2. INTRODUCTION
BIO-TECHNOLOGY
⢠Bio means living organisms
⢠Technology is the use of various techniques, instruments,
processes to develop new products for human benefits
⢠Biotechnology refers to the use of living organism i.e. plants,
animals, microorganisms or their parts in developing useful
products utilizing various techniques.
⢠It may also be any technological application that uses
biological systems, living organisms or derivatives thereof, to
make or modify products or processes for specific use
3. PHARMACEUTICAL BIOTECHNOLOGY
⢠Pharmaceutical refers to the science of preparing drug delivery
systems (dosage forms) of chemical, therapeutic, diagnostic or
biological substance. It involves all the processes of developing,
analyzing, stabilizing and marketing of prepared DDSs
(pharmaceuticals).
⢠Pharmaceutical biotechnology can be the use of biological
systems (e.g. cells or tissues) or biological molecules (e.g.
enzymes or antibodies) for/in the manufacture of pharmaceutical
products.
⢠Or it involves all technologies needed to produce
biopharmaceuticals (other than (non-genetically modified)
animal- or human blood-derived medicines).
4. HISTORY OF BIOTECHNOLOGY
⢠Wine from grapes was process of fermentation by yeast.
⢠It is developed from ages without any understanding of
underlying biotechnological aspects.
⢠Only a little description was first demonstrated in 1870 by
Louis Pasteur.
⢠Chemical conversions in living cells are performed by
chemical precursors (enzymes) and according to him these
traditional cell processes should be consider biochemical
conversions.
5. CONT.âŚ
⢠Decades after Pasteur's discovery, now the catalytic role of
enzymes for most biochemical conversions became apparent.
⢠Based on that enzymes became tools for the control and
optimization of the traditional processes by process of catalysis.
⢠The concept, arise in the molecular biology in around 1950, that
DNA encodes proteins and in this way controls all cellular
processes, becomes the basis of new era in biotechnology.
⢠The development of the recombinant DNA technology in 1970âs,
allowed biotechnologists to control gene expression in the
organisms used for biotechnological manufacturing that allowed
new ways for the introduction of foreign DNA into all kinds of
organisms. So genetically modified organisms were constructed.
6. TABLE: PHARMACEUTICALS TRADITIONALLY OBTAINED BY
DIRECT EXTRACTION FROM BIOLOGICAL SOURCE MATERIAL.
MANY OF THE PROTEIN-BASED PHARMACEUTICALS MENTIONED ARE NOW
PRODUCED BY GENETIC ENGINEERING
Substance Medical application
Blood products (e.g. coagulation factors) Treatment of blood disorders such as haemophilia A or B
Vaccines Vaccination against various diseases
Antibodies Passive immunization against various diseases
Insulin Treatment of diabetes mellitus
Enzymes Thrombolytic agents, digestive aids, debriding agents
(i.e. cleansing of wounds)
Antibiotics Treatment against various infections agents
Plant extracts (e.g. alkaloids) Various, including pain relief
7. TYPES OF PHARMACEUTICAL
BIOTECHNOLOGY PRODUCTS
⢠Various terms are now used to describe these products such as
âbiologicâ, âbiopharmaceuticalâ and âproducts of pharmaceutical
biotechnologyâ or âbiotechnology medicinesâ.
⢠âBiologicâ refers to any pharmaceutical product produced by
biotechnological manufacturer, âbiologicâ generally refers to
medicinal products derived from blood, as well as vaccines,
toxins and allergen products.
8. TYPES OF PHARMACEUTICAL
BIOTECHNOLOGY PRODUCTS
⢠âBiopharmaceuticalâ describe a class of therapeutic proteins
produced by modern biotechnological techniques, specifically
via genetic engineering or, in the case of monoclonal antibodies,
by hybridoma technology.
⢠Although the majority of biopharmaceuticals or biotechnology
products now approved or in development are proteins
produced via genetic engineering, these terms now also
encompass nucleic-acid-based, i.e. deoxyribonucleic acid
(DNA)- or ribonucleic acid (RNA)-based products, and whole-
cell-based products.
9. GENE EXPRESSION
⢠Nucleic Acid: Either of the two acids (DNA/RNA) present in the
nucleus and in some cases in the cytoplasm of all living cells. Main
function in heredity and protein synthesis
⢠Nucleotide: A nucleotide consists of a nitrogenous base (purine double
ringed, adenine & guanine or pyrimidine single ringed cytosine,
thymine & uracil) plus a molecule of sugar and one of phosphoric acid.
