdonotspecifytheelementsofthepattern, indeedassignidentitiestotheseelements.Mutations inthesegenesresultinthedevelopmentoftheelementsofthespecifiedpatternwithinappropriateidentities.Thebestexampletodescribethesegenesis Antennapedia(Antp).Asthenamesuggests,adominantmutationinthisgenetransformstheantennal structuresontheheadtoanadditionalsecondleg. Generally,normalAntpisrequiredinthesecondthoracicsegmenttoinitiatethecascadeofeventsthatlead tothedevelopmentofleg.Perhaps,thisinvolvesthe regulationofseveralgenes.Hence,thehomeoticgenes areconsideredasmastercontrollersofdevelopmental programming(AbbottandKaufman,1986).

Mostofourknowledgeonhomeoticsisduetothe pioneeringworksofEdLewisonbithoraxcomplex (BX-C)(Lewis,1978).Thehomeoticgenesconsistof 180nucleotideconsensussequencecalledthehomeobox.Thehomeoboxcorrespondstoa60aminoacid domain,namely,thehomeodomain,whichisinvolved intheDNAbinding.Thesehomeobox-containing (HOX)genesarenotlimitedtoDrosophilabutlater

havebeenfoundandstudiedinmanyotherorganisms rangingfrominvertebrates tovertebrates,including mammals( Ruddleetal.,1994;Santinietal.,2003). HOXgenesoccurinclusters,andinterestingly,not onlythesegenesbutalsothesynteny(relativegene order)withintheclusterisconserved(Ruddleetal., 1994).Moreover,theorderofHOXgenesonthechromosomeissameastheorderofthesegmentsthat theyaffectalongtheanterioposterioraxis.Genetic analysisrevealedtheexistenceofposteriordominance:genesactingattheanteriorareregulatedby theirposteriorneighbors.Forexample,BX-Ccomprisesregionsencodingthreehomeodomaingenes calledUltrabithorax(Ubx),Abdominal-A(AbdA), andAbdominal-B(AbdB)andanoncodingRNA, iab-8-ncRNA( Gummallaetal.,2012;Lewis,1978).In thiscase,iab-8-ncRNArepressesAbdA(Gummalla etal.,2012 )andbothAbdAandAbdBrepressUbx ( Lewis,1978 ).Inadditiontotheintrahomeoticregulation,thesehomeoticgenesarealsoregulatedby segmentationgenes.Forexample,hunchback(gap gene)isknowntolimittheUbxexpression ( Wuetal.,2001 )andmutationsinpair-rulegenes influencetheexpressionofhomeoticgenes.Atpresent,however,theknowledgeofhowsegmentation genesregulatehomeoticgeneexpression/repression islimited.Nevertheless,asdiscussedsofar,studies onDrosophilahavetremendouslycontributedtothe understandingofdevelopmentinmetazoananimals. Theeventsassociatedwithpatternformationin Drosophilaareschematicallydepictedin Fig.1.4

Drosophilagenome

D.melanogaster hasfourpairsofchromosomes:an X/Ypairandthreeautosomeslabeled2,3,and4.The fourthchromosomeisquitetiny.Thesizeofthe genomeis165millionbasepairsandcontainsanestimated14,000genes(Adamsetal.,2000)(bycomparison,thehumangenomehas3400millionbasepairs andmayhaveabout25,000genes,Internationalhuman genomeconsortium,2004). Drosophila genomecontains aconsiderableamountofnonprotein-codingDNA sequencesthatareinvolvedinthecontrolofgene expression.Thedeterminationofsexin Drosophila occursbytheratioofXchromosomestoautosomes.

History

HistoricalperspectiveofDrosophila contributionstobiotechnology

Theseedsformodernbiotechnologyresearchin Drosophilaweresownbythe1970s.In1974,random clonesforDrosophila,thefirstforanyorganism,were generatedintheD.S.HognesslaboratoryatStanford University.Byearly1975,clonelibrariesrepresenting theentiregenomeweregeneratedandscreensfor clonescarryingspecificsequenceswiththenewly developedmethodofcolonyhybridizationwerein place.Inearly1979,cloningofagene,ultrabiothorax, wasachievedforthefirsttime.Bylate1980,many mutantalleleshadbeenclonedandshowntobethe consequenceofchromosomalbreakageortransposable elementinsertion.Subsequently,theavailabilityof transposableelementvectorsonlyaddedtothegrowth ofDrosophilaasamodelinthefieldofbiotechnology. Theuseoftransposableelementsforgeneratingtransgenicflieshasrevolutionizedgenemanipulationin DrosophilaandpioneeredthedevelopmentofapowerfularrayoftechniquesinDrosophila,manyofwhich wereultimatelyadaptedtoothermetazoans.These methodsrangefromenhancertraps(1987),large-scale insertionalmutagenesis(1988),site-specificrecombinationforgeneratingchromosomalrearrangements (1989)tothehighlypopularbinarysystemsforcontrollingectopicgeneexpression(1993).By1999,over 1300geneswerecloned,sequencedandfunctionswere characterizedusingthelossoffunctionphenotypes. Theseenormoustoolsweresomeaningfulthatmost researchersdidnotevenconsiderthewhole-genome sequencingofDrosophila.Ultimately,whenthe Drosophilagenomewassequencedin2000,itgave anothervalueadditiontothefieldofbiotechnology, thewhole-genomeshotgunapproach,forgenome sequencing.Subsequently,thevastresourcesmade availableinrecentyearsbytheresearchcommunity

FIGURE1.4 Schematicdepictionofclassesofgenesassociated withpatternformationin Drosophilamelanogaster

forgenomewiseectopicexpressionandknockdown (RNAi),bothinvitroandinvivo,haveenhancedthe utilityoftheDrosophilamodel.Recenteffortsbyfly researchershavedecipheredthestageaswellas tissue-specificexpressionaswellaslocalizationofthe majorityofthegenesinDrosophila.Thefully sequencedgenomecoupledwiththesevariousgenetic andmoleculartoolsledtotheupsurgeofDrosophila asamodelforbasicaswellastranslationalresearch.

