Download BSc in Applied Biotechnology 5 BO0055 ‑ PLANT AND ANIMAL

PROGRAM
BSc in Applied Biotechnology
SEMESTER
5
SUBJECT
BO0055 - PLANT AND ANIMAL BIOTECHNOLOGY
BOOK ID
B0962
SESSION
Winter 2015
No
Q 1
Question/Answer key
Marks Total Marks
10
Describe the gene transfer methods in animal cells.
( Unit 4 ; Section 4.4 )
A 1
Gene transfer methods in animal cells.
The gene transfer to animal cells can be achieved essentially via three routes:
1.Direct DNA transfer:
Direct DNA transfer is the physical introduction of DNA directly into cell. It
involves microinjection and particle bombardment.
A)Microinjection: It is a simple mechanical process in which an extremely fine
micro needle penetrates the cell membrane and sometimes the nuclear envelope
and releases its contents.
Microinjection is normally performed under a specialized optical microscope setup
called a micromanipulator.
B)Particle Bombardment: It is direct delivery method, initially developed for the
transformation of plants.
This involves coating small metal particles with DNA and then accelerating them
into target tissues using powerful force; such as a blast of high pressure gas or an
electric discharge through water droplet.
2.Transfection :
Transfection includes a number of physical and chemical techniques to persuade
cells to take up DNA from their surroundings like DNA/Calcium phosphate
co-precipitation method, chemical transfection methods, Phospholipids as gene
delivery vehicles and Electroporation.
In DNA/Calcium phosphate co-precipitation method the process of transfection
involves the admixture of isolated DNA (10-100 μg) with a solution of calcium
chloride under conditions which allow fine co-precipitate of DNA/calcium
phosphate to be formed. Cells are then incubated with the precipitated DNA
either in solution or in tissue culture dish.
3.Transduction: The gene is transferred by packaging the DNA inside animal
viruses
In viral transduction, the adsorption and penetration are the principal events.
In enveloped viruses the entry of gene is by fusing with the plasma membrane
and via endosomes at the cell surface
In transduction, the non-enveloped viruses may cross the plasma membrane
directly or may be taken up into endosomes.
Q 2
10
Explain the methods used for creating transgenic animals.
A 2
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( Unit 5 ; Section 5.3 )
1
Methods used for creating transgenic animals:
The three principal methods used for the creation of transgenic animals are:
• 1. DNA microinjection
• 2.
Embryonic stem cell-mediated gene transfer and
• 3.
Retrovirus-mediated gene transfer.
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• 1) 1. DNA Microinjection
This method involves the direct microinjection of a chosen gene construct
from another member of the same species or from a different species, into the
pronucleus of a fertilized ovum.
It is one of the first methods that proved to be effective in mammals.
The introduced DNA may lead to the over or under-expression of certain
genes or to the expression of genes entirely new to the animal species.
The insertion of DNA is, however, a random process, and there is a high
probability that the introduced gene will not insert itself into a site on the host DNA
that will permit its expression.
The manipulated fertilized ovum is transferred into the oviduct of a recipient
female, or foster mother that has been induced to act as a recipient by mating
with a vasectomized male.
A major advantage of this method is its applicability to a wide variety of
species.
• 2) 2. Embryonic Stem Cell-mediated Gene Transfer
This method involves prior insertion of the desired DNA sequence by
homologous recombination into an in vitro culture of embryonic stem (ES) cells.
Stem cells are undifferentiated cells that have the potential to differentiate into
any type of cell (somatic and germ cells) and therefore to give rise to a complete
organism.
These cells are then incorporated into an embryo at the blastocyst stage of
development. The result is a chimeric animal.
ES cell-mediated gene transfer is the method of choice for gene inactivation,
the so-called knock-out method.
This technique is of particular importance for the study of the genetic control
of developmental processes.
This technique works particularly well in mice.
It has the advantage of allowing precise targeting of defined mutations in the
gene via homologous recombination.
• 3) 3. Retrovirus-mediated Gene Transfer
To increase the probability of expression, gene transfer is mediated by means
of a carrier or vector, generally a virus or a plasmid.
Retroviruses are commonly used as vectors to transfer genetic material into
the cell, taking advantage of their ability to infect host cells in this way.
Offspring derived from this method are chimeric, i.e., not all cells carry the
retrovirus.
Transmission of the transgene is possible only if the retrovirus integrates into
some of the germ cells.
For any of these techniques the success rate in terms of live birth of animals
containing the transgene is extremely low.
Providing that the genetic manipulation does not lead to abortion, the result is
a first generation (F1) of animals that need to be tested for the expression of the
transgene.
Depending on the technique used, the F1 generation may result in
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chimeras.
When the transgene has integrated into the germ cells, the so-called germ
line chimeras are then inbred for 10 to 20 generations until homozygous
transgenic animals are obtained and the transgene is present in every cell.
At this stage embryos carrying the transgene can be frozen and stored for
subsequent implantation.
Q 3
Describe any 4 screening techniques used for identification of specific clones.
A 3
( Unit 7 ; Section 7.4 )
Screening techniques used for identