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FAISALABAD

MAJOR BIOTECHNOLOGY
CENTERS
JAMSHORO

National Biosafety Guidelines

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 endusers.
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.

Regulated
material

Scope of
Guidelines

Guidelines
consist on 2
Parts

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
Awarding
exempt status for Laboratory work/field work with
exempt
genetically modified organisms, if there is
sufficient
information/grounds
available
to status
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 Permission
data/evidence becomes available after the for
deregulation
approval, which warrants its deregulation.
To standardize processing, separate format for Format for
application/proposal for obtaining permission to Application/
undertake laboratory genetic manipulation work,
field trial and commercialization have been proposal
designed.

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

COMPOSITION AND FUNCTIONS OF VARIOUS


Na

BIOSAFETY COMMITTEES

t
Co iona
mm l B
io s
it t
afe
ee
t
(N
BC y
),

BIOSAFETY COMMITTEES

Institutional
Biosafety Committee
(IBC)

ry
o
is
v
l Ad tee
a
it
ic
n
m
ch om
e
T
C

National Biosafety Committee (NBC),


Secretary, Ministry of Environment
Member (Biosciences), Pakistan Atomic Energy
Commission Chairpersons of concerned Institutional
Biosafety Committees Representatives of Provincial
Governments
Representatives of Government of AJK
Chairman, PARC
Representative Ministry of Food & Agriculture
Representative Ministry of Health
Representative Ministry of Science and Technology
Representative Ministry of Education
Director-General, Department of Plant Protection

Chair person
Member
Members
Members
Member
Member
Member
Member
Member
Member
Member
Member

Director-General, Pakistan EPA

Memebr

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.

NBC
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
Committees
and

efforts of Institutional Biosafety


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


release
of Genetically Modified

work and commercial


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
Committees
the various

and provide to Institutional Biosafety


notifications and assessment forms,
biosafety guidelines, related documents and assorted signs for
facilities.

To coordinate
agencies

efforts between pertinent government

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, the
National 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.
high-level laboratories and containment facilities
onToainspect
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.

Institutional
Biosafety
Committees
Purpose & Objectives

Purpose
Design and present training for the
Institutional Biosafety Committee (IBC)
members that meets the requirements of
the NBC Guidelines and prepares them
for expanded efforts in the review of
Recombinant DNA (rDNA) applications.

Objectives
Provide a basic introduction to rDNA research who do
not regularly work with rDNA.
Review the roles and responsibilities of the IBC in
oversight of rDNA research, including the various types
of research covered by the NBC Guidelines, the
biosafety levels of the Guidelines, and the typical review
and approval processes that apply to various forms of
rDNA research, exclusive of University policies and
procedures.
Apply practical guidance for the review of research
applications utilizing general research examples and
case studies as well as specific University principal
investigator applications provided by the University
Biosafety Officer and IBC Chair.

Agenda

Definitions

Biosafety oversight

Research involving recombinant DNA

Responsibilities under the NBC


Guidelines for Research Involving
Recombinant DNA Molecules

IBC

protocol review

Definitions

Definitions

Biohazard:
An agent of biological origin that
has the capacity to produce
harmful effects on humans; i.e.
microorganisms,
toxins
and
allergens derived from those
organisms, and allergens and
toxins derived from plants or
animals.

Definitions
Biosafety:
Applying a combination of
laboratory practices and
procedures, laboratory
facilities, and safety
equipment when working
with potentially infectious
microorganisms.

Definitions

Risk Assessment:
Addressing laboratory
activities involving infectious
or potentially infectious
material and implementing
measures to reduce the
workers and environments
risk of exposure to an agent to
an absolute minimum.

Risk Assessment
t
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/E
Reservoir of pathogen
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v
ice
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ct
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Pr
r
Portal of escape
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a
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r pm
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s
Transmission
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E
tio
a
iz
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Route of entry/
m
e
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I
c
n
infectious dose
la
l
i
ve
r
Su

Chain of Infection

sk
Ri

t
en
m
ss
se
As

Susceptible host

Incubation period

Definitions
Biosecurity:
Protection of highconsequence microbial
agents and toxins, or
critical relevant
information, against theft
or diversion by those who
intend to pursue
intentional misuse.