Principle

Drosophilaisawell-studiedandhighlytractable geneticmodelsystemtodecipherthemolecular mechanismsunderlyingvariousbiologicalprocesses. Thecompletionofgenomesequencingandannotation discoveredthehighdegreeofconservationoffundamentalbiologicalprocessesbetweenDrosophilaand mammals.Thishaspromptedthebiotechnologiststo utilizeDrosophilatounderstandthemolecularbasisof humandiseases.TheeaseatwhichDrosophilatransgenicscanbecreatedwasalsoinstrumentalinthesuccessofthismodelforunderstandinghumandiseases. Usingaplethoraofmoleculartoolsavailablefor Drosophila,biotechnologistsgeneticallymanipulated Drosophilabyeitherinsertingthehumangenesinthe flygenomeormodifyingthefunctionofhumandisease orthologsinDrosophila.

Methodology

Giventhefocusofthischapter,herewedescribe onlythoseDrosophila-basedmethodsessentialforthe germ-linetransformationofDrosophilatogenerate transgenics.

CulturingofDrosophila

D.melanogaster isrearedonstandardDrosophila foodmediumat22 C 6 1 C.Stocksareusuallymaintainedinvials(upto20 30flies/vial),andexperimentalculturesaremaintainedinbottles(astheypermit thegrowthoffliesinlargenumbers).

PreparationofDrosophilafoodmedium

Materialsrequired

Glassorplasticvials(70 90mmheightwith 25 30mmouterdiameter)

Roundflatbottomhalf-pintglassorplasticbottles

Agar-agar,maizepowder,sugar,yeast, methylparaben,andpropionicacid

Therecipetoprepare1Lofflyfoodisasfollows:

Agar-agar8g

Maizepowder15g

Sucrose100g

Bakerydryyeast100g

10%Benzoicacid5mL

Propionicacid8mL

Water1000mL

Oneliterofwaterisaddedtoa2-L(glassorstainlesssteel)beaker,andthesameistobekeptonahot plate.Sugarisaddedslowlytothewater,thebeakeris coveredwithaglassplateandthewaterisheated.In themeantime,thesolidingredients(agar-agar,maize powder,anddryyeast)shouldbemixedandtobe addedtowateronceitstartstoboil,withconstant stirring.Thecontentsareboiledfor15 20minutes. Subsequently,measuredquantities(asabove)of10% benzoicacidandpropionicacidareaddedwiththoroughstirring.Theheateristurnedoffandfoodis broughttothetableandcanbepoured(3 5mL)into thevialdependingupontherequirement.Thefood shouldbeallowedtocoolandsolidifybeforeplugging thevialswithcotton.Acoupleofyeastgranules shouldbeaddedtothesevialsandneedtoleavethem overnight.Nowthesefoodvialsarereadyforusage. Thesameisthecasewithbottlesexceptthatthequantityoffoodwillbeproportionallyhigher.

Handlingofflies

Drosophila,whichbelongstoaclassofinsects,tends tofly.Thereforethesefliesneedtobeputtosleepfor sexingofmalesandfemalesandtosetup/carryoutthe experiment,dependingupontherequirement.Several methodsareavailabletoputfliestosleep.Theseinclude exposureoffliestoether,chilling(orcooling),CO2,or nitrogen,thelatterthreebeingleastharmful.Ofthese threechoices,coolingislittlebitmessybutisthesimplestwithouttherequirementforanysophisticated equipmentandneedsmerelyiceandpetridishes.In addition,itistheonlymethodwhichwillnotaffectfly neurology.Theremainingtwomethodsrequirecommerciallyavailablegascylindersandcontrollerstoprovidea regulatedsupplyofthegastoincapacitatetheflies.

Flydisposal

Thisisaveryessentialstepwhenusingflies.A bottleorbeakerwith(new/used)oil(whosedensity isheavierthanwater,suchasmineraloil),referredto

asflymorgue,isgenerallyused.Theanesthetizedbut unusedfliesshouldbedumpeddirectlyintothefly morgue.Generally,theydrowntothebottombut needtobeensured(especiallyinthecaseofoldmorgue).Discardingoffliesinthemorgueisaimedat minimizationofstockcontaminationandtokeepthe labenvironmentflyfree.Theoldvialsandbottles containingfliesshouldbeautoclavedtokilltheflies, priortodiscarding.

Eggcollection

Abundantbatchesofeggssynchronizedinageare requiredforexperiments.Ingeneral,200 300adults fromfreshculturesshouldbetransferredintobottles orcollectionchamberscontainingtinypetriplatescontainingflyfood(orgrapejuice-agarfood).Tooptimize eggcollections,fliesarestarvedfor4 6hoursunder lightinemptybottles.Subsequently,fliesaretransferredtocollectionchambersandkeptindark.The firsthour’scollectionshouldbediscarded,toavoid thoseeggsretainedbyfemalesinanticipationoffresh food.Thereafter,eggcollectionplatescanberemoved andreplacedwithnewonesat30-minuteintervals.

