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FAISALABAD
MAJOR BIOTECHNOLOGYCENTERS
JAMSHORO
National Biosafety Guidelines
Recognizing the revolutionary economic potential of the new biotechnology in agriculture, health, industry, environment and energy sectors; and appreciating the concerns that mixing of genes from unrelated organisms might create natural imbalance that is not yet adequately understood; and considering the fears that manipulated genes or products thereof if allowed to move around freely in nature, may pose potential hazards; and realizing the apprehensions that certain transgenic organisms may be harmful or become harmful to economic plants, animals and human being; and discharging the obligations of the Convention of Biological Diversity (CBD) and Cartagena Protocols to which Pakistan is a signatory; the Minister of Environment, constituted a National Biosafety Expert Committee to deliberate on these issues to regulate the safe release of Genetically Modified Organisms (GMOs) and products thereof.These guidelines have been prepared keeping in view the guidelines prepared by UNIDO, FAO, WHO, UNEP, and all the developed and developing countries with modification to suit our unique and specific socio-economic and
geographic environment.
Recognizing Economic Potential
UNIDO, FAO,WHO, UNEP etc
National Biosafety Guidelines
The objective of these guidelines is to prevent unintentional negligence leading to misuse and irresponsibility by laboratory workers/researchers as well as the end-users.
To define the bounds guide lines / regulatory jurisdiction of these guide lines , biotechnology has been defined as processes using living organisms or parts thereof to make or modify products; and to improve plants, animals, or microorganisms for specific uses. “Recombinant DNA” has been defined as molecules developed outside living cells by joining natural or synthetic DNA segments to DNA that can replicate in a living cell and those DNA molecules that result from the replication of
such DNA.
prevent unintentional negligence
Biotechnology
National Biosafety Guidelines
For the purposes of these guidelines, regulated material includes all genetically modified material modified materials ( D N A & R N A preparations , viroids , viruses cells and organisms, modified or constructed through genetic engineering), derivatives thereof and wastes or by-products of genetic engineering practices (containing viable organisms or otherwise).
8. The scope of these guidelines embraces all works related to gene manipulation employing recombinant DNA technology for all purposes including the development of transgenic plants, animals and microorganisms; production of vaccines; industrial manufacturing of genetically modified organisms and products thereof, and their release into the environment for field trials as well as for commercial uses.
9. The Guidelines consist of two parts; the first part relates to regulated work in laboratory research and field trials; and the second part deals with procedures for approvals which must be obtained to deregulate the regulated materials to allow their free movement and commercial uses.
Guidelines consist on 2 Parts
Scope of Guidelines
Regulated material
National Biosafety Guidelines
All regulated laboratory research works are classified into (a) Minimal risk, (b) Low risk, (c) Considerable level of risk and laboratory containment conditions are accordingly prescribed. For regulated fieldwork, comprehensive containment conditions have been prescribed separately for genetically modified microorganisms plants and animals.
The mechanism of monitoring and implementation of the proposed guidelines is built on three tiers as specified in the Biosafety Rules, 2005, namely a National Biosafety Committee (NBC); a Technical Advisory Committee (TAC); and Institutional Biosafety Committees (IBCs) at the institutional levels. The NBC headed by the Secretary, Ministry of Environment will be responsible to oversee all laboratory work, field trials and allow commercial releases of GMOs and their products.
Enforcement of various clauses of the National Biosafety Guidelines will be administered by the three monitoring implementation bodies, as per legal authority under Clause 7(g) of the Pakistan Environment Protection Act 1997.
Risks
Tires of BGLs
National Biosafety Guidelines
The IBC may recommend to NBC for awarding exempt status for Laboratory work/field work with genetically modified organisms, if there is sufficient information/grounds available to consider the work as having no risk and NBC may consider for formal approval.
Permission for deregulation granted by the NBC can be withdrawn if sufficient technical data/evidence becomes available after the approval, which warrants its deregulation.
To standardize processing, separate format for application/proposal for obtaining permission to undertake laboratory genetic manipulation work, field trial and commercialization have been designed.
Awarding exempt status
Permission for deregulation
Format for Application/proposal
National Biosafety Guidelines
A separate application format has also been designed for the movement of regulated materials and/or exempt status.
Instructions for the preparation of applications as well as for its assessment have also been prescribed to streamline and standardize the procedures for assessment and evaluation.