⢠A nucleotide is the basic building block of nucleic acids. RNA and DNA
are polymers made of long chains of nucleotides.
⢠Gene: A gene is the basic physical and functional unit of heredity.
Genes are made up of DNA. Genes carry instructions to make proteins.
However, many genes do not code for proteins. In humans, genes vary
in size from a few hundred DNA bases to more than 2 million bases.
10.
11. ⢠Genetic information, chemically determined by DNA structure, that is
transferred during cell division to daughter cells by (DNA replication)
⢠There are two process of replication (RNA polymerases):
⢠Transcription expressed by conversion of DNA into mRNA
⢠It involves use of enzyme transcriptase.
⢠Triplet of nucleotides code for mRNA (specify a particular amino acid).
This copying is known as Codon.
⢠Translation followed by conversion of mRNA into protein.
⢠In eukaryotes, mature mRNA molecules must leave the nucleus and
travel to the cytoplasm, where the ribosomes are located. On the other
hand, in prokaryotic organisms, ribosomes can attach to mRNA while it
is still being transcribed. In this situation, translation begins at the 5'
end of the mRNA while the 3' end is still attached to DNA
12. PROTEIN SYNTHESIS
⢠Ribosome is composed of two subunits: the large (50S) subunit and the
small (30S) subunit
⢠Each subunit exists separately in the cytoplasm, but the two join together on
the mRNA molecule.
⢠The ribosomal subunits contain proteins and specialized RNA molecules
specifically, ribosomal RNA (rRNA) and transfer RNA (tRNA). The tRNA
molecules are adaptor molecules, they have one end that can read the
triplet code in the mRNA through complementary base-pairing, and another
end that attaches to a specific amino acid.
⢠The mRNA and aminoacyl-tRNA complexes are held together closely, which
facilitates base-pairing. The rRNA catalyzes the attachment of each new
amino acid to the growing chain.
13. During transcription, the enzyme RNA polymerase (green) uses DNA as a template to
produce a pre-mRNA transcript (pink). The pre-mRNA is processed to form a mature
mRNA molecule that can be translated to build the protein molecule (polypeptide)
encoded by the original gene.
14.
15. GENE SPLICING
⢠Intron:
⢠A segment of a gene situated between exons that is removed by
spliceosome before translation of mRNA and does not function in coding
of protein synthesis. They have to be removed because they facilitate
genetic shuffling or rearrangement of portion of genes which encodes
various unit of function, thus creating new genes with new combination of
properties.
⢠Exons:
⢠Segment of a gene that is involved in coding of protein synthesis
16.
17.
18.
19. GENETIC ENGINEERING
⢠The deliberate modification of an organismâs genetic information
by directly changing its nucleic acid genome is called Genetic
Engineering or Recombinant DNA technology or gene cloning.
⢠Transgenic Organisms
⢠If genetic material from another species is added to the host, the
resulting organism is called Transgenic Organism.
⢠Cisgenic Organisms
⢠If genetic material from same species or specie that can naturally
breed with the host is used, the resulting organism is called
Cisgenic Organism.
20. TOOLS OF GENETIC ENGINEERING
⢠These are three tools used to carry process of recombinant
DNA (rDNA) technology.
⢠1. Enzymes
⢠2. Vectors
⢠3. cDNA Clone
21. 1. ENZYMES 2. VECTORS
⢠Four enzymes are used for
preparing recombinant DNA
1. Endonucleases (Restriction
Endonucleases)
2. DNA Ligases
3. Reverse Transcriptases
4. Alkaline Phosphatases
⢠Vectors are DNA molecules which
carry a foreign DNA fragment when
inserted into them.
⢠There are 4 major types of vectors:
1. Plasmids
2. Bacteriophages and other viruses
3. Cosmids
4. Artificial Chromosomes
22. RESTRICTION ENDONUCLEASES
⢠They can cut DNA at specific locations (can cleave a specific
DNA sequence).
⢠They cleave phosphodiester bond in DNA. DNA can be marked
specifically at particular site by a characteristic pattern of
methylated or glucosylated nucleotides.
