Applications of BiotechnologyApplications of Biotechnology

Recombinant DNA Recombinant DNA Products and OrganismsProducts and Organisms

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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

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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