18. These biosafety guidelines have been developed on the basis of technical information presently available and may change in the future as more know-how becomes available. Therefore, revision of these guidelines will be a continuous process.
19. These biosafety guidelines will be supplemented with annexures explaining/elaborating concepts, procedures and protocols required in monitoring/implementation of the guidelines.
Movement of material
Instructions
Modification
in BGLs
Annexures
National Biosafety Guidelines
COMPOSITION AND FUNCTIONS OF VARIOUS BIOSAFETY COMMITTEES
National Biosafety
Committee (NBC),
Institutional Biosafety Committee
(IBC)
Technical Advisory
Committee
BIOSAFETY COMMITTEES
National Biosafety Committee (NBC),
Secretary, Ministry of EnvironmentMember (Biosciences), Pakistan Atomic Energy Commission Chairpersons of concerned Institutional Biosafety Committees Representatives of Provincial GovernmentsRepresentatives of Government of AJKChairman, PARCRepresentative Ministry of Food & Agriculture Representative Ministry of HealthRepresentative Ministry of Science and Technology Representative Ministry of EducationDirector-General, Department of Plant Protection
Director-General, Pakistan EPA
Chair person Member Members Members Member Member Member Member Member Member Member MemberMemebr
Functions of National Biosafety Committee.
• Establish standards and procedures for risk assessment and labeling of living modified organisms, substances or cells and products thereof.
• Consider application(s) for import, export or commercial release of living modified organisms, and on the recommendations of Technical Advisory Committee allow release or reject applications after reviewing the risk assessment carried out in accordance with the biosafety guidelines, the procedures established under clause (a) and any other reliable information.
• Ban or restrict import, export, sale, purchase or trading of any living modified organism causing or likely to cause risk to public health, safety or environment.
•Cooperate with other relevant federal or provincial authorities overseeing the import and release of living organisms and formulate guidelines for the identification, inspection and regulation of transgenic species exotic organisms and others.
•Develop linkages with foreign biosafety committees and relevant agencies to ensure that genetic manipulation practices in Pakistan address international biosafety concerns and observe universal codes of conduct.
•Restrain on the advice of Technical Advisory Committee any person, authority or institute involved with genetic manipulation experiments of potential hazards.
•To facilitate exchange of technical expertise to various research institutions and regulatory agencies in setting up appropriate experimental conditions.
NBC
•To coordinate efforts of Institutional Biosafety Committees and inform and educate the public on biosafety issues and on proposed national policies.
•To facilitate all levels of supervision of genetic manipulation work by assisting other regulatory bodies including Institutional Biosafety Committees, in establishing pertinent codes disciplines and guidelines for the appraisal of biohazards and the management of bio- safeguards.
•To ensure that laboratory, field work and commercial release of Genetically Modified Organisms and their products conforms to the National Biosafety Guidelines.
NBC
•To inform the various institutions engaged in genetic manipulation work about new developments in biosafety so as to avoid exposure of laboratory personnel, the community or the environment to undue risks.
•To prepare Documents and provide to Institutional Biosafety Committees the various notifications and assessment forms, biosafety guidelines, related documents and assorted signs for facilities.
•To coordinate efforts between pertinent government agencies and private organizations to maintain safety levels in biotechnological work and to prepare them for biological emergencies.
NBC
•To certify high-level laboratories, plant glass houses and animal houses intended for use in high-risk work. Upon request by the institution, and at the earliest convenience, theNational Biosafety Committee may inspect a facility and either issue certification, or recommend additional precautions, if elements of the facility are determined to be inadequate •To support the types of risk or hazard companying work requiring such physical containment.
•To inspect high-level laboratories and containment facilities on a regular basis. The National Biosafety Committee may inspect laboratories and facilities of containment level C2, PH2. and C2A, as specified in the bio safety guidelines, equivalent or higher at any time subsequent to certification without prior notice.
NBC
• To keep information of commercial significance confidential from public domain if so requested in writing by applicant, person or institution or organization.
• To inspect systems equipment and instruments governing ambient biosafety levels in genetic manipulation laboratories.
• To monitor the safety related aspects of on going research projects and achievements involving genetically engineered organisms/hazardous substances or cells and products thereof.
