An Introduction What is Gene Transfection Generation of genetically modified animals Genetic modification of germ cells Breeding modified individuals To combine gene cutting or expression tools ID: 1009813
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1. Neural Gene TransfectionAn Introduction
2. What is Gene Transfection?Generation of genetically modified animalsGenetic modification of germ cellsBreeding modified individualsTo combine gene cutting or expression toolsAcute modulation of Gene ExpressionRequires transfection / transduction
3. What is Gene Transfection?Moving exogenous DNAIncluding gene sequencesUsually coding for proteinsinto Living Cellsin order to modify cellular Gene Transcriptionto thereby modify Cell Function
4. Cellular TransfectionNucleic Acids can not diffuse through lipophilic cell membranesAlso true for some drugsDue to hydrophilic or charge propertiesChemical facilitationPhysical stimulationMay increase efficiency
5. Cellular TransfectionCell culture – in vitroFirst successful gene transfer – 1976-78Reveals much about cell functionWhat genes are not normally active in these cellsEach type of cell is unique relative to gene expressionHow to increase/inhibit gene expressionVisualizing transfected cells
6. Cellular TransfectionIn vivo – elucidating system functionwith in situ cellular communication maintainedBiochemical milieu is normal - completeSystemic deliveryCirculating nucleic acids have short half-lifeDirect injectionStill requires help to cross plasma membrane
7. Neural TransfectionSystemic DeliveryBlood-Brain-Barrier (BBB)Isolates brain from cranial vasculatureGlial tight-junctions surrounding vasculatureShort ½–life protection neededIndiscriminant delivery to all brain regionsExpression may be modified by promoter tools
8. Neural Transfection in vivoDirect Injection into specific Brain RegionsBypassing BBBTargeting Neuronal subtypesEven within a regionGlutamate, GABA, Monoamine, NeuropeptideMay elucidate neurocircuit function
9. Methods for Neural TransfectionChemically mediated transfectionExternal carriers with specific chemical propertiesPhysical enhancement of DNA movementReversible alterations in plasma membraneor endocytosisVirally mediated transductionIntrinsic transfection ability of viruses
10. Chemically Mediated TransfectionFormation of Complexes with nucleic acidsGood gene packaging, low toxicity Low efficiency
11. Chemically Mediated TransfectionLipids (lipofection)hydrophilic domain + lipophilic tailNanoparticles High stability & protectionPolymersMacromolecules of repeated units
12. Lipid Mediated TransfectionLipoplex – DNA + hydrophilic domainLipid vectors suffer from short ½-lifeRapid degradation from phagocytesCationic lipid protects DNAFrom cleavage by endonucleasesIn circulation
13. Lipid Mediated Transfectionadded Protection and StabilizationPolyethylene glycol (PEG)Pegolation1,2 dioleoylphosphatidyl-ethanolamineDoPE
14. Lipid Mediated Transfection PEG or DoPEIncreases fusion with cell membraneEndosome lipocomplexFacilitates movement of DNAbetween endosomes & cell nucleusinfluence 3o structure of lipoplex
15. Lipid Mediated Transfection Nanoemulsions (LNEs)Dispersion of nanoparticles of lipidsLiquid phase due to surfactantHigher efficiency than liposomesin vitro or in vivo delivery
16. Lipid Mediated Transfection Solid Lipid Nanoparticles (SLNs)Lipid nanospheres Outer hydrophilic shell - lipid bilayerInner core – long-chained lipidsSolid and Stable at body ToMay cross the BBB
17. Lipid Mediated TransfectionEfficient in vivo gene transfectionBy intracranial (ic) intracerebroventricular (icv)or intranasal (in) delivery
18. Nanoparticles & TransfectionHigh stabilityGold nanoparticlesSilica nanoparticlesFullerenesPolymersMany others
19. Gold assisted TransfectionCoated with cationic moleculesBind nucleic acids & endocytosisEfficient delivery of DNAVery low toxicityCross the BBB – enhanced by PEG etcOptical properties – region localization
20. Silica TransfectionOxide of siliconCoated with organic amino acidsInteract with nucleic acidsProtect from endonucleasesPhagocytosis internalizationNeuroinflammation after intranasal
21. Transfection by FullerenesSpherical (SF) Cylindrical nanotubes (CNTs)Cationic charges – stable 2x DNAProtective against nucleasesSFs can cross BBBCNTs poorly soluble in watercross plasma membrane - functionalized with peptides and conjugated to DNABucky Balls
22. Polymer nanoparticlesPEI = polyethylenimineChitosansTMC = trimethylated chitosanDendrimersPAMAM = polyamidoamine
23. Polymer mediated TransfectionPositively charged surfaceInteracts with negative DNACan be pegolatedCan be modified by peptides
24. Physical EnhancementElectroporationCreates temporary H20-filled holes in membraneapplication of electric-field pulsesSonoporationUltrasound stimulated temporary poresMagnetic-assisted TransfectionDirects magnetic nanoparticles containing DNA
25. PhysicalEnhancement
26. Physical EnhancementElectroporationMostly used in vitroUsed in vivo and ex vivo on embryosSonoporation+ microbubbles increases efficiencyMagnetic-assisted TransfectionDirects magnetic nanoparticles containing DNA
27. Physical EnhancementElectroporationMostly used in vitroUsed in utero and exo utero on embryos
28. Sonoporation EnhancementMostly in vitroBubbles maycontain cationic lipids to bind DNAMostly in vitroLow efficiency
