BIOTEK

• BIOTECHNOLOGY – technologies that involvethe use of living organisms or products fromliving organisms in order to benefit humansusually for medical, agricultural and industrialapplication

Science based technologies, such asrecombinant DNA techniques, biochemistry,molecular and cellular biology, microbiology,and genetics

Impact on society

• Biotechnology has made it possible to detect,and in some cases treat, diseases such assickle-cell anemia, and cystic fibrosis• Genetic tests used to track down criminals inassault cases based on the uniqueness of theirDNA• Genetic counselling provides advice onheritable diseases, and genetic screening ofworkers in possible risk industries

Concerns about biotechnology and geneticengineering– Novel DNA– Antibiotic resistance genes– New proteins– Escape of genes in the environment– Reduction in biodiversity

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

Biotech and health

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.

PCR amplification of MTHFR exon 4

M M 　　 CCCC 　 　 CTCT TTTT

198 bp198 bp

175 bp175 bp

Gel Electrophoresis

Enzyme digestion (HinfI)

G A G C CCC (wild type)

Ala198 bp198 bp

TT (mutant) G A G T CVal

~175 bp~175 bp~23 bp~23 bp

G　A　N　T　CC　T　N　A　G

PCR amplification of RET exon 2

Gel Electrophoresis

Enzyme digestion (EagI)

294 bp294 bp

~207 bp~207 bp~87 bp~87 bp

C　G　G　C　C　GG　C　C　G　G　C

PCR-RFLP untuk polimorfisme G135A gena RET

SMN2 Exon 8

SMN2 Exon 8

3 5 7 8 9 11 19 20 21 22 23 24 C+ C-Mk

B

C

ASMN1 Exon 7

Vietnamese SMA Patients

NAIP Exon 5

SMN2 Exon 7

SMN2 Exon 8

Insulin

Cloning organism

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.

• 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.

• 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.

• 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.

Retrovirus-mediated gene delivery ex vivo

• 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 can’t reproduce)

• (a) creates a space for insertion of an expression cassette • (b) The modified MMLV acts as a vector for the expression

cassette; although replication-defective, 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 patient’s 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.

Adenovirus-mediated gene delivery in vivo.

• 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).

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.

Biotechnology in agriculture

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

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

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

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

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

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

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

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.

• 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.

• Utilization of marginal lands– produce plants that are more tolerant to

drought, salt and heat stresses, toxic heavy metals

Problems with rDNA technology

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

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

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

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

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”

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

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.

Super weeds

• Herbicide resistant GM crops• Insect resistant GM crops• Gene flow between trangenics and traditional

maize in Mexico• Gene flow - natural process

New viruses

• Recombination through horizontal gene transfer

• Naturally occurring viruses and spread to new hosts

Regulating Biotechnology

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

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

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

NBC - NCST

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

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 >1% GM material in the foods