NBC
Institutional Institutional Biosafety Biosafety
CommitteesCommitteesPurpose & Purpose & ObjectivesObjectives
PurposePurpose
Design and present training for the Design and present training for the Institutional Biosafety Committee (IBC) Institutional Biosafety Committee (IBC) members that meets the requirements of members that meets the requirements of the NBCthe NBC Guidelines Guidelines and prepares them and prepares them for expanded efforts in the review of for expanded efforts in the review of Recombinant DNA (rDNA) applications.Recombinant DNA (rDNA) applications.
ObjectivesObjectives•• Provide a basic introduction to rDNA research who do Provide a basic introduction to rDNA research who do
not regularly work with rDNA.not regularly work with rDNA.
•• Review the roles and responsibilities of the IBC in Review the roles and responsibilities of the IBC in oversight of rDNA research, including the various types oversight of rDNA research, including the various types of research covered by the NBC Guidelines, the of research covered by the NBC Guidelines, the biosafety levels of the Guidelines, and the typical review biosafety levels of the Guidelines, and the typical review and approval processes that apply to various forms of and approval processes that apply to various forms of rDNA research, exclusive of University policies and rDNA research, exclusive of University policies and procedures.procedures.
•• Apply practical guidance for the review of research Apply practical guidance for the review of research applications utilizing general research examples and applications utilizing general research examples and case studies as well as specific University principal case studies as well as specific University principal investigator applications provided by the University investigator applications provided by the University Biosafety Officer and IBC Chair.Biosafety Officer and IBC Chair.
•• DefinitionsDefinitions•• BiosafetyBiosafety oversightoversight•• Research involving recombinant DNAResearch involving recombinant DNA•• Responsibilities under the Responsibilities under the NBC NBC Guidelines for Research Involving Guidelines for Research Involving Recombinant DNA MoleculesRecombinant DNA Molecules••IBC protocol reviewIBC protocol review
AgendaAgenda
DefinitionsDefinitions
Biohazard:Biohazard:An agent of biological origin that An agent of biological origin that has the capacity to produce has the capacity to produce harmful effects on humans; i.e. harmful effects on humans; i.e. microorganisms, toxins and microorganisms, toxins and allergens derived from those allergens derived from those organisms, and allergens and organisms, and allergens and toxins derived from plants or toxins derived from plants or animals.animals.
DefinitionsDefinitions
Biosafety:Biosafety:
Applying a combination ofApplying a combination oflaboratory practices andlaboratory practices andprocedures, laboratory procedures, laboratory facilities, and safety facilities, and safety equipment when working equipment when working with potentially infectious with potentially infectious microorganisms.microorganisms.
DefinitionsDefinitions
DefinitionsDefinitions
Risk Assessment:Risk Assessment:Addressing laboratory Addressing laboratory activities involving infectious activities involving infectious or potentially infectious or potentially infectious material and implementing material and implementing measures to reduce the measures to reduce the workerworker’’s and environments and environment’’s s risk of exposure to an agent to risk of exposure to an agent to an absolute minimum.an absolute minimum.
Chain of InfectionChain of InfectionReservoir of pathogenReservoir of pathogen
Portal of escapePortal of escape
TransmissionTransmission
Route of entry/Route of entry/infectious doseinfectious dose
Susceptible hostSusceptible host
Incubation periodIncubation period
Risk AssessmentRisk Assessment
Practices/Equipment
Practices/Equipment
Personal Protective
Personal Protective
Equipment (PPE)
Equipment (PPE)
Immuniza
tions
Immuniza
tions
Surveillance
Surveillance
Risk Assessment
Risk Assessment
Biosecurity:Biosecurity:Protection of highProtection of high--consequence microbial consequence microbial agents and toxins, or agents and toxins, or critical relevant critical relevant information, against theft information, against theft or diversion by those who or diversion by those who intend to pursue intend to pursue intentional misuse.intentional misuse.
DefinitionsDefinitions
DefinitionsDefinitions
Select Agents:Select Agents:Pathogens and toxins considered to have the Pathogens and toxins considered to have the
potential to pose a severe threat to human, potential to pose a severe threat to human, animal, or plant health and safety.animal, or plant health and safety.
