applications of biotechnology recombinant dna products and organisms
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Applications of BiotechnologyApplications of Biotechnology
Recombinant DNA Recombinant DNA Products and OrganismsProducts and Organisms
Applying Your KnowledgeApplying Your KnowledgeApplying Your KnowledgeApplying Your Knowledge
A.A. An enzyme that cleaves DNA at specific An enzyme that cleaves DNA at specific sequences is a __________ .sequences is a __________ .
B.B. A sequence of DNA that is complementary to A sequence of DNA that is complementary to the gene of interest is a _________. the gene of interest is a _________.
C.C. A small, independently replicating DNA A small, independently replicating DNA molecule is a ___________. molecule is a ___________.
1.1. ProbeProbe2.2. CloneClone3.3. PlasmidPlasmid4.4. Restriction EnzymeRestriction Enzyme
Recombinant DNA ProductsRecombinant DNA Products
PharmaceuticalPharmaceutical Used for Used for
Factor VIIIFactor VIII Blood ClottingBlood Clotting
Human Growth Human Growth HormoneHormone
Pituitary Pituitary DwarfismDwarfism
InsulinInsulin DiabetesDiabetes
InterferonInterferon CancerCancer
Tissue Plasminogen Tissue Plasminogen ActivatorActivator
Heart Attack Heart Attack
VaccineVaccine Hepatitis BHepatitis B
Optimizing the Yield Optimizing the Yield of a Gene Productof a Gene Product
• Selecting the VectorSelecting the Vector• Selecting the Host CellSelecting the Host Cell• Selecting the DNA Delivery MethodSelecting the DNA Delivery Method• Constructing the Recombinant DNA Constructing the Recombinant DNA
MoleculeMolecule• Maximizing Gene ExpressionMaximizing Gene Expression• Collecting the Gene ProductCollecting the Gene Product
VectorsVectorsPlasmidsPlasmidsShuttle vectorsShuttle vectorsVirusesVirusesArtificial ChromosomesArtificial ChromosomesExpression VectorsExpression Vectors
Selecting the VectorSelecting the Vector
AdvantagesAdvantagesVectorVector DisadvantagesDisadvantages
PlasmidPlasmid
CosmidCosmid(from (from bacterial virus)bacterial virus)
VirusVirus
Can carry foreign DNA Can carry foreign DNA into bacteria and yeastinto bacteria and yeast
Must remain small in Must remain small in size to increase uptake size to increase uptake and avoid damageand avoid damage
Can carry large Can carry large DNA fragmentsDNA fragments
Technically more Technically more difficult to construct than difficult to construct than recombinant plasmidrecombinant plasmid
Can reach plant and Can reach plant and human cellshuman cells
Disabled virus may Disabled virus may regain disease-regain disease-causing propertiescausing properties
Selecting the Host CellSelecting the Host Cell
•Genetics is well Genetics is well knownknown•Plasmids andPlasmids and viruses are well viruses are well knownknown•High reproductiveHigh reproductive rate rate
•Can cause diseaseCan cause disease
•Endotoxins fromEndotoxins from
cell wallcell wall
•Poor protein exporterPoor protein exporter
•Cannot remove intronsCannot remove introns
AdvantagesAdvantagesProkaryoticProkaryoticHostHost
DisadvantagesDisadvantages
BacillusBacillus
E. coliE. coli
•Actively exports Actively exports proteinsproteins• Plasmids andPlasmids and viruses are well viruses are well knownknown
•Cannot remove intronsCannot remove introns
Selecting the Host CellSelecting the Host Cell
•Genetics is well Genetics is well knownknown• Non-pathogenic Non-pathogenic • Proteins of greater Proteins of greater complexity can complexity can be made be made •Removes intronsRemoves introns
AdvantagesAdvantagesEukaryotic Eukaryotic HostHost
DisadvantagesDisadvantages
mammalianmammalian
yeastyeast
•Avoids toxicAvoids toxic bacterial proteinsbacterial proteins•Allows proper export, Allows proper export, protein foldingprotein folding and modificationand modification
•EXPENSIVE!EXPENSIVE!
•May not be able to May not be able to modify or export modify or export complex mammalian complex mammalian proteins proteins
Applying Your KnowledgeApplying Your Knowledge
1.1. PlasmidPlasmid2.2. CosmidCosmid3.3. VirusVirus
A.A. Best for large sequences of DNA (1-3)Best for large sequences of DNA (1-3)
B.B. Best for proper modification of complex Best for proper modification of complex proteins (4-6)proteins (4-6)
C.C. Best for entry into specific cell types (1-3)Best for entry into specific cell types (1-3)
D.D. Inexpensive organism that can remove Inexpensive organism that can remove introns (4-6)introns (4-6)
4.4. YeastYeast5.5. mammalian cellmammalian cell6.6. E. coliE. coli
Microinjection: Placing DNA in each
cell with a thin needle (animal cells,
plant protoplasts)
DNA Delivery Methods
Virus: Binds to a receptor on the cell surface and injects DNA into the cell(bacteria, plants,
animals)
Liposome: Sphere of lipids
that is compatible with plasma membrane
(animal cells, plant protoplasts
Additional DNA Delivery MethodsAdditional DNA Delivery Methods
Method Method How it Works How it Works Used For/InUsed For/InChemicals that open Chemicals that open holes in membranes holes in membranes
Temporary holes admit DNA Temporary holes admit DNA Bacterial cellsBacterial cells
Animal cellsAnimal cells
Plant protoplastsPlant protoplasts
ElectroporationElectroporation Electric shock causes Electric shock causes temporary holes to admit DNAtemporary holes to admit DNA
Animal cellsAnimal cells
Plant protoplastsPlant protoplasts
Particle Particle BombardmentBombardment
DNA-coated metal pellets are DNA-coated metal pellets are propelled into cells with “gene propelled into cells with “gene gun” gun”
Plant cellsPlant cells
Constructing the Recombinant DNA MoleculeConstructing the Recombinant DNA Molecule
Coding Region
Plasmid Sequences-origin of replication-antibiotic resistance
Promoter
Term
inat
or
Ribosome Ribosome Binding SiteBinding Sitefor mRNAfor mRNA
PromoterPromoter
- Binding site for - Binding site for RNA polymerase RNA polymerase- Initiation of - Initiation of transcriptiontranscription
TerminatorTerminator
-Site where -Site where transcription transcription endsends
Expressing the Gene: Production of Expressing the Gene: Production of
ProteinProtein
Increased Increased YieldYield
GoalGoal Possible MethodsPossible Methods
• To efficiently To efficiently produce as much produce as much protein as possible protein as possible
• Increase plasmidIncrease plasmid copy number copy number•Increase Increase promoter promoter efficiency efficiency•Increase mRNA Increase mRNA binding to binding to ribosome ribosome
Collecting the Gene ProductCollecting the Gene Product
• less protein less protein breakdown breakdown
• No cell can beNo cell can be protease-freeprotease-free
AdvantagesAdvantages DisadvantagesDisadvantagesAvoiding ProteinAvoiding ProteinLoss usingLoss using
Protease-deficient Protease-deficient BacteriaBacteria
Fusion to nativeFusion to native proteinsproteins
Accumulation of Accumulation of aggregatesaggregates
•proteins not proteins not degradeddegraded
•avoids proteaseavoids protease activityactivity
• May be technicallyMay be technically difficult difficult•Must remove nativeMust remove native protein protein
•Aggregates difficult Aggregates difficult
to extract without to extract without breakdown breakdown
Collecting the Gene ProductCollecting the Gene Product
• Used to increase Used to increase export of a protein export of a protein
•A signal sequence A signal sequence directs a ribosome to directs a ribosome to attach to rer to attach to rer to complete synthesis of complete synthesis of the protein the protein •Protein product is Protein product is delivered into rer for delivered into rer for transport within the transport within the cell to the outside cell to the outside
How it WorksHow it Works
Addition of Addition of a Signal a Signal SequenceSequence
UsesUses
Used to Treat: Used to Treat:
DwarfismDwarfism
Human Growth Human Growth HormoneHormone
Functions:Functions:-Increases cell -Increases cell uptake of amino uptake of amino acids acids -Promotes use-Promotes use of fat of fat
Advantages: Decreased danger of diseaseAdvantages: Decreased danger of diseaseLess expensive, available in larger quantitiesLess expensive, available in larger quantities
Pharmaceutical ProductsPharmaceutical Products
SizeSize191 Amino Acids191 Amino Acids
Library?Library?