Dechorinationofeggs

Topreparetheeggsformicroinjection,theoutercoveringofegg,namelychorion,shouldberemoved.For chemicaldechorination,eggsarewashedfromtheegg collectionplatetoanetwellusingeggwashbuffer (0.03%TritonX-100,0.4%NaCl)andthenetwellcontainingtheeggsisplacedinto2%sodiumhypochlorite (bleach)for2minutes.Thereafter,theeggsarethoroughlywashedwitheggwashbufferandcollectedonto anagarbedorontoapetriplate.Usingafinebrush,the eggsarearrangedinaverticallylinearfashionandtransferredtoaslideusingadouble-sticktape(Scotch665). Afteroptimaldehydration,theeggsarecoveredwitha layerofhalocarbonoil.Later,DNAisinjected(througha fineglassneedle)intotheseeggsundertheinverted phasecontrastmicroscopeusingmicromanipulator.

PreparationofDNAforinjection

Togeneratethetransgenic,thegeneofinterestshould firstbeclonedintoavectorusedforgerm-linetransformation.ForDrosophila,P-element-basedvectors(e.g., pPUASTandpPCASPER)thatcanintegratethegeneof interestintotheflygenomearequitecommonlyused. Dependingupontherequirement,thecompletegeneor thecodingregioncanbeclonedintotheP-elementvectors.Oncethegeneiscloned,theplasmidDNAis extracted.ThesuccessfultransformationrequiresDNA

atanoptimalconcentrationof1 μg/μLandshouldbe endotoxin-free.Thereafter,theplasmidDNAismixed withahelperplasmidDNAataratioof3:1:helper encodesfortransposaserequiredforthetranspositionof thetransgeneintotheflygenome.Subsequently,the mixedDNAisprecipitatedusingethanolandresuspendedintheinjectionbuffer(5mMKCl;0.1mM NaPO4 bufferpH7.5).

Protocols

Protocolforgerm-linetransformationin

Drosophila

Theprotocolgivenbelowisadaptedfromthoseof Prof.JohnBelote,UniversityofSyracuse,UnitedStates, andProf.Heifetz,UniversityofJerusalem,Israel.This isschematicallyrepresentedinFlowChart1.1.

Materialsrequired

Therequiredmaterialsareasfollows:plasmidpurificationkit;helperplasmid(generally, Δ 2 3);injectionneedle:glasswithdiameterofopeningofthe tipapproximately0.5 μm;flystrain:whiteeyeof

Preparation of plasmid DNA

Cloning of gene Prepare needles Expand Drosophila stock

Load to needle

Fit to injector

Micro-injector

Collect fertilized eggs

Dechorionate eggs

Align on slides

Cover with halocarbon oil

Inject DNA into embryo under the inverted microscope

Leave 18°C for 24 h

Collect larvae, transfer to fresh food

10–12 days

Isolate adult males/females

Crossed with noninjected counterparts

Marker based selection of transgenics (eye color)

Stabilization of transgenics

D.melanogaster (w1118);petridishes,grapejuice,agar, potassiumchloride,sodiumphosphate(monobasic anddibasic),double-sticktape(Scotch665),tweezers (fineneedle,sharp;Sigma),fineneedles,halocarbon oil,permittedfoodcolor(redorgreen);andnetwells.

Procedure

A. PreparationofDNA

i. Preparetheendotoxin-freeDNAofthegene cloneusingaplasmidmaxi-prepkit.Wide varietiesofkitsarecommerciallyavailableand followthemanufacturer’sinstructionsto preparetheDNA.

ii. Ina0.6-mLeppendorftube,mix15 μgof plasmidofinterestwith5 μgofhelperplasmid, ethanol-precipitatethemixtureusing1/10 volumeof3MNAOACandtwotimesvolume of100%ethanol.Leavetubesovernightat 20 C.Nextmorning,centrifugethetubesat 14,000rpmfor30minutes,washthricewith 70%ethanol(eachfor3minutesat13,000rpm), andultimatelyresuspendthepelletin50 μL injectionbuffer(5mMKCl;0.1mMNaPO4 bufferpH7.5).Thiscanbestoredat 20 Ctill furtheruse.

iii. Arecentstudy(Sonaneetal.,2013)hasshown thattheuseofnanoparticles(NPs)todeliver DNAthroughmicroinjectioncanleadtothe reductionofrequiredDNAquantityby10foldswithoutcompromisingontheefficiency ofgerm-linetransformation.

B. Collectionofflyembryos

i. Use4 6daysoldadultfliesfromfreshcultures forembryocollection.

ii. Place200 300 w1118 adultsinbottlesor collectionchamberscontainingtinypetridishes withgrapejuice-agarmedium.

iii. Placethebottleindark.

iv. Discardthefirsthour’scollectionplateand placeafreshone.

v. Replaceeggcollectionplateswithnewonesat 30minuteintervalsandusetheembryosfor injection.

C. Removalofchorionfromembryos

i. Washtheembryosfromtheeggcollectionplate toanetwellusingeggwashbuffer(0.03% TritonX-100,0.4%NaCl)

ii. Placethenetwellcontainingtheembryosin 2%sodiumhypochlorite(bleach)for2minutes.

iii. Washtheembryosthoroughlywitheggwash bufferandcollectontoanagarbedorontoa petridish.

D. Preparationofembryosforinjection

i. Cutasmallstripofdouble-sticktape(Scotch 665)andstickinthecenterofaslide.

ii. Usingafinebrush,arrangetheembryosina verticallylinearfashiononagarbed.

iii. Transfertheembryostotheslidebyreversing theagarbedontotheedgeofthedouble-stick tape.Finalorientationoftheembryosshould besuchthattheposteriorendoftheembryo shallhangoffthetape.

iv. Observethequalityanddevelopmentalstage oftheembryo.Discardthoseembryoswith poorqualityoragedembryos(visualizationof syncytialblastoderm).

v. Placetheslideinapetridishcontainingthe desiccantfor4 5minutes(optimaldehydration timesaretobedetermineddependinguponthe relativehumidityoftheinjectionroom).

xvi. Afteroptimaldehydration,covertheembryos withalayerofhalocarbonoilusingaglass/ plasticpipette.