29. MagnetofectionMagnetic Nanoparticles (MNPs)MNPs contain iron oxide (Fe2O3, FeO, Fe3O4)core + additional organic or inorganic coatingliposomal, polymers, peptides, ligands + receptorsBinds DNA wellEndocytosis leaves membrane intactSimple and highly efficient – still mostly untried
30. Virally Mediated TransductionNanometric infective agentsProtein CapsidProtects Genetic MaterialLipid envelope from cell membraneSometimes protects capsidAll cells can be infectedDependent on capsid or envelope = tropism
31. Virally Mediated TransductionGenetic material carried by a virusCan be modifiedor mostly substitutedBy a Synthetic ConstructCapsid can also be modifiedProducing the desired specific tropism
32. Virus Types Pirated for TransfectionLentiviruses: slow virusRetroviruses like HIVAdeno-associated VirusesInfects humans, but doesn’t cause diseaseAdenoviruses – Adenoid infection/tonsillitisHerpes Simplex Viruses Genital herpes, cold sores, chicken pox/shingles
33. Virus TypesPiratedforTransfection
34. Virus Types UsedAll bind receptorsLentivirusesDelivers RNAAdeno-associated Viruses AAVSingle-stranded DNAAdenoviruses & Herpes Simplex VirusesDeliver double stranded DNA
35. LentivirusesAble to infect fully differentiated cellsRely on retrotranscription: RNA→DNAReverse transcriptaseDerived from pathogenic agentsSafer vectors substitution of envelope proteinEfficient gene delivery in vivoInjected intracranially
36. AdenovirusesNon-integrating naked virusesEfficiently transfect dividing cellsor non-dividing cellsHigh virulencePotent toxicity in the CNSHelper virus carries some informationTo reduce toxicity
37. AAV VectorsNever associated with any pathology most used vector for gene transfection in vivoSmall packing capacity (5 kb)Increased by clever molecular tricksDividing DNA package into 2 AAV vectorsAAV serotypes have distinctive tropismNeurotropism: AAV1, 2, 4, 5, 8, 9, and AAVrh10
38. AAV VectorsStable infection – may last yearsAAV9 and AAVrh10 transported along axonsCapsid gene shufflingGenerates different cap proteins Increases tropism flexibilitySome AAVs cross the BBB
39. Herpes Simplex VectorsLarge genome –thus- large capacity152 kb – can carry 30kb experimental DNAHigh Tropism for CNSNo immunogenic responseMay activate during replicationDeleting some HSV DNA → saferComplex envelope – difficult development
40. How to make use of viral vectors?Viral genes must be replacedAt least partlySubstituting a gene packageTo accomplish specific experimental goalsThe gene transfection cassetteThis cassette must contain elements that regulate and drive expression
41. Virally delivered Gene packagesFor Coordinated control of gene expressionManipulating gene expressionUsing Transcription Factor regulatory elementsFound in the gene’s promoterRequires cis-acting elements in both 5’ and 3’ untranslated region (UTR)of the expression cassette
42. Transfection Gene CassetteTargeting Neuron Specific PromotersTo promote gene expression in neurons onlyNot in gliaDifferent cassettes could target Glia5’ UTR cis-regulatory elements: Core promoter, enhancer, silencers, insulatorsMatrix attachment regions, locus control regions
43. Neural Transfection Cassette5’ cis-regulatory elements: (upstream of gene)such as a promoterare Binding Sitesfor trans-acting factorssuch as Transcription FactorsUsing those found only in neuronsAllows for Neural Transfection Cassettes
44. Targeting Specific NeuronsThe brain is huge and complex
45. Targeting Specific NeuronsThe brain is huge and complexEvery region has specific functionsAnd specific cell types to accomplish thoseSpecific neuronal types have specificPhenotypesCreated by specific proteins
46. Targeting Specific NeuronsSpecific Proteins have unique genesWith unique regulatory systemsDistinctive promotersDistinctive trans-acting systems to drive themTherefore, each neural cell typeCan be targeted specifically
47. Building a cassette to hit Specific NeuronsFind a promoter for a protein Uniquely found in those neuronsOr that protein may be the focus of investigatione.g. Orx2 receptor in our researchNeuron specific promotersDo not specify cell typesNeuron specific enolase, synapsin-1, PDGF, TH, DBH
48. Neuron specific promotersNeuron specific enolase (NSE)synapsin-1 (SYN)Platelet-derived Growth Factor (PDGF)Tyrosine Hydroxylase (TH –catecholamines)DA β Hydroxylase (DβH – NE, Epi neurons)Ca++ Calmodulin Kinase (CamK, excitatory)
49. Building a cassette to hit Specific NeuronsCell specific promoters may be too largeFor viral vectorsConstitutive viral promoters Drive expression in all cells (CMV)Smaller hybrid promotersEfficiently induce strong specific expression
50. Building a cassette to drive Specific GenesSpecific promoter drives Gene expressionCassette promoter and gene need not Come from the same systemThis pairing may not promote Strong expressionOr any expression
51. Enhancing Promoter – Gene couplingStrong expression may require an enhancerTranscriptional activationHuman cytomegalovirus (CMV)Immediate-early gene enhancerHighest activity of all viral enhancersInserted 5’ to cellular promoter
52. Enhancing Promoter – Gene couplingWoodchuck Hepatitis Virus Post-transcriptional regulatory element (WPRE)cis-acting RNA elementAuguments transgene espressionPowerfully increases mRNA accumulationMost effective inserted 3’ UTR
53. See what you’ve transfectedAdd a gene for a fluorescence proteinGFPYFPmCherry
54. What a Cassette might look likeCMVSYNDβHCREBGFPWPRE5’3’enhancerenhancerNeuron specificpromoterCell type specificpromotergeneColor gene