Vector?Vector?
Host?Host?
cDNAcDNA
plasmidplasmid
bacterialbacterial (after removing (after removing signal sequence) signal sequence)
Used to Treat: Used to Treat:
HemophiliaHemophilia
Factor VIIIFactor VIII
Function:Function:
-Blood Clotting-Blood Clotting
Advantages: Easier to obtainAdvantages: Easier to obtain Free from contaminationFree from contamination
Pharmaceutical ProductsPharmaceutical Products
SizeSize
-2332 Amino Acids-2332 Amino Acids
-26 introns-26 introns
-25 carbohydrate -25 carbohydrate attachment sites attachment sites on the protein on the protein
Library?Library?
Vector?Vector?
Host?Host?
cDNAcDNA
virusvirus
mammalianmammalian (hamster) (hamster)
Transgenic AnimalsTransgenic Animals
• Transgenic = containing one or Transgenic = containing one or more genes from another species in more genes from another species in every cell of the organismevery cell of the organism
• TypesTypes1. “Pharm” Animals1. “Pharm” Animals2. Models of Human Diseases2. Models of Human Diseases
3. Gene Targeted Animals3. Gene Targeted Animals
““Pharm” AnimalsPharm” Animals
• Animals that produce pharmaceuticals Animals that produce pharmaceuticals and release them in milk or semenand release them in milk or semen
ProductProduct ActionAction
GoatsGoats tPA = tissuetPA = tissue
Plasminogen Plasminogen ActivatorActivator
Breaks up blood Breaks up blood clots to treat clots to treat Heart AttacksHeart Attacks
PigsPigs HemoglobinHemoglobin Blood SubstituteBlood Substitute
SheepSheep Alpha-1-Alpha-1-antitrypsinantitrypsin
Hereditary Hereditary emphysemaemphysema
Models of Human Diseases Models of Human Diseases
• Transgenic Mice act as Models of Transgenic Mice act as Models of Human Diseases for ResearchHuman Diseases for Research
Human GeneHuman Gene Mouse Model ForMouse Model For
BRCA1BRCA1 Breast CancerBreast Cancer
HDHD Huntington’s DiseaseHuntington’s Disease
HGHHGH Human Patients Human Patients Receiving Human Receiving Human Growth HormoneGrowth Hormone
Procedure for Gene TargetingProcedure for Gene Targeting
Gene TargetingGene Targeting• Introducing a different form of a gene to Introducing a different form of a gene to
replace a host genereplace a host gene
““knockout”knockout” Inactivation of a gene by gene Inactivation of a gene by gene targetingtargeting---mouse with ADA gene knocked out as ---mouse with ADA gene knocked out as
a model for SCID a model for SCID
““knock in” knock in” Introducing a gene with altered Introducing a gene with altered function by gene targetingfunction by gene targeting---mice with---mice with
human adult hemoglobinhuman adult hemoglobin
human fetal hemoglobinhuman fetal hemoglobin
human sickle cell hemoglobinhuman sickle cell hemoglobin
Applying Your KnowledgeApplying Your Knowledge
1.1. Knockout AnimalKnockout Animal2.2. ““Pharm” Animal Pharm” Animal 3.3. Model of Human DiseaseModel of Human Disease4.4. Knock in AnimalKnock in Animal
A.A. Animal that secretes medicine in its milkAnimal that secretes medicine in its milk
B.B. Mouse with a human BRCA 1 gene addedMouse with a human BRCA 1 gene added(no mouse genes altered) (no mouse genes altered)
C.C. Mouse with its ADA gene inactivatedMouse with its ADA gene inactivated