E. Preparationofneedleforinjection

i. Dependinguponthemicroinjectorbeingused, eitherpullglassneedlesusinganeedlepuller orbuycommerciallyavailableglassneedles meantforDrosophilainjections.Inbothcases, diameteroftheopeningofthetipisexpected tobeapproximately0.5 μm.

ii. Mix15 μLofDNApreparedforinjectionwith 4 μLoffoodcolor(greenorred,depending uponavailability).

iii. Loadtheneedlewith2 5 μLofDNAeitherby usingapipetteorthroughcapillaryaction (particularlyincaseofpulledneedles).

iv. Connecttheloadedneedletotheholder connectedtoamicromanipulatorora syringe.Incaseofpulledneedles,ensureto breakthetipoftheneedlebyrubbingthesame againstthecornerofthecoverglass,placedon aslide.

v. Placeaslidecontainingadropofhalocarbon oilandensurethattheneedleisinthesame verticalfocalplaneasembryos.

F. Injectionofembryos

i. Lifttheneedleverticallyandreplacetheslide containingthehalocarbonoilwiththeslide containingembryos.

ii. Positiontheneedleslightlyawayfromthe middleoftheposteriortipoftheembryoand penetratetheembryo.Afterpenetration,draw theneedleasbackaspossible,withoutleaving theembryo,anddeposittheDNAinthe posterior-mostregionoftheembryocytoplasm.

iii. Injectalltheembryosontheslideinasimilar manner.

G. Postinjectioncareofembryos

i. Removethenoninjectedembryostoavoidfalse negatives.

ii. Placetheslidewithinjectedembryosinapetri platecontainingthemoisttissuepaper.

iii. After24 28hours,transferhatchedlarvae usingafineneedletoavialcontainingfresh food.

iv. Monitorthedevelopmentoftheselarvaeand crosseacheclosedadulttotheoppositesex partnerfrom w1118 stock.

v. After10 12days,screenforthered/orange/ yellow-eyedflies,whicharetransformants.

H. Generationofstabletransgenicflies

i. Collectunmatedtransformantsandcrosstothe oppositesexpartnerfrom w1118 stock.

ii. Collectunmatedheterozygousmalesand femalesresultingfromthiscrossandplacethe sameinfreshvials.

iii. Intheresultantprogeny,isolateunmated homozygousmalesandfemales(willhavedark eyecolor)fromheterozygousflies(eyecolor lighterthanhomozygotes)andsetupacrossof homozygotes.

iv. Cross-checktheresultantprogenyforsimilareye colorinallfliestoensurethatthelineisstable.

Ethicalissues

Biologicalresearchinvolvingorganismsrequires ethicalclearance.Therationaleofintroducingethical issuesliesmajorlyonthejudicioususeoftheanimals andtheirhumanetreatmentbefore,during,andafter theexperiments.Thereforeitisnowmandatoryina researchinstitutionorinauniversity,wherebiological researchispursued,tohaveanimalethicsandhuman ethicscommitteeinplace.Thesecommitteeslookinto thedetailsoftheprogramthatwillbeundertakenby theresearchersforthepossibleuseoflaboratoryanimals(essentiallymammalssuchasmice,rats,guinea pigs)andforthehumansamples.Noresearchdataon animals/humansarepublishednowadaysinjournals iftherespectiveresearchersdonotprovidethedetails oftheethicalclearancedata/number.

Whileintoxicologyandotherrelatedfields,researchersmajorlydependonthedatageneratedonlaboratory animalstryingtoextrapolatethedatatohumans.Several factorsthatremainashurdlesforthegenuineextrapolationistheintraspeciesvariationandcompoundingeffects resultingfromoneexperimenttoanother.

Smalleranimalshaveplayedpivotalrolestowardour present-dayunderstandingof fundamentalfacetsofbiology.Inparallel,informationgeneratedintheseorganismsduetoeaseofhandling,theirisogenicconditions negatingintraspeciesvariation,simpleyetfunctionally homologoustissuearchitecturetohighermammalsand moreimportantlyinthepostgenomicera,thegene

homologyexistingbetweenthesmallerorganismslike Drosophila, Caenorhabditiselegans,etc.,tomammalsand humanshaveledtoimportantdiscoveriesthathaverelevancetohumans.Inthiscontext,limitedethicalconcerns areraisedfortheuseofsmallerorganismsespecially invertebratesandlowervertebratesinbiologicalexperimentsandtesting.EuropeanCentrefortheValidationof AlternativeMethodshasrecommendedseveralsuch organismstopromotethebasicprinciplesof3Rs(refine, replacement,andreduce)inbiologicalresearchandtesting(Ballsetal.,1995).

Translationalsignificance

HowdoesDrosophilaresearchovertheyearshave madesignificantinroadsintheareaofbiotechnology? Thisisapertinentquestionthatcanbeasked.Overthe years,severalsignificantinformationpertainingtogeneticsanddevelopmenthavebeengeneratedusing Drosophilawhichhasadvancedourknowledge. Dependinguponthetypeofplasmidused,thegerm-line transformationcanleadtothegenerationoftransgenics wheretransgenesarerandomlyintegratedintothe genomeorintospecificsites(site-specificintegration) withinthegenome.Thelatterwouldhelptoovercome thevariationsbetweentransgenicsarisingduetoinsertionatrandomsiteswithinthegenome.Byexploiting thesame,researchershavegeneratedagenome-wide collectionoftransgenicstoknockdownorectopically expressgenesinastage-and/ortissue-specificmanner. Inaddition,inthelastcoupleofyears,researchershave developedDrosophilareagentsusefulforgenerating DrosophilageneknockoutsbyemployingCRISPR/CAS9 technology,whichhasbeenwidelyusedwithmammalianmodels.Bymanipulatingthegenomethroughgermlinetransformation,duringthelastdecade,numberof Drosophilamodelsusefulforunderstandingseveral humandiseaseshavebeendeveloped(seeClinicalsignificancesectionfordetails).Inaddition,transgenic Drosophilaarealsobeingusedinthefieldoftoxicology. TransgenicDrosophilacontainingLacZunderthecontrol ofhsp70promoterhasextensivelycontributedtothe understandingofstressresponsemechanismunderlying heavymetaland/orpesticidetoxicity(reviewedin Guptaetal.,2010).Further,transgenicDrosophilamodelshavebeenutilizedtoassessthechemical-mediated reproductivetoxicity(Misraetal.,2014a,b)andinnate immuneresponse(Pragyaetal.,2015).

Clinicalsignificance

Modelorganismsmadeinvaluablecontributionsfor studiesofhumandiseasemechanismsduetothehigh

degreeofconservationoffundamentalbiological processesthroughouttheanimalkingdom.Inthiscontext,itisveryimportanttorememberthathuman organizationismuchmorecomplexthanthatof Drosophila.Despitesharingthefundamentalprocesses,Drosophilaandhumansmightstilldifferin mechanismsthroughwhichtheseprocessesareregulated/implemented.Consequently,researcherssensiblydevelopedDrosophila-basedmodelsforhuman diseasesassociatedwithmutationsinoneortwogenes orforthosediseaseswheretheessentialpathways areconserved.Thesemodelsareexcellentforunderstandingthediseaseprogression,pathogenesis,and underlyingmechanisms.Further,thesemodelsarealso usefulindrugdiscovery,althoughcautionshouldbe exercisedbeforeextrapolatingthedosedataand/or deliveryroutetohumanuse.Nevertheless,Drosophilabasedmodelsforhumandiseasepresentaquickand inexpensiveapproachforfirst-tierscreeningofalarge numberofmoleculesduringthedrugdiscoveryprocess.Below,wehaveprovidedafewexamplesof Drosophila-basedmodelsforhumandiseases.

Drosophila-basedmodelsforunderstanding humanneurodegenerativediseases

Neurodegenerativediseases(NDs),asthename suggests,ariseduetoprogressivelossofspecificneuronalpopulations.Humangeneticstudiesledtothe identificationofgenesassociatedwithcertainNDsbut mechanisticunderstandingofpathwaysandprocesses underlyingthediseaseremainedincompletedueto ethicaland/ortechnicalconstraints.Inthiscontext, Drosophila,withitscomplexbehavior,including learningandmemory,drivenbyasophisticatedbrain andnervoussystem,providedexcellentmodelsfor decipheringsignalingpathwaysandunderstanding thecellularprocessesdefininghumanND(reviewed in Hirth,2010; LuandVogel,2009).Forexample, Drosophila-basedmodelsareavailableforAlzheimer’s disease(AD),themostcommonND,characterized byage-dependentgradualimpairmentinmemory andcognitiveabilities.Inhumans,selectiveatrophy ofthehippocampusandthefrontalcerebralcortex, amyloid β (Aβ)plaquedeposition,aggregationofthe microtubule-associatedproteintauarethehallmarks ofADpathology.TheAβ plaqueismainlycomposed ofAβ-40andAβ-42peptides,theproductsofendoproteolysisoftheamyloidprecursorprotein(APP) throughsecretases.Autosomal-dominantmutations anddefectivetraffickingofAPPaffecttheonsetand/ orprogressionofAD;bothcircumstancespromote thegenerationofamyloidAβ peptides.Drosophila genomecontainshomologsforhumanAD-related

genes:APP,presenilin,andtau.Thereforeresearchers employedDrosophilaasamodelsystemforAD research.DrosophilamodelsofAPP-mediatedADdo simulatecertaincharacteristicsofhumanADpathology,includingAβ plaquedeposition.Inadditionto providinginsightstothepathology,thesemodelsare alsousefultoidentify/targetthemodulatorsofAD pathologythroughgeneticorpharmacologicalinterference.Likewise,Drosophilamodelsareavailablefor Parkinson’sdisease(PD)too.PDisthesecondmost commonneurodegenerativedisordercharacterizedby impairedmotorskills:uncontrollabletremor,imbalance,slowmovement,andmusclerigidity.PDpathologyisassociatedwithprogressivelossofdopamine neuronsinthesubstantianigraparscompactaofthe ventralmidbrain.FormationofLewybodies,mainly composedofsynucleinandubiquitin,amongotherproteinsisthepathologicalhallmarksofPD.Severalgenes havebeenassociatedwithPDinhumans:alphasynuclein,parkin,ubiquitincarboxy-terminalhydrolase L1(UCHL1),phosphataseandtensinhomologue (PTEN)-inducedkinase1,(PINK-1),DJ-1,leucine-richrepeatkinase2(LRRK2),high-temperaturerequirement proteinA2(HTRA2),glucocerebrosidase,polymerase gammaandtau.Mutationsinalpha-synucleinand ParkinhavebeenassociatedwithPDinhuman. Exceptforalpha-synuclein,Drosophilacarriesthe homologsoftheremainingPD-associatedgenes. Accordingly,analysisofDrosophilamutations/transgenesofPD-associatedgenehomologsprovidedvaluableinsightstoPDpathogenicmechanismsandtargets ofPD-relatedgenes.Lackofalpha-synucleinhomolog didnotdeterresearchersfromusingDrosophila tostudymechanismunderlyingalpha-synucleinrelatedPDanditstargets.Byutilizingthepowerful genetictoolsavailablewithDrosophila,researchers expressedhumanalpha-synucleininDrosophilato investigateitsfunctionalpropertiesinPDandtoelucidateitstargets.Interestingly,metabolomichallmarks ofDrosophilawithParkinson-likesymptomsarecomparabletothoseofPDpatients(Shuklaetal.,2016). ThesefindingshighlighttheutilityofDrosophilaas amodeltounderstandthepathologicalintricacies underlyingNDs.