•• VirusesViruses•• BacteriaBacteria•• FungiFungi•• ToxinsToxins
DefinitionsDefinitions
Responsible Official (RO) and Responsible Official (RO) and Alternate Responsible Official (ARO)Alternate Responsible Official (ARO)An individual designated by the entity, which An individual designated by the entity, which acts on their behalf and has the authority and acts on their behalf and has the authority and control to ensure compliance with the control to ensure compliance with the regulationsregulations
–– Approved by the Respective Department Approved by the Respective Department –– Familiar with regulation requirementsFamiliar with regulation requirements
DefinitionsDefinitions
Recombinant DNA Recombinant DNA MoleculesMoleculesUnder the current Under the current NBC NBC Guidelines, NIH GuidelinesGuidelines, NIH Guidelines, , these are molecules these are molecules constructed outside of living constructed outside of living cells by joining natural or cells by joining natural or synthetic DNA segments to synthetic DNA segments to DNA molecules that can DNA molecules that can replicate in a living cell, or replicate in a living cell, or molecules that result from molecules that result from their replication.their replication.
DefinitionsDefinitions
NBC Guidelines for Research Involving NBC Guidelines for Research Involving Recombinant DNA MoleculesRecombinant DNA Molecules(NBC Guidelines)(NBC Guidelines)A document created in 2005 that outlines principles for A document created in 2005 that outlines principles for the safe conduct of research employing recombinant the safe conduct of research employing recombinant DNA technology. The NBC Guidelines detail practices DNA technology. The NBC Guidelines detail practices and procedures for the containment of various forms and procedures for the containment of various forms of recombinant DNA research, for the proper conduct of recombinant DNA research, for the proper conduct of research involving genetically modified plants and of research involving genetically modified plants and animals, and for the safe conduct of human gene animals, and for the safe conduct of human gene transfer research.transfer research.
•• National Biosafety Center (NBC)National Biosafety Center (NBC)•• National Institutes of Health (NIH)National Institutes of Health (NIH)
–– Office of Biotechnology ActivitiesOffice of Biotechnology Activities•• Centers for Disease Control (CDC)Centers for Disease Control (CDC)•• Occupational Safety & Health Administration Occupational Safety & Health Administration
(OSHA)(OSHA)•• Environmental Protection Agency (EPA)Environmental Protection Agency (EPA)•• US Dept of Agriculture (USDA)US Dept of Agriculture (USDA)•• US Dept of JusticeUS Dept of Justice•• US Dept of TransportationUS Dept of Transportation•• US Dept of CommerceUS Dept of Commerce•• World Health Organization (WHO)World Health Organization (WHO)•• Community Activist GroupsCommunity Activist Groups
External OversightExternal Oversight
•• Institutional Biosafety Committee (IBC)Institutional Biosafety Committee (IBC)•• Biosecurity and Biosafety ProgramsBiosecurity and Biosafety Programs•• Emergency Response PlanEmergency Response Plan•• Standard Operating Procedures (SOPStandard Operating Procedures (SOP’’s)s)•• Laboratory Inspections (internal and Laboratory Inspections (internal and
external; CDC, USDA, AAALAC, etc.)external; CDC, USDA, AAALAC, etc.)•• Training and DocumentationTraining and Documentation•• Institutional Animal Care and Use Committee Institutional Animal Care and Use Committee
(IACUC)(IACUC)•• Institutional Review Board (IRB)Institutional Review Board (IRB)
Internal OversightInternal Oversight
IBCIRB
IACUC
Institutional CommitteesInstitutional Committees
•• Relationship not prescribed in the Relationship not prescribed in the NBC GuidelinesNBC Guidelines
•• Institutions should determine best Institutions should determine best way for these committees to way for these committees to interact and share informationinteract and share information
IBCIBC’’s, IACUCs, IACUC’’s, and IRBs, and IRB’’ss
Introduction to Introduction to Research Involving Research Involving
rDNA Molecules rDNA Molecules
ObjectivesObjectives
•• Understand basic processes involved in Understand basic processes involved in gene expressiongene expression
•• Become familiar with characteristics and Become familiar with characteristics and construction of recombinant gene transfer construction of recombinant gene transfer vectorsvectors
•• Learn to recognize the characteristics of Learn to recognize the characteristics of gene transfer systems that have implications gene transfer systems that have implications for their safe usefor their safe use
•• Gene ExpressionGene Expression•• Recombinant Gene ExpressionRecombinant Gene Expression•• Gene NonGene Non--ExpressionExpression•• Viral Vector TechnologyViral Vector Technology
OutlineOutline
Gene ExpressionGene Expression