Definitions
Select Agents:
Pathogens and toxins considered to have the
potential to pose a severe threat to human,
animal, or plant health and safety.
Viruses
Bacteria
Fungi
Toxins

Definitions
Responsible Official (RO) and
Alternate Responsible Official (ARO)
An individual designated by the entity, which
acts on their behalf and has the authority and
control to ensure compliance with the
regulations
Approved by the Respective Department
Familiar with regulation requirements

Definitions
Recombinant DNA
Molecules
Under the current NBC
Guidelines, NIH Guidelines,
these are molecules
constructed outside of living
cells by joining natural or
synthetic DNA segments to
DNA molecules that can
replicate in a living cell, or
molecules that result from
their replication.

Definitions
NBC Guidelines for Research Involving
Recombinant DNA Molecules
(NBC Guidelines)
A document created in 2005 that outlines principles for
the safe conduct of research employing recombinant
DNA technology. The NBC Guidelines detail practices
and procedures for the containment of various forms
of recombinant DNA research, for the proper conduct
of research involving genetically modified plants and
animals, and for the safe conduct of human gene
transfer research.

External Oversight
National Biosafety Center (NBC)
National Institutes of Health (NIH)
Office of Biotechnology Activities

Centers for Disease Control (CDC)


Occupational Safety & Health Administration
(OSHA)
Environmental Protection Agency (EPA)
US Dept of Agriculture (USDA)
US Dept of Justice
US Dept of Transportation
US Dept of Commerce
World Health Organization (WHO)
Community Activist Groups

Internal Oversight

Institutional Biosafety Committee (IBC)


Biosecurity and Biosafety Programs
Emergency Response Plan
Standard Operating Procedures (SOPs)
Laboratory Inspections (internal and
external; CDC, USDA, AAALAC, etc.)
Training and Documentation
Institutional Animal Care and Use Committee
(IACUC)
Institutional Review Board (IRB)

Institutional Committees

IBC

IACUC

IRB

IBCs, IACUCs, and IRBs


Relationship not prescribed in the
NBC Guidelines
Institutions should determine best
way for these committees to
interact and share information

Introduction to
Research Involving
rDNA Molecules

Objectives
Understand basic processes involved in
gene expression
Become familiar with characteristics and
construction of recombinant gene transfer
vectors
Learn to recognize the characteristics of
gene transfer systems that have implications
for their safe use

Outline

Gene Expression
Recombinant Gene Expression
Gene Non-Expression
Viral Vector Technology

Gene Expression

Molecular Biology
An attempt to explain cellular
processes by understanding the
interactions of the molecules involved
Requires manipulation of genes and
their expression in different situations
Recombinant DNA and gene transfer
are enabling technologies for
molecular biology

Basic Processes of Gene


Expression
Replication
synthesis of an exact duplicate nucleic acid
(maintenance of the genetic information)

Transcription
making an RNA copy of a DNA molecule

Translation
converting RNA sequence into an amino
acid sequence

The Flow of Genetic Information

Deoxyribonucleic Acid (DNA)


Genetic material for most organisms
Long strands of nucleotide bases
Four different bases (A, G, C, T)
Uniqueness due to specific sequence of
bases
Two strands associate through hydrogen
bonds between complementary bases
A bonds with T, C bonds with G

DNA Replication
Because of base pairing,
sequence of bases on one
strand determines
sequence of the other
strand
Each strand in a molecule
can be a template to make
a copy of the molecule

Ribonucleic Acid (RNA)


There are some Understandable chemical
differences between DNA and RNA
Genetic material for some viruses
Messenger RNA (mRNA): an information
exchange molecule
Other RNAs
Protein synthesis (rRNA, tRNA)
Catalysis (RNAase P)
Regulatory functions (miRNA)

Messenger RNA (mRNA)


An information exchange molecule
The information is stored in the sequence of
bases in the strand

mRNAs are translated to make a protein


In eukaryotic cells a messenger RNA
codes for only one protein

Reading Frame
Sequence of bases in mRNA is read in
groups of three for translation
Referred to as codons

A sequence can be presented into


codons in three ways:
AGC TAG CTA GCT AGC TAG CTA
A GCT AGC TAG CTA GCT AGC TA
AG CTA GCT AGC TAG CTA GCT A
This is referred to as reading frame

Proteins
Long linear polymers of molecules
called amino acids
Information for synthesis of proteins is
contained in the nucleic acid
Proteins have a variety of functions
Catalysis of chemical reactions (enzymes)
Structural (histones, cytoskeletal proteins)
Regulatory (transcription factors, growth
factors)