Drosophilaasamodelforunderstandinghuman metabolicdisorders

Diabetesandobesityarethemostcommonmetabolicdisordersencounteredinthepresent-daylifestyle.Todate,obesityconcernsarelimitedmainlyto developednations,althoughdevelopingcountriesare catchingupquickly.Further,obesityinthemajority casesleadstodiabetes.Diabetes,arisingmainlyfrom

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Rabbi Joseph Albo criticised Maimonides’ thirteen articles of faith (םירקיע). Whilst recognising all of them as true, he would make a difference between fundamental principles (םירקיע) and secondary beliefs (םישרש). The former are all those dogmas by which Judaism falls and stands, without which Jewish faith cannot be imagined; the latter are those principles which are actually true, but Judaism can be conceived without them. To the former he counts, e.g., the belief in the existence of God, to the latter the belief that to Him alone prayer is to be offered. For Judaism cannot be conceived without the belief in God’s existence, but could be conceived without the belief that only God is to be prayed to. Albo further finds fault with Maimonides for not having embodied in the Creed the belief in man’s free-will, in the truth of the Biblical account of the miracles, in the Creatioexnihilo, and the like. To these objections Maimonides would reply, that the articles enumerated by him were all actually fundamental, the question whether Judaism could be imagined without this or that principle being of no importance whatever; and that the dogmas named by Albo as omitted, were implied in the Thirteen Principles. According to Albo there are three fundamental principles (םירקיע): Existence of God, Revelation, and Reward and Punishment. The first includes four articles (םישרש): Unity of God, His Incorporeality, Eternity, and Perfection; the second implies three: God’s Omniscience, Divine inspiration, and Divine messengers (prophets); the third only one: Providence. Albo’s criticisms on Maimonides have passed away without effect. The Principles [174]of Faith as formulated by Maimonides have found their way into the daily Prayer-book in prose and poetry, and have since formed an essential element in every text-book of Jewish religion. Modern theologians erroneously quote Albo in favour of rejecting some of the articles, because he speaks of three fundamental principles; but they forget that Albo never rejects any of the thirteen principles; he only insists on making a difference between those which are more and those which are less fundamental.

OntheFirstPrinciple, pp. 22 sqq.

Maimonides does not mention the term Creator except in the beginning of each paragraph as a substitute for “God.” He employs the philosophical term “First Cause” in defining the existence of God. In the sixty-ninth chapter of the first book of “The Guide” we find the explanation thereof. He says: “The philosophers, as you know, call God the First Cause (הָלּﬠ and הבּס); but those who are known by the name Mutakallemim (Mohammedan theologians) are very much opposed to the use of that name, and prefer to call Him ‘Maker’ (לﬠוֹפּ), believing that there is a great difference whether we use the one term or the other. They argue thus: Those who say that God is the Cause, implicitly assume the coexistence of the Cause with that which was produced by that Cause, and believe that the universe is eternal, and that it is inseparable from God. Those, however, who say that God is the Maker do not assume the coexistence of the maker with his work; for the maker can exist anterior to his work; we cannot even imagine how a maker can be in action unless he existed before his own work. This is an argument advanced by persons who do not distinguish between the potential and the actual. For there is no difference whether we say ‘cause’ or ‘maker;’ ‘cause’ as a mere [175]potentiality precedes its effect; and ‘cause’ as actuality coexists with its effect. The same is the case with ‘maker;’ so long as the work is not done, he is a maker potentially, and exists before his work; he is an actual maker when the work is done, and then he coexists with his work.”

“The reason why the philosophers called God ‘the Cause’ and did not call Him ‘the Maker’ is not to be sought in their belief that the universe is eternal, but in other principles, which I will briefly explain to you. Everything owes its origin to the following four causes: the substance, the form, the agens, the final cause. The philosophers believe—and I do not differ from them—that God is the agens, the

form, and the final cause of everything; in order to express this, they call God ‘the Cause’ of all things. Every one of these three causes leads, through a chain of causes, to God as the First Cause.” Maimonides further points out in this chapter that the choice of the term by no means decides the question whether the universe has had a beginning or not.

Maimonides has been severely criticised by his successors for the absence of the belief in “Creation from nothing” from the Creed. In “The Guide” Maimonides distinctly states that the arguments for “Creation from nothing” and the arguments against it are equibalanced, and that for this reason he follows the literal interpretation of the Scripture as regards Creation. Were the arguments in favour of the eternity of the universe stronger, he would not have found any difficulty in interpreting Scripture accordingly. Such being the view of our great philosopher, he could not make the belief in Creation part of the Creed, or declare that all who denied the Creation from nothing were unbelievers.