•• An attempt to explain cellular An attempt to explain cellular processes by understanding the processes by understanding the interactions of the molecules involvedinteractions of the molecules involved
•• Requires manipulation of genes and Requires manipulation of genes and their expression in different situationstheir expression in different situations
•• Recombinant DNA and gene transfer Recombinant DNA and gene transfer are enabling technologies for are enabling technologies for molecular biologymolecular biology
Molecular BiologyMolecular Biology
Basic Processes of Gene Basic Processes of Gene ExpressionExpression
•• ReplicationReplication–– synthesis of an exact duplicate nucleic acid synthesis of an exact duplicate nucleic acid
(maintenance of the genetic information)(maintenance of the genetic information)•• TranscriptionTranscription
–– making an RNA copy of a DNA moleculemaking an RNA copy of a DNA molecule•• TranslationTranslation
–– converting RNA sequence into an amino converting RNA sequence into an amino acid sequenceacid sequence
The Flow of Genetic InformationThe Flow of Genetic Information
Deoxyribonucleic Acid (DNA)Deoxyribonucleic Acid (DNA)
•• Genetic material for most organismsGenetic material for most organisms•• Long strands of nucleotide Long strands of nucleotide ““basesbases””
–– Four different bases (A, G, C, T)Four different bases (A, G, C, T)–– Uniqueness due to specific sequence of Uniqueness due to specific sequence of
basesbases–– Two strands associate through hydrogen Two strands associate through hydrogen
bonds between complementary basesbonds between complementary bases–– A bonds with T, C bonds with GA bonds with T, C bonds with G
DNA ReplicationDNA Replication
•• Because of base pairing, Because of base pairing, sequence of bases on one sequence of bases on one strand determines strand determines sequence of the other sequence of the other strandstrand
•• Each strand in a molecule Each strand in a molecule can be a template to make can be a template to make a copy of the moleculea copy of the molecule
Ribonucleic Acid (RNA)Ribonucleic Acid (RNA)
•• There are some Understandable chemical There are some Understandable chemical differences between DNA and RNAdifferences between DNA and RNA
•• Genetic material for some virusesGenetic material for some viruses•• Messenger RNA (mRNA): an information Messenger RNA (mRNA): an information
exchange moleculeexchange molecule•• Other RNAsOther RNAs
–– Protein synthesis (Protein synthesis (rRNArRNA, , tRNAtRNA))–– Catalysis (Catalysis (RNAaseRNAase P)P)–– Regulatory functions (Regulatory functions (miRNAmiRNA))
•• An information exchange moleculeAn information exchange molecule–– The information is stored in the sequence of The information is stored in the sequence of
bases in the strandbases in the strand•• mRNAs are translated to make a proteinmRNAs are translated to make a protein•• In eukaryotic cells a messenger RNA In eukaryotic cells a messenger RNA
codes for only one proteincodes for only one protein
Messenger RNA (mRNA)Messenger RNA (mRNA)
•• Sequence of bases in mRNA is read in Sequence of bases in mRNA is read in groups of three for translationgroups of three for translation–– Referred to as codonsReferred to as codons
•• A sequence can be presented into A sequence can be presented into codons in three ways:codons in three ways:
AGC TAG CTA GCT AGC TAG CTAAGC TAG CTA GCT AGC TAG CTAA GCT AGC TAG CTA GCT AGC TAA GCT AGC TAG CTA GCT AGC TAAG CTA GCT AGC TAG CTA GCT AAG CTA GCT AGC TAG CTA GCT A
•• This is referred to as reading frameThis is referred to as reading frame
Reading FrameReading Frame
•• Long linear polymers of molecules Long linear polymers of molecules called amino acidscalled amino acids
•• Information for synthesis of proteins is Information for synthesis of proteins is contained in the nucleic acidcontained in the nucleic acid
•• Proteins have a variety of functionsProteins have a variety of functions–– Catalysis of chemical reactions (enzymes)Catalysis of chemical reactions (enzymes)–– Structural (histones, cytoskeletal proteins)Structural (histones, cytoskeletal proteins)–– Regulatory (transcription factors, growth Regulatory (transcription factors, growth
factors)factors)
ProteinsProteins
•• Region of nucleic acid that contains the Region of nucleic acid that contains the base sequence information to encode a base sequence information to encode a protein (or RNA)protein (or RNA)
•• In eukaryotic cells coding regions are In eukaryotic cells coding regions are often interrupted with nonoften interrupted with non--coding coding regions that have to be removed in regions that have to be removed in mRNAmRNA–– Called intervening sequences or intronsCalled intervening sequences or introns
•• Genes are preceded by a promoterGenes are preceded by a promoter–– Starting signal for the synthesis of an mRNAStarting signal for the synthesis of an mRNA
What Is A Gene?What Is A Gene?