⢠It produces the DNA with staggered single stranded ends. Those
ends that are staggered act as cohesive or sticky ends during
recombination, making them suitable for splicing with segments
of foreign source DNA that has been excised using the same
endonucleases.
23. ⢠Type I Restriction Endonucleases
⢠It cleaves DNA at a random distance from the recognition site in the DNA
nucleotide sequence.
⢠Type II Restriction Endonucleases
⢠It cleaves the DNA at defined position close or within its recognition site.
⢠Type III Restriction Endonucleases
⢠Cleaves outside its recognition sequence and require two sequences in
opposite orientation within same DNA
⢠Type IV Restriction Endonucleases
⢠Cleaves modified (e.g. methylated) DNA
24.
25. ⢠DNA Ligase: It can attach segments of double stranded DNA. Catalyzes
the formation of phosphodiester bond.
⢠Reverse Transcriptase: It synthesizes cDNA from mRNA.
⢠Alkaline Phosphatase: This enzyme digests 5-phosphoryl group on DNA.
⢠If plasmid is used as a vector for foreign DNA, a major problem arises when
the plasmid DNA is treated with restriction enzyme, the cohesive end of the
cut plasmid instead of joining with the foreign DNA, joined together to form a
circular plasmid again.
⢠To overcome this problem, the cut plasmid is treated with alkaline
phosphatase enzymes.
⢠The foreign DNA fragment is not treated with this enzyme and hence 5â end
of foreign DNA join the 3 end of plasmid.
⢠So alkaline phosphatase help in producing recombinant DNA molecules.
26. VECTORS
⢠Plasmids: Plasmids are small extra chromosomal molecules of DNA that
replicate independently of the bacterial chromosomes
⢠They are normally covalently closed, circular, super coiled molecules.
⢠Plasmids do not normally contain the genetic information for the essential
metabolic activities of microorganism, but they generally contain genetic
information for specialized features, such as resistance to heavy metals and
antibiotics.
⢠Plasmids are excellent vectors for small DNA fragment and hence are
important tool for genetic engineering.
⢠Plasmid pBR322 which was created specifically for use in cloning DNA is a
frequently used cloning vector that can replicate in E.Coli.
⢠Plasmid contain roughly thousand or hundred of thousand base pairs
27.
28.
29. PHAGE VIRUS
⢠All phages contain nucleic acid (DNA or RNA) present in head structure
or capsid.
⢠The head act as a protective covering for nucleic acid.
⢠The tail is attached to the head. The tail is a hollow tube through which
nucleic acid passes during infection.
⢠The bacteriophages attach to specific chemical sites on the surface of
bacterium and the viral nucleic acid penetrates the host cell.
⢠Once the viral nucleic acid enters the bacterium, bacterial metabolism is
stopped by the destruction of the host Genome.
⢠First viral DNA and then viral coat protein appears, which then assembles
and mature particles are produced (Lytic Cycle).
30.
31. COSMIDS
⢠Cosmids (hybrid DNA) are plasmids that contain lambda phage cos sites and
can be packed into phage capsids. Cosmid is not a naturally found in living
cells. It is constructed vector.
⢠The lambda genome contains a recognition sequence called a cos site at
each end.
⢠When the genome is to be packed in a capsid it is cleaved at one cos site
and the linear DNA is inserted into capsid until the second cos site has
entered.
⢠Thus any DNA inserted between the cos site is packed.
⢠Cosmids contain several restriction sites and antibiotic resistance genes.
⢠They are packed in lambda capsids for efficient injection into bacteria, but
they can also exist as plasmids within bacterial hosts E.g. Col E1 cosmid is a
typical cosmid used in genetic engineering
32. ⢠Lambda is a temperate bacteriophage with a genome size of about 48.5 kilo
base pairs. It has a specific susceptibility for E coli. Its entire DNA sequence
is known. The term temperate means it enjoys one of two life cycles. Lytic
cycle replicate many times, produces more phage. It can also take up
lysogenic growth meaning that it migrates its DNA into bacterial cell.
⢠Lambda genome exists as a linear double stranded molecule with a single
stranded complementary ends.
⢠The points at which the segments of bacteriophage lambda are cut are
called as Cos (Cohesive sites).
⢠The Cos sites are the only part of lambda that is included in the plasmid of a
cosmid vector. No viral gene is included in the vector when the plasmid is
introduced to E-coli.
33.