What Is A Gene?
Region of nucleic acid that contains the
base sequence information to encode a
protein (or RNA)
In eukaryotic cells coding regions are
often interrupted with non-coding
regions that have to be removed in
mRNA
Called intervening sequences or introns

Genes are preceded by a promoter


Starting signal for the synthesis of an mRNA

Recombinant Gene
Expression

Recombinant Gene Expression


Over-expression for purification
Bacterial expression (E. coli)
Insect cell expression (baculovirus)
Mammalian cell expression (vaccinia virus)
Generally done in cultured cells

Expression in a cell to exert an effect


Tailored to the cell system being studied
Can be in cultured cells or in vivo

Applications of rDNA
Research tools
Protein production
Transient expression studies
Viral replicons
Stable cell lines
Transgenic animals
Knock-down/knock-out animals

Gene therapy
Transgenic animals for protein
production

Applications of rDNA
Pharmaceutical compound screening
Biochemical assays
Cell-based assays

Vaccine production
Sub-unit vaccines
Virus-like particles

Transgenic plants
Crop improvement
Disease/pest-resistant plants

What is cDNA?
cDNA: a DNA copy of an mRNA
containing the protein coding domain of
a gene
Generally do not use genomic DNA
clones for recombinant gene expression
due to size and splicing requirements
mRNA (+)
5

AAAA 3
5
3

mRNA (+)

AAAA 3
5
(-) strand DNA
5
3

ds DNA
3
5

Expression Cassettes
Minimum requirements for constructing
a recombinant gene for expression:
Promoter
cDNA (transgene)
Termination signal (poly A site)

Expression cassette

Escherichia coli K-12


E. coli is a normal resident of the intestinal
tract
The K-12 isolate

Ineffective at colonizing the human gut


Much rDNA work with K-12 considered lower risk
Many common laboratory strains derived from K12

Useful tool in molecular biology

Easy to propagate in artificial media


Short generation time
Can easily generate large numbers of cells

Plasmid DNA
Small (~2-20 kbp) circular DNA
molecules
Replicate in bacterial cells independently
of the host cell chromosome
Carry genes which render host resistant to
antibiotics
Some plasmids can be swapped among
different species of bacteria (Broad host
range)

Are exploited for use as cloning and


gene expression vectors

How Do We Exploit Plasmids?


Restriction enzymes
Recognize and cut DNA at specific
sequences

Pieces of cut DNA can be mixed and resealed to form new plasmids.
Large amounts of new plasmids can be
made in E. coli.

Why is E. coli So Important?


Transcriptional regulation is wellunderstood
Potential for very high expression levels
Excellent plasmid DNA factory
Easy to introduce plasmid DNA into cells
Easily cultured to high cell densities
Plasmid DNA is easily isolated from cells

Elements of a Basic
Expression Vector
Promoters are tailored
to the type of cell:
bacteria
Multiple cloning sites
yeast
mammalian
plant
Gene of interest
insect
Promoter

Needed for
growth in E.
coli

Plasmid
origin of
replication

Termination signals are


tailored to prokaryotes or
eukaryotes:
eukaryotic cells require a poly
A signal
Termination signal

Antibiotic
resistance gene
for selection

Gene Delivery Technologies for


Eukaryotic Cells
Chemical transfection
Calcium phosphate
Liposomes
Electroporation
Micro-injection
Ballistic barrage
Virus-like particles
(VLP)
Bacteria/bacteriophage
Viral-mediated

Chemical
methods
Physical
methods
Biological
methods

Transient Gene Expression


Plasmids transfected into cells express
proteins for a short time (1 to 3 days)
If there are no elements on a plasmid to
allow DNA to be maintained or
replicated, the input DNA is degraded
and diluted out of the culture by cell
division
When a plasmid is transfected into cells
for short-term expression, it is referred
to as transient gene expression

Stable Expression
Long-term expression requires
maintenance of input DNA
Cassette that expresses a protein that
inactivates a toxic drug (dominant
selectable marker)
Enables cells to survive in presence of
drug
Allows selection of rare cells that have
recombined plasmid DNA into their
genome

These are referred to as stable cell lines or


stably transfected cells

Expression Vector for Stable


Expression
Transgene

The neomycin
phosphotransferase
gene inactivates a
toxic drug (G418 or
geneticin)

Plasma DNA Delivery


Advantages

Limitations

Few limitations on
DNA size
Non-infectious
Chemically defined
Lack of immune
response

Inefficient delivery
Low expression
levels
Short term
expression
Toxicity
Different methods for
different cells