Recombinant Gene Recombinant Gene ExpressionExpression
Recombinant Gene ExpressionRecombinant Gene Expression
•• OverOver--expression for purificationexpression for purification–– Bacterial expression (Bacterial expression (E. coliE. coli))–– Insect cell expression (baculovirus)Insect cell expression (baculovirus)–– Mammalian cell expression (vaccinia virus)Mammalian cell expression (vaccinia virus)–– Generally done in cultured cellsGenerally done in cultured cells
•• Expression in a cell to exert an effectExpression in a cell to exert an effect–– Tailored to the cell system being studiedTailored to the cell system being studied–– Can be in cultured cells or Can be in cultured cells or in vivoin vivo
•• Research toolsResearch tools–– Protein productionProtein production–– Transient expression studiesTransient expression studies–– Viral repliconsViral replicons–– Stable cell linesStable cell lines–– Transgenic animalsTransgenic animals–– KnockKnock--down/knockdown/knock--out animalsout animals
•• Gene therapyGene therapy•• Transgenic animals for protein Transgenic animals for protein
productionproduction
Applications of rDNAApplications of rDNA
•• Pharmaceutical compound screeningPharmaceutical compound screening–– Biochemical assaysBiochemical assays–– CellCell--based assaysbased assays
•• Vaccine productionVaccine production–– SubSub--unit vaccinesunit vaccines–– VirusVirus--like particleslike particles
•• Transgenic plantsTransgenic plants–– Crop improvementCrop improvement–– Disease/pestDisease/pest--resistant plantsresistant plants
Applications of rDNAApplications of rDNA
•• cDNA: a DNA copy of an mRNA cDNA: a DNA copy of an mRNA containing the protein coding domain of containing the protein coding domain of a genea gene
•• Generally do not use genomic DNA Generally do not use genomic DNA clones for recombinant gene expression clones for recombinant gene expression due to size and splicing requirementsdue to size and splicing requirements
5′ AAAA 3′mRNA (+)
3′ 5′5′ AAAA 3′mRNA (+)
(-) strand DNA5′3′ 5′3′
ds DNA
What is cDNA?What is cDNA?
•• Minimum requirements for constructing Minimum requirements for constructing a recombinant gene for expression:a recombinant gene for expression:–– PromoterPromoter–– cDNA (transgene)cDNA (transgene)–– Termination signal (poly A site)Termination signal (poly A site)
Expression cassetteExpression cassette
Expression CassettesExpression Cassettes
•• E. coliE. coli is a normal resident of the intestinal is a normal resident of the intestinal tracttract
•• The KThe K--12 isolate12 isolate–– Ineffective at colonizing the human gut Ineffective at colonizing the human gut –– Much rDNA work with KMuch rDNA work with K--12 considered lower risk12 considered lower risk–– Many common laboratory strains derived from KMany common laboratory strains derived from K--
1212•• Useful tool in molecular biologyUseful tool in molecular biology
–– Easy to propagate in artificial mediaEasy to propagate in artificial media–– Short generation timeShort generation time–– Can easily generate large numbers of cellsCan easily generate large numbers of cells
Escherichia coli Escherichia coli KK--1212
•• Small (~2Small (~2--20 kbp) circular DNA 20 kbp) circular DNA moleculesmolecules–– Replicate in bacterial cells independently Replicate in bacterial cells independently
of the host cell chromosomeof the host cell chromosome–– Carry genes which render host resistant to Carry genes which render host resistant to
antibioticsantibiotics–– Some plasmids can be swapped among Some plasmids can be swapped among
different species of bacteria (different species of bacteria (““Broad host Broad host rangerange””))
•• Are exploited for use as cloning and Are exploited for use as cloning and gene expression vectorsgene expression vectors
Plasmid DNAPlasmid DNA
•• Restriction enzymesRestriction enzymes–– Recognize and cut DNA at specific Recognize and cut DNA at specific
sequencessequences•• Pieces of cut DNA can be mixed and rePieces of cut DNA can be mixed and re--
sealed to form new plasmids.sealed to form new plasmids.•• Large amounts of new plasmids can be Large amounts of new plasmids can be
made in made in E. coliE. coli..