34. ⢠CHARACTERISTIC FEATURES:
⢠It is a circular double stranded DNA.
⢠It contains a complementary single strand regions, these regions are abbreviated
as Cos site.
⢠The cosmids DNA dose not participate in the multiplication of phage particles.
⢠Advantages:
⢠The cosmids transfer somewhat larger foreign gene into the bacterial cell.
⢠The cosmids pick up even long size genes. Hence it is used for the entire
genome of organisms.
⢠Loading capacity of cosmid is usually 40,000-45,000 base pairs.
⢠Disadvantages:
⢠Each cosmid requires 2 Cos sites for the successful packing of recombinant
cosmids.
⢠The packing fails when the distance of separation exceeds 54000 base pairs or
when it is less than 38,000 base pairs.
35. ARTIFICIAL CHROMOSOMES
⢠Artificial chromosomes are DNA molecules or fragment
assembled in vitro from defined genetic base pairs, which
guarantee stable maintenance of large DNA fragments with the
properties of natural chromosomes.
⢠The mapping and analysis of complex genomes require vectors
that allow cloning and work with large DNA fragments. Lambda-
based vectors have many advantages but a comparatively small
size of inserts (maximum 48â49 kb) complicates the use of these
vectors for the mapping of complex genomes.
⢠ACs can carry human DNA fragments as large as 1Mb
⢠Types: BAC, YAC, PAC, HAC
36. YEAST ARTIFICIAL CHROMOSOMES
⢠Yeast artificial chromosomes (YACs) are genetically engineered
chromosomes derived from the DNA of the yeast, Saccharomyces
cerevisiae.
⢠Yeast artificial chromosome (YAC) vectors allow the cloning, within yeast
cells, of fragments of foreign genomic DNA that can approach 500kbp in
size.
⢠The YAC was devised and first reported in 1987 by David Burke.
⢠YAC is built using an initial circular DNA plasmid, which is typically cut into
a linear DNA molecule using restriction enzymes; DNA ligase is then used
to ligate a DNA sequence or gene of interest into the linearized DNA,
forming a single large, circular piece of DNA.
39. HUMAN ARTIFICIAL CHROMOSOME
⢠A human artificial chromosome (HAC) is a micro-chromosome that can act
as new chromosome in a population of human cells.
⢠That is, instead of 46 chromosomes, the cell could have 47 with the 47th
being very small, roughly 6-10 mega bases (Mb) in size instead of 50-250
Mb for natural chromosomes, and able to carry new genes introduced by
human researchers.
⢠Ideally, researchers could integrate different genes that perform a variety of
functions, including disease defense.
⢠These chromosomes are derived from endogenous chromosomes.
⢠Generated by natural fragmentation of chromosomes, telomere-directed
chromosome breakage, or radiation-induced chromosome breakage,
containing an endogenous functional centromere.
⢠The chromosomes can then be transferred into other cell lines by
microcell-mediated chromosome transfer (MMCT).
40. PROCESS OF GENETIC ENGINEERING
⢠Process of genetic engineering consists of following steps
1. Identification and isolation of the gene of interest
2. Selection of appropriate vector (micro-organism)
3. Cloning the gene of interest
4. Amplifying the gene to produce many copies (PCR)
5. Transformation of Plasmid into micro-organism
6. Identification of micro-organisms containing plasmid
7. Multiplying the plasmid in bacteria and recovering the cloned construct
8. Transfer of the construct into the recipient tissue
9. Expression of gene in recipient genome
41. IDENTIFICATION, ISOLATION OF GENE OF INTEREST
⢠The gene of interest is isolated from the host tissues
⢠Usually micro-organisms canât process human genes due to the process gene splicing or
alternative gene splicing
⢠So cDNA is constructed from the mRNA (reverse transcriptase) utilized to form desired
protein (e.g. Insulin)
⢠cDNA can be isolated from tissue DNA by process of Gel Electrophoresis
⢠Gel electrophoresis is a technique used to separate DNA fragments according to their size
⢠Gel Electrophoresis consist of gel (2% agarose gel or 20% polyacrylamide gel) loaded in a
container indentations (wells) with DNA at one end of gel and an electric current is applied to
pull them through the gel
⢠DNA fragments are negatively charged, so they move towards the positive electrode.