However strange this argumentation of Maimonides may appear, and however arbitrary his treatment of Scriptural teaching, his view is not without justification. It seems [176]strange that, in spite of all his reverence for the Bible, he should have entrusted himself entirely to the guidance of his own reason, and forced, as it were, the Bible by peculiar interpretations to follow his reasoning. In truth, however, the method of Maimonides is neither strange nor arbitrary. There is no doubt that figurative language is extensively used in the Scriptures, especially in the poetical and the prophetical books. Whether a certain expression or phrase was to be understood in its literal meaning or in a figurative sense must be learnt from the context; in some cases—as, e.g., in the exhortation, “Ye shall circumcise the foreskin of your heart” (Deut. x. 16)—the figurative sense is accepted by all, whilst in other cases opinions are divided.

Our decision in favour of the one interpretation or the other is based on our conviction that the Bible contains nothing but truth. When we discover a contradiction between a Biblical statement and the dictates of our reason, we are sure that we have erred either in the right understanding of the words of the Bible or in our reasoning. On finding the mistake in our reasoning we abandon what we have hitherto considered as fully established; but so long as we are unable to discover where our reasoning is faulty, we either suspend our judgment for the present and consider the question as one of the problems which we have not yet been able to solve satisfactorily, or, whenever possible, we attempt to reconcile by figurative interpretation the teaching of the Bible with the results of our research. Maimonides is therefore justified in saying that so long as reason does not decide against the teaching of the Bible in its literal sense he would adhere to the latter, and only if reason were to decide against the Creatioexnihilo, he would follow reason and interpret Scripture accordingly.

It cannot be denied that Maimonides travelled here on rather slippery ground, and set a dangerous example when [177]he admitted that he would interpret Scripture according to his preconceived view of the world’s beginning. But, on the other hand, it must be owned that many passages of the Bible admit of a figurative interpretation, and the reader must follow his own reason and discretion in deciding in each particular case which of the two interpretations is the correct one. Maimonides has not made excessive use of this license.

Saadiah in his EmunothVe-deothdevotes the first chapter to the problem of the Creation. It is headed שודח “Creation,” and examines thirteen different opinions as to the origin of the universe. In the conclusion of this chapter he makes the following remarks: “Perhaps some one might ask in what manner something was produced from

nothing. To this we reply as follows: If we were able to understand this, we should not have ascribed the creative act to God alone. But we declare God as the only Creator, because we can form no idea as to the manner in which something is created from nothing. Those who desire us to show them how to do this, desire, in fact, that we should make them and ourselves creators. We only conceive in our mind the fact of the Creation, but cannot form an idea or image of the process itself.… There may be some who think little of the universe, and wonder that this should be the result of all the power and wisdom of God. We reply that He created as much as, according to His knowledge, would be within the range of man’s observation and perception, and would be sufficient to teach man the existence of God.… How can we conceive the idea that the universe counts only 4633 years? But the universe has been created, as we believe, and must have had a beginning at a certain time. Suppose we had been living in the year 100; we should then not have been surprised: why should we be surprised now?” The question as to the purpose for which the universe [178]was created, Saadiah makes three attempts to answer. Maimonides, however, in “The Guide,” more correctly, shows that the question is unanswerable and superfluous. For, whatever purpose we assume, we must always further inquire what is the purpose of this purpose, and so on adinfinitum, till we arrive at the answer, it was the Will of God. If the prophet declares that God “hath not created it in vain, but hath formed it for dwelling,” he likewise says implicitly it was the WillofGodthat the earth should be for a dwelling.

The question, however, arises whether the Biblical account of the Creation harmonises in all its parts with the results of scientific research. To prove the existence of harmony between the two discordant elements has been since days of old the task which theologians proposed to themselves; philosophic culture forced them to accept the doctrines of a certain school of thought as established

truths, whilst religious feeling would not allow them to abandon the teaching of the inspired writers. But the search after this harmony was superfluous, and the harmony found was illusory. For, whilst the teaching of the Bible remains unchanged, the systems of philosophy and science, like everything human, have no claim to permanency; each system has its season; it begins to shine, and rises higher and higher; and when it has reached the zenith, it begins steadily to decline till it disappears beneath the horizon of science. So long as Aristotle and Ptolemy were dominant, theologians exerted themselves to show that the account contained in the first chapter of Genesis fully harmonises with Aristotle and Ptolemy. When these princes were dethroned, and their places were occupied by others, the old harmony was gone, and a new method had to be invented. Maimonides has clearly pointed out how the conflict between reason and faith, where it existed, could best be brought to a conclusion. [179]Such of the laws of nature as have been established by human acumen and human observation have been discovered in the phenomena of existing nature; but the phenomenon of creation has never been observed in nature from which we could learn the laws of creation.

In the seventeenth chapter of the Second Book of “The Guide” Maimonides says as follows: “Everything produced comes into existence from non-existence; even when the substance of a thing has been in existence, and has only changed its form, the thing itself which has gone through the process of genesis and development, after having arrived at its final state, has properties different from those which it possessed at the commencement of the transition from potentiality to reality or before that time.… It is quite impossible to infer from the qualities which a thing possesses after having passed through all the stages of its development what its condition was at the moment when this process commenced; nor does the condition of a thing at that moment show what its previous

condition had been. If you make this mistake, and attempt to prove the nature of a thing in potential existence by its properties when actually existing, you will fall into great confusion; you will reject evident truth and admit false opinions.… If philosophers would consider this well, and reflect on it, they would find that it represents exactly the dispute between Aristotle and ourselves. We, the followers of Moses, our teacher, and of Abraham, our father, believe that the universe has been produced from nothing, and has developed in a certain manner, and that it has been created in a certain order. The Aristotelians oppose us, and found their objections on the properties which the things in the universe possess when in actual existence and fully developed. We admit the existence of these properties, but hold that these properties themselves have come into existence from absolute non-existence. [180]The arguments of our opponents are thus refuted; they have demonstrative force only against those who hold that the nature of things as at present in existence proves the Creation. But this is not my opinion.”