How Do We Exploit Plasmids?How Do We Exploit Plasmids?
•• Transcriptional regulation is wellTranscriptional regulation is well--understoodunderstood
•• Potential for very high expression levelsPotential for very high expression levels•• Excellent plasmid DNA factoryExcellent plasmid DNA factory
–– Easy to introduce plasmid DNA into cellsEasy to introduce plasmid DNA into cells–– Easily cultured to high cell densitiesEasily cultured to high cell densities–– Plasmid DNA is easily isolated from cellsPlasmid DNA is easily isolated from cells
Why is Why is E. coliE. coli So Important?So Important?
Antibiotic Antibiotic resistance gene resistance gene for selectionfor selection
Plasmid Plasmid origin of origin of replicationreplication
Gene of interestGene of interestPromoterPromoter Termination signalTermination signal
Promoters are tailored Promoters are tailored to the type of cell:to the type of cell:bacteriabacteriayeastyeastmammalianmammalianplantplantinsectinsect
Multiple cloning sitesMultiple cloning sites
Termination signals are Termination signals are tailored to prokaryotes or tailored to prokaryotes or eukaryotes:eukaryotes:eukaryotic cells require a poly eukaryotic cells require a poly A signalA signal
Needed for Needed for growth in growth in E. E. colicoli
Elements of a Basic Elements of a Basic Expression VectorExpression Vector
•• Chemical transfectionChemical transfection–– Calcium phosphateCalcium phosphate–– LiposomesLiposomes
•• ElectroporationElectroporation•• MicroMicro--injectioninjection•• Ballistic barrageBallistic barrage•• VirusVirus--like particles like particles
(VLP)(VLP)•• Bacteria/bacteriophageBacteria/bacteriophage•• ViralViral--mediatedmediated
Chemicalmethods
Physicalmethods
Biologicalmethods
Gene Delivery Technologies for Gene Delivery Technologies for Eukaryotic CellsEukaryotic Cells
•• Plasmids transfected into cells express Plasmids transfected into cells express proteins for a short time (1 to 3 days)proteins for a short time (1 to 3 days)
•• If there are no elements on a plasmid to If there are no elements on a plasmid to allow DNA to be maintained or allow DNA to be maintained or replicated, the input DNA is degraded replicated, the input DNA is degraded and diluted out of the culture by cell and diluted out of the culture by cell divisiondivision
•• When a plasmid is transfected into cells When a plasmid is transfected into cells for shortfor short--term expression, it is referred term expression, it is referred to as transient gene expressionto as transient gene expression
Transient Gene ExpressionTransient Gene Expression
•• LongLong--term expression requires term expression requires maintenance of input DNAmaintenance of input DNA–– Cassette that expresses a protein that Cassette that expresses a protein that
inactivates a toxic drug (dominant inactivates a toxic drug (dominant selectable marker)selectable marker)
–– Enables cells to survive in presence of Enables cells to survive in presence of drugdrug
–– Allows selection of rare cells that have Allows selection of rare cells that have recombined plasmid DNA into their recombined plasmid DNA into their genomegenome
•• These are referred to as stable cell lines or These are referred to as stable cell lines or stably transfected cellsstably transfected cells
Stable ExpressionStable Expression
Transgene
The neomycin The neomycin phosphotransferase phosphotransferase gene inactivates a gene inactivates a toxic drug (G418 or toxic drug (G418 or geneticin)geneticin)
Expression Vector for Stable Expression Vector for Stable ExpressionExpression
AdvantagesAdvantages
•• Few limitations on Few limitations on DNA sizeDNA size
•• NonNon--infectiousinfectious•• Chemically definedChemically defined•• Lack of immune Lack of immune
responseresponse
LimitationsLimitations
•• Inefficient deliveryInefficient delivery•• Low expression Low expression
levelslevels•• Short term Short term
expressionexpression•• ToxicityToxicity•• Different methods for Different methods for
different cellsdifferent cells
Plasma DNA DeliveryPlasma DNA Delivery