Because all DNA fragments have the same amount of charge per mass, small fragments
move through the gel faster than large ones
⢠When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands,
each representing a group of same-sized DNA fragments (Ethidium Bromide)
43. SELECTION OF VECTOR (MICRO-ORGANISM)
⢠The cDNA to be cloned is to be linked to a vector DNA, that serves as a
vehicle for carrying foreign DNA into a suitable host cell, such as the
bacterium Escherichia coli (short replication time 20 min).
⢠The vector plasmid is removed by centrifugation of E.coli in density gradient
cesium chloride solution. Plasmid remains in upper layers and DNA goes to
bottom layers. In order to clone cDNAs within bacterial hosts two types of
vectors are common, namely
⢠(a) The DNA segment to be cloned in, introduced into the bacterial cell by
first joining it to a plasmid and secondly, causing the bacterial cells to take
up the plasmid from the medium, and
⢠(b) The DNA segment is joined to a portion of the genome of the bacterial
virus lambda (Îť) which is subsequently allowed to infect a culture of bacterial
cells. Thus, a huge quantum of viral progeny are produced, each of which
contains the foreign DNA segment (20-48kb).
44. CONSTRUCTION OF HYBRID DNA
⢠The cDNA and selected vector (e.g. plasmid of E. coli) are first
methylated at the specific points from where gene transfer is required
and then both treated with same restriction endonucleases type IV.
⢠Both DNAs will be converted to sticky ends containing cos single
strands.
⢠Plasmid (vector) strand is treated with Alkaline Phosphatase to digest 5â
phosphate group so that it canât rejoin to form vector again
⢠cDNA and vector are mixed and treated with DNA ligases to form
double stranded helix hybrid DNA (rDNA) by forming the phospho-di-
ester linkage with chain of plasmid.
45.
46. AMPLIFYING DNA: POLYMERASE CHAIN REACTION (PCR)
⢠Hybrid DNA constructed is now converted to multiple copies (1 billion)
using in-vitro amplification technique known as PCR.
⢠Oligonucleotide Primers (at least one complementary), DNA Polymerases,
and Nucleotide bases are added in this process.
⢠It involves following three steps to construct one million copies in just 20
cycles.
I. Denaturation of DNA Fragment:
⢠The target DNA containing sequence to be amplified is heat-denatured
(around 94°C for 15 sec to 60 sec) to separate its complementary strands,
this process is called melting of target DNA.
47. II. Annealing of Primers:
⢠Primers are added in excess and the temperature is lowered to about 50-68°C
for 60 sec., as a result the primers form the hydrogen bonds and anneal to the
DNA on both sides of the DNA sequence.
III. Primer Extension:
⢠Finally different nucleoside triphosphate (dATP, dCTP, dGTP, dTTP) and a
thermo-stable DNA polymerase (Taq polymerase from Thermus aquaticus
and Vent polymerase from Thermococcus litoralis) are added to the reaction
mixture, it helps in polymerization process of primers and, therefore, extends
the primers (at 72-74°C) resulting in synthesis of multiple copies of target
DNA sequence.
⢠After completion of all these steps in one cycle, again the second cycle is
repeated following the same process. If 20 such cycles occur, then about one
million copies of target DNA sequence are produced.
48.
49. TRANSFORMATION OF HYBRID DNA (PLASMID)
TO E.COLI
⢠The process of re-introduction of hybrid DNA into bacterium is called
transformation. The E.coli cells devoid of plasmid in second step is used.
⢠There are many renowned techniques used for transformation
1. Chemical method
⢠Chemical substances, such as polyethylene glycol (PEG), polyvinyl alcohol
(PVA), and calcium phosphate [Ca3 (PO4)2] predominantly increase the
uptake of DNA by plant cells as well as micro-organisms.
⢠Calcium chloride heat soaking method involves the instant (sudden) heating
(45°C) and the cooling to freezing (0°C) in one minute that result in pore
size extension of E.coli cellular membrane and allows a larger plasmid to be
uptake.
50. 2. ELECTROPORATION
⢠Introduction of rDNA into the cells by exposing them critically for
specific very short durations directly by the electrical pulses of
high-voltage field strength which perhaps induced transient pores
in the plasma lemmaâ.