This reasoning holds good with regard to the modern theory of Evolution. We may be able to discover numerous facts in evidence of this theory, we may well conceive the idea of a protoplasm developing into a whole system of worlds, and yet our belief in the truth of the Biblical account of the Creation is not shaken in the least. The laws of Evolution are theresultof the creative act of the Almighty, and not its causes; they include nothing that could disprove the correctness of the theory of Creatioexnihilo.

Rabbi S. R. Hirsch, in his “Commentary on Genesis” (i.) says: “The word תישארב ‘in the beginning,’ teaches that nothing preceded the act of Creation; that there was a Creatioexnihilo. This truth forms the foundation of the faith which the Divine Law is intended to establish in our hearts. The opposite theory is the doctrine of the

eternity of the substance, a theory which leaves to the Creator nothing but the function of giving form to the substance that has existed already from eternity, and which has been the basis of the heathen belief up to the present day.… The first word of the Torah dispels the darkness of this false belief; and the words, ‘The opening of thy word giveth light’ (Ps. cxix. 130), have in the Midrash correctly been applied to the word תישארב. Everything, the matter and the form of all beings, is the result of the free will of the Creator, who continues to rule matter and form, and to determine both the natural forces and the laws of their action. For it is His free will that created matter, endowed it with certain forces, and fixed the laws by which the forces impress the different forms on it.” [181]

The idea of development and evolution is not entirely excluded from the account of the Creation. Not in one moment or in one day was the universe produced, but in six days by successive creations of a systematic order. In Mishnah Aboth (v. 1) this is expressed in the following way: “By ten words (תורמאמ) the universe was created, although this could have been done by one word.” Commentators have variously attempted to explain this fact, and to show that the order observed in the Creation was determined by the nature of the things themselves. Thus Ibn Ezra believes that the successive creations were the results of the continued action of light and heat.17 But it is by no means necessary to reconcile the Biblical account with every theory that happens to be considered by some scholar or school as the right one. There may be found in nature and in the working of the natural laws some facts analogous to certain acts of the creation; but a perfect equality of two such incongruent things as the creation from nothing and development of created beings is impossible. By forcing the text of the Bible into such harmony we deprive the account of its poetry and beauty, and weaken the force of its teaching.

Science teaches that millions of millions of years must have elapsed before the earth received its present form; that it took millions of years before the light of certain stars could reach the earth. In all these calculations one important factor is ignored, viz., that for every development something must be given, which is subject to the process of developing; to determine in what condition that something was, when it passed from the passive state of creation to the active state of developing, is a problem for the solution of which there is no analogy in nature. He who could create a germ endowed with all the natural forces required for [182]development and differentiation into the great variety of forms which we perceive at present, must certainly have been able to create the things actually endowed with these forms. Thus, also, the various strata of the earth, whatever forms they contain, cannot with certainty be described as the results of development; they may just as well have come directly from the hand of the Creator.

Maimonides (The Guide, xxx.) says in reference to this question: “You should also know the dictum of our Sages—‘All the beings of the work in the beginning (תישארב השעמ) were created in their full height, their fully developed reason, and endowed with the best of properties.’ Note this, for it involves an important principle. The work of the Creation went on for six days; every day brought to light a new force, a new result of a creative action, but on the seventh day ‘God declared18 the work which He had done as finished,’ as endowed with the properties and forces required for their further development” (The Guide, I. lxvii.).

Science has proved, it is maintained, that the earth does not form the centre of the universe, and that man does not form the principal object in nature, in opposition to the teaching of the Scriptures that the earth is the centre round which the whole universe revolves, and that man on earth is the lord of the creation. Whatever view the

authors of the Biblical books held as regards the systems of the universe, whether they placed the earth in the centre or not, whether all the stars and systems of stars existed, in their opinion, only for the sake of the earth or for the benefit of man, their object was to address man, to instruct him, and to teach him the omnipotence, wisdom, and goodness of God. For [183]this reason the account of the Creation is given in such a manner that man should be able to reproduce in his mind the work of each day of the Creation, to view it from his standpoint, and to recognise the benefits each day’s work bestowed on him. The fact that other beings are benefited at the same time, and that the benefit they derive is likewise part of the Creator’s design, is by no means denied by those who believe that the well-being of man was included in the design of the Creator. It is part of our duty of gratitude to ascribe the benefits we enjoy to their Author. The prophets and the inspired singers knew well the place which weak and mortal man occupies in the universe; but they did not ignore the dignity and importance with which the Creator endowed him in spite of all his weakness and apparent insignificance. “Whatisman,” exclaims the Psalmist, “that thouartmindfulofhim?andthesonofman,thatthouvisitesthim? Andyetthouhastmadehimalittlelowerthanangels,andhast crownedhimwithgloryandhonour” (Ps. viii. 5, 6).

OntheFifthPrinciple, p. 44.

The principle that no other being but God is worthy of being addressed in prayer implies the belief that God can fulfil our petitions. We believe in the efficacy of prayer. It is true that when we communicate our wishes to the Most Holy, our just Lord and our loving Father, we are eoipsoreminded to examine our desires, whether they contain anything unholy, anything unjust or ignoble. Prayer to God has therefore the salutary effect of purifying, refining, and ennobling our heart. It banishes evil thoughts, and thus saves