Staff Site Universitas Negeri Yogyakarta

(1)

(2)

• _{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

(3)

Science based technologies, such as recombinant DNA techniques,

biochemistry,

molecular and cellular biology, microbiology,

(4)

(5)

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}

(6)

Concerns about biotechnology and genetic

engineering – Novel DNA

– Antibiotic resistance genes – New proteins

(7)

• _{Many countries are actively reviewing the}

safety and ethics of biotechnology research

and its applications

• _{Some countries have established}

research

guidelines, biosafety framework

(8)

(9)

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

(10)

PCR amplification of MTHFR exon 4

Enzyme digestion (HinfI)

(11)

PCR amplification of RET exon 2

Gel Electrophoresis Enzyme digestion (EagI)

294 bp

(12)

SMN2 Exon 8 SMN2 Exon 8

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

B

C

A SMN1 Exon 7

Vietnamese SMA Patients

NAIP Exon 5 SMN2 Exon 7

(13)

(14)

(15)

(16)

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

(17)

• _{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

(18)

• _{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}

(19)

• _{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

(20)

(21)

• _{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

(22)

• _{(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

(23)

(24)

• 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

(25)

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}

(26)

(27)

Reasons for developing transgenic plants

– To improve agricultural, horticultural and ornamental value of a crop plant – Can act as a living bioreactor

(28)

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

(29)

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

(30)

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

(31)

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

(32)

Papaya Resistant to viral

infection

• _{Resistance to papaya}

ringspot virus (PRSV)

• _{Production of papaya}

for human

consumption, either fresh or processed

• _{Cornell University,}

(33)

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,

(34)

Benefits of the New

Technology

• _{Higher yields & lower pesticide usage}

– Provide indirect benefits for

(35)

• _{More Nutritious Foods}

– increasing the levels of essential

amino acids, vitamins, bio-available iron and to reduce toxicity

– more nutritious harvested products

(36)

• _{Utilization of marginal lands}

– produce plants that are more tolerant to drought, salt and heat stresses,

(37)

Problems with rDNA

technology

• _{Instability in transgene expression} • _{Interruption or silencing of existing}

genes

• _{Activation of silent genes}

(38)

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}

(39)

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}

(40)

Food safety

• _{Unsafe for human consumption ?}

– Allergic reaction to new proteins – 60% of processes foods in

supermarkets in the USA contain a GM ingredient

(41)

Allergic reaction

• _{Cry9C a protein in Starlink corn}

• _{Insecticidal protein shares properties}

with proteins that are known food allergen

(42)

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}

(43)

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

(44)

Super weeds

• _{Herbicide resistant GM crops} • _{Insect resistant GM crops}

• _{Gene flow between trangenics and}

traditional maize in Mexico

(45)

New viruses

• _{Recombination through horizontal}

gene transfer

• _{Naturally occurring viruses and}

(46)

Regulating Biotechnology

• _{Cartagena Protocol} • _{CODEX Alimentarius}

(47)

Cartagena Protocol

• _{Trade related treaty}

• _{Regulation on the transboundary}

movement of living modified organisms

• _{Risk assessment system based on}

(48)

CODEX Alimentarius

• _{Human health risk analysis of GM foods} • _{Case-by-case assessment}

• _{Evaluate direct and unintentional} effects

• _Toxicity

• _{Allergenicity}

(49)

NBC - NCST

• _{Permission to import GMOs} • _{Risk assessment}

• _{Environmental impact assessment} • _{Monitor imports and uses of}

(50)

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}