⢠Low voltage long-pulsed method: 300-400 V cmâ 1 for 10-15 ms
⢠High voltage short-pulsed method: 1000-1500 V cmâ 1 for 10 Âľs
⢠low-voltage long-pulses technique give rise to relatively high rates
of transient transformation; whereas, high-voltage short-pulses
method produce usually high rates of stable transformation
⢠almost 50% protoplast viable survival may invariably give the
highest rates of stable transformation.
51. 3. PARTICLE GUN DELIVERY
⢠This method essentially made use of a 1-2 ¾m tungsten or gold
particles, precoated with the DNA.
⢠Adequately accelerated (specific device) to such a degree of
velocities that gainfully enable their entry right into either the
plant cells or the nuclei
⢠There are two most effective and equally successful devices
that usually aid in causing acceleration of the particles
⢠(a) utilization of pressurized helium gas, and
⢠(b) utilization of the released electrostatic energy by a droplet of
water after exposure to a very high voltage.
52. VERIFY MICROORGANISM CARRYING VECTOR
(PLASMID)
⢠In order to verify that our hybrid DNA is successfully transformed into
vector e.g. E.coli, microbiological assay technique can be used to verify
plasmid containing E.coli.
⢠Microbiological methods are based on the measurement and evaluation of
zones of inhibited bacterial growth or promoted growth due to resistance
⢠Prepare Muller Hilton agar media and sterilize using autoclave
⢠Introduce the inoculum of transformed E.coli in agar media and shake well
⢠Half fill already sterilized, cooled petri plates with medium and allow to cool,
harden
⢠Dig Micro-wells in media at appropriate distance and add tetracycline disks
in prepared wells (E.coli containing plasmid is resistant to tetracycline)
⢠Incubate for 24 to 48 h at 37â to check for zone of inhibition or promotion
53. EXPRESSION OF GENE
⢠The uptake and integration of a transgene does not guarantee
that the gene will express itself in the new genetic environment.
⢠The inoculum of E.coli is microinjected (transformed) in a
controlled condition to animal (mice) or any plant that do not
have that expressive gene i.e. Insulin
⢠The blood samples of animal collected and tested for antibodies
produced against insulin
⢠The presence of antibodies verify that the gene is expressive,
quality can be accessed by microtiter techniques and verifying
against antibody bank.
54. GENE SILENCING
⢠Interruption or suppression of the expression of a gene at
transcriptional or translational levels is called as gene silencing. As
the most of the emphasis of genetic engineering efforts had been
traditionally to increase the expression of gene. Especially the useful
ones gene regulation and gene silencing has helped in this regards.
1. Gene silencing can take both at the transcriptional as well as
translational levels.
2. Transcriptional gene silencing takes place due to modification of
DNA mainly by methylation of histones by acetylation.
3. These changes remarkably affect the transcription of those stretches
of DNA, usually by reducing the transcription initiation at the
promoter.
55. 4. Post transcriptional gene silencing takes place at the RNA level.
Here specific transcripts of genes are degraded by the special
complex called Dicer. Dicer consists of Ribonuclease.
5. Dicer molecules quickly degrade specific transcripts there by
reducing their levels and bringing about the silencing even
through the transcription continues normally.
6. Because post transcriptional gene silencing is involved in
targeting individual transcripts thus it can be used efficiently to
silence the required genes and to understand their functions in
the body.
7. This technique can also be used for silencing disease causing
gene. E.g. cancer.
56. APPLICATIONS OF GENETIC ENGINEERING
1. Many of the genetic diseases like hemophilia, sickle cell anemia etc., can
be treated using gene therapy.
2. Itâs due to genetic engineering that industrial production of vaccines like
insulin, interferons is possible which is a real blessing for mankind.
3. By using a recombinant technology, scientists have inserted genes into
bacteria for the production of human growth hormone. This hormone is
used to treat dwarfism. In 1986 the hormone becomes commercially available
as Protropin.
4. Urokinase a clot dissolving enzyme is produced from genetically engineered
bacteria which is a savior for angina patients.
5. Since microbial cells have much higher metabolic rates, genes of desired
enzymes (of commercial values) could be introduced into plasmid of bacteria.
E.g. yeasts are being engineering to yield enzymes for cheese industry.
57. 6. Attempts are being made to engineering plants with bacterial genes that
trap Nitrogen (N2) and convert it to a form plants. This would eliminate the
necessity of nitrogen fertilizers.
