BIOTEK

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BIOTEK
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• BIOTECHNOLOGY technologies that involve
• the use of living organisms or products from
• living organisms in order to benefit humans
• usually for medical, agricultural and industrial
• application

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• Science based technologies, such as
• recombinant DNA techniques, biochemistry,
• molecular and cellular biology, microbiology,
• and genetics

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Impact on society

• Biotechnology has made it possible to detect,
• and in some cases treat, diseases such as
• sickle-cell anemia, and cystic fibrosis
• Genetic tests used to track down criminals in
• assault cases based on the uniqueness of their
• DNA
• Genetic counselling provides advice on
• heritable diseases, and genetic screening of
• workers in possible risk industries

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• Concerns about biotechnology and genetic
• engineering
• Novel DNA
• Antibiotic resistance genes
• New proteins
• Escape of genes in the environment
• Reduction in biodiversity

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• Many countries are actively reviewing the
• safety and ethics of biotechnology research
• and its applications
• Some countries have established research
• guidelines, biosafety framework
• Ban work on stem cell research

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Biotech and health
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PCR RFLP (Restriction Fragment Length
Polymorphisms)
A combination of PCR restriction method to
detect SNP (single nucleotide polymorphsim)
The sample is first run in a restriction digest
to cut the DNA, then gel electrophoresis is
performed on this digest. In the case of MTHFR
C677T polymorphism, single band of 198 bp denotes
CC genotype, two bands of 198 and 175 bp denote
CT genotype and single band of 175 bp denotes TT
genotype.
After gel electrophoresis, DNA can be visualized
by staining with ethidium bromide, an
intercalating agent and fluorescent dye.
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PCR amplification of MTHFR exon 4
Enzyme digestion (HinfI)
CC (wild type)
G A G C C
198 bp
Ala
175 bp
23 bp
Val
Gel Electrophoresis
M CC CT TT
198 bp
175 bp
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PCR-RFLP untuk polimorfisme G135A gena RET
PCR amplification of RET exon 2
Enzyme digestion (EagI)
294 bp
207 bp
87 bp
Gel Electrophoresis
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Vietnamese SMA Patients
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C
C-
Mk
SMN1 Exon 7
A
SMN2 Exon 7
SMN2 Exon 8
B
SMN2 Exon 8
SMN2 Exon 8
NAIP Exon 5
C
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Insulin
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Cloning organism
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Human Gene Therapy

• repair the damage caused by a genetic deficiency
  through introduction of a functional version of
  the defective gene.
• a cloned variant of the gene must be incorporated
  into the organism in such a manner that it is
  expressed only at the proper time and only in
  appropriate cell types.

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• A basic strategy in human gene therapy involves
  incorporation of a functional gene into target
  cells.
• The gene is typically in the form of an
  expression cassette consisting of a cDNA version
  of the gene downstream from a promoter that
  drives expression of the gene.

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• A vector carrying such an expression cassette is
  introduced into target cells, either
• ex vivo via gene transfer into cultured cells in
  the laboratory and administration of the modified
  cells to the patient
• or in vivo via direct incorporation of the gene
  into the cells of the patient.

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• A replication-deficient version of Maloney murine
  leukemia virus can serve as a vector for
  expression cassettes up to 9 kb in size.
• Recombinant, replication-deficient adenoviruses
  enter target cells via specific receptors on the
  target cell surface the transferred genetic
  information is expressed directly from the
  adenovirus recombinant DNA and is never
  incorporated into the host cell genome.

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Retrovirus-mediated gene delivery ex vivo
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• Retroviruses are RNA viruses that replicate
  their RNA genome by first making a DNA
  intermediate.
• The Maloney murine leukemia virus (MMLV) is the
  retrovirus used in human gene therapy.
• Deletion of the essential genes gag, pol, and env
  from MMLV makes it replication-deficient (so it
  cant reproduce)

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• (a) creates a space for insertion of an
  expression cassette
• (b) The modified MMLV acts as a vector for the
  expression cassette although replication-defectiv
  e, it is still infectious. Infection of a
  packaging cell line that carries intact gag, pol,
  and env genes allows the modified MMLV to
  reproduce
• (c), and the packaged retroviral viruses can be
  collected and used to infect a patient (d).
• In the cytosol of the patients cells, a DNA copy
  of the viral RNA is synthesized by viral reverse
  transcriptase, which accompanies the viral RNA
  into the cells. This DNA is then randomly
  integrated into the host cell genome, where its
  expression leads to production of the expression
  cassette product.

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Adenovirus-mediated gene delivery in vivo.
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• Adenoviruses are DNA viruses.
• The adenovirus genome (36 kb) is divided into
  early genes (E1 through E4) and late genes (L1 to
  L5)
• (a) Adenovirus vectors are generated by deleting
  gene E1 (and sometimes E3 if more space for an
  expression cassette is needed)
• (b)deletion of E1 renders the adenovirus
  incapable of replication unless introduced into a
  complementing cell line carrying the E1 gene
• (c)Adenovirus progeny from the complementing cell
  line can be used to infect a patient. In the
  patient, the adenovirus vector with its
  expression cassette enters the cells via specific
  receptors
• (d) Its linear dsDNA ultimately gains access to
  the cell nucleus, where it functions extra
  chromosomally and expresses the product of the
  expression cassette (e).

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DNA fingerprinting

• DNA fingerprinting is used to link small amounts
  of biological material
• human genome contains non-coding repetitive DNA
  sequences, the length of which varies from
  individual to individual.
• Short tandem repeats (STRs) thus exist in which
  dinucleotides (e. g., -T-X-) are frequently
  repeated.
• Each STR can occur in five to 15 different
  lengths (alleles), of which one individual
  possesses only one or two.

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Biotechnology in agriculture
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• Reasons for developing transgenic plants
• To improve agricultural, horticultural and
  ornamental value of a crop plant
• Can act as a living bioreactor
• Means of studying the action of genes

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Crops and traits

• Canola Herbicide tolerance modified seed fatty
  acid content
• Maize Herbicide tolerance resistance to corn
  root worm resistance to European corn borer
• Melon Delayed ripening
• Papaya Resistance to viral infection
• Potato Resistance to Colorado potato beetle
  resistance to potato leafroll luteovirus
• Rice Herbicide tolerance Beta-carotene
• Soya bean Herbicide tolerance modified seed
  fatty acid content
• Squash Resistance to viral infection
• Tomato Delayed ripening delayed softening

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Delayed ripening in Tomato

• Introduction of a gene that results in
  degradation of a precursor of the plant hormone,
  ethylene
• Production of tomatoes for human consumption,
  either fresh or processed
• Agritope Inc. USA

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Resistance to Colorado potato beetle

• Introduction of a toxin gene from a bacteria that
  results in insect resistance in potato
• Production of potatoes for human consumption and
  livestock feed including potato process residue
• Monsanto Company, USA

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Herbicide tolerance Corn

• Glyphosate herbicide Tolerance
• Production of Z. mays for human consumption (wet
  mill or dry mill or seed oil), and meal and
  silage for livestock feed
• Monsanto Company, USA

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Papaya Resistant to viral infection

• Resistance to papaya ringspot virus (PRSV)
• Production of papaya for human consumption,
  either fresh or processed
• Cornell University, USA UWI, Jamaica

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Virus resistant tomato

• Insertion of a mutant gene from the virus to
  prevent replication
• Production of tomatoes for human consumption,
  either fresh or processed
• UW-Madison, Hebrew Univ., UWI

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Benefits of the New Technology

• Higher yields lower pesticide usage
• Provide indirect benefits for consumers and the
  environment through lower usage of pesticides and
  there are higher yields due to reduced pest
  losses.

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• More Nutritious Foods
• increasing the levels of essential amino acids,
  vitamins, bio-available iron and to reduce
  toxicity
• more nutritious harvested products that keep
  much longer in storage and transport.

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• Utilization of marginal lands
• produce plants that are more tolerant to
  drought, salt and heat stresses, toxic heavy
  metals

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Problems with rDNA technology

• Instability in transgene expression
• Interruption or silencing of existing genes
• Activation of silent genes
• Expression of anti-nutrients

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Unanticipated effects in transgenic crops

• Canola overexpression of phytoene synthase
  resulted in X500 increase in levels of a and
  b-carotene
• Maize the stems of Bt maize contain more lignin
• Potato expressing kanamycin showed changes in
  phynotypic and yield performance

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Concerns

• Food safety
• Allergic reaction Cry9C protein in Starlink
  corn
• Use of antibiotic marker genes
• Negative impact non-target species Bt corn
• Creation of super weeds - gene flow
• Creation of new viruses - recombination

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Food safety

• Unsafe for human consumption ?
• Allergic reaction to new proteins
• 60 of processes foods in supermarkets in the
  USA contain a GM ingredient
• Soy, corn, canola and some fresh vegetables

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Allergic reaction

• Cry9C a protein in Starlink corn
• Insecticidal protein shares properties with
  proteins that are known food allergen
• Not licensed for human consumption
• Taco Bell Home originals

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Antibiotic marker genes

• Antibiotics kanamycin, hygromycin,
  tetracycline, ampicillin
• Used to identify plants carrying the transgene
• Presence of the gene in the gut could enter gut
  organism, increase resistance
• WHO report that antibiotic genes are safe

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Impact on non-target species

• Pollen from GM corn contains the insecticidal Bt
  toxin thought to be a danger to the monarch
  butterfly
• Milkweed with pollen from Bt corn plants could
  kill monarch catapillars that fed on them
• Six recent studies finds that pollen from
  varieties of Bt corn most commonly grown inthe
  USA do not contain enough toxin to harm monarchs.

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Super weeds

• Herbicide resistant GM crops
• Insect resistant GM crops
• Gene flow between trangenics and traditional
  maize in Mexico
• Gene flow - natural process

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New viruses

• Recombination through horizontal gene transfer
• Naturally occurring viruses and spread to new
  hosts

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Regulating Biotechnology

• Cartagena Protocol
• CODEX Alimentarius
• National Biosafety Committee-NCST

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Cartagena Protocol

• Trade related treaty
• Regulation on the transboundary movement of
  living modified organisms
• Risk assessment system based on science intended
  to assure the safety of products to be imported

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CODEX Alimentarius

• Human health risk analysis of GM foods
• Case-by-case assessment
• Evaluate direct and unintentional effects
• Toxicity
• Allergenicity
• Changes in nutritional properties
• Gene stability

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NBC - NCST

• Permission to import GMOs
• Risk assessment
• Environmental impact assessment
• Monitor imports and uses of transgenic crops

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European Union vs. USA

• EU ban the importation of GM foods for sale
• Strong support by consumers
• Call for labeling of products derived from GMO
• Label if there is gt1 GM material in the foods