7. By using gene expression, silencing and tracking researchers have
revolutionized in the field of recombinant technology. Now they are able
to find out more genes and are able to remove single deleterious allele.
8. Genetic engineering is being used to create genetically modified food and
genetically modified crops. These crops are resistant to herbs, sherbs, and
fungal viruses.
9. In materials science, genetically modified virus has been used to construct
a more environment friendly lithium ion battery.
APPLICATIONS OF GENETIC ENGINEERING
58. GENE THERAPY
⢠Many of the human diseases results just due to a single faulty gene.
Examples of such diseases include sickle cell anemia; cystic fibrosis etc.
correcting the illness by substituting an abnormal gene by a normal one is
the bases of the gene therapy.
⢠So gene therapy is the insertion of gene into an individual cells and tissues
to treat a disease such as hereditary disease in which a deleterious mutant
allele is replaced with a functional one.
59. TYPES OF GENE THERAPY
⢠There are two types of gene therapy.
⢠Somatic gene therapy:
⢠In somatic gene therapy, the diseased organs are targeted but the change
happened through this one, is not transferred to the next generation.
⢠Germ line gene therapy:
⢠In germ line gene therapy defects in gametes or fertilized eggs are
targeted.
60. GENE TRANSFER TECHNIQUES
⢠Genes can be delivered during gene therapy in two ways.
⢠In vivo
⢠Ex vivo
⢠In vivo method: In this method the delivery agent is introduced within the
body of the patients e.g. suspension containing vector is targeted directly
into the patient either systemically or directly into targeted tissue e.g.
malignant tumor.
⢠Ex vivo method: In ex vivo method some of the target or infected cells
e.g. stem cells, myoblasts fibroblasts or usually blood is withdrawn and
then treated with the vector. After the gene therapy is complete, the treated
cells are returned to the patientâs body which then proliferate and spread to
the entire body taking with them to the introduced gene.
61. VECTORS IN GENE THERAPY
⢠For most of the in vivo methods in gene therapy, viral vectors are used.
These viruses are crippled (replication defective) i.e. they are not able to
reproduce. Following are the some of the examples for the viral vectors.
⢠Retroviruses (RNA tumor viruses)
⢠Adenoviruses (DNA tumor viruses)
⢠Adeno associated viruses (DNA tumor viruses)
⢠Herpes simplex viruses (DNA tumor viruses)
⢠Herpes simplex virus specifically infects nerve cells and thus can be used
for gene therapy to those cells and can be used as DNA tumor viruses.
62. GOALS OF GENE THERAPY
⢠Gene replacement: Replacing a mutant gene that cause disease or
abnormality with a healthy copy of gene (normal).
⢠Gene editing or correction: The abnormal gene can be repaired through
selective reverse mutation which returns the gene to its normal function.
⢠Knocking out: Inactivating or knocking out a mutant gene that is
functioning improperly.
⢠Gene augmentation: Gene therapy can be used to increase the activity of
specific gene or mediate their action.
⢠Introducing a new gene: By using gene therapy a new gene can be in
corporate into the body to help fight a disease
63. LIMITATION AND PROBLEMS OF GENE THERAPY
⢠Multi gene disorder: Conditions and disorders that arise from mutations in a
single gene are the best candidates for gene therapy. But itâs unfortunate for
a gene most commonly occurring disease like HBP, arthritis, heart diseases
etc, are due to the defects or mutation in more than one gene. Multi gene
problems are difficult to handle treat effectively using the gene therapy.
Thatâs why it is said that gene therapy is still in its infancy.
⢠Problems with viral vectors: Viral vectors may give arise to a number of
problems like toxicity immune and inflammatory response gene control and
targeting issue. No doubt we used crippled vectors but there is always fear
that the viral vector may recover its ability to cause diseases.
64. ⢠Short lived nature of gene therapy: Long term benefits from gene therapy
cannot be achieved, if there is requirement that the therapeutic DNA must
be long lived, stable and this DNA must be functional for a longer period of
time. In current scenario gene therapy is not a permanent cure for the
diseases having long treatment cycle.
⢠Immune Response: Itâs a well-known phenomenon that whenever there is
entry of a foreign substance into human tissues there is always stimulation
of immune response to attack the invader. So the risk of failure of gene
therapy due to stimulation of immune system is a potential risk. Also due to
immune systemâs enhanced response to invader it is difficult for gene
therapy to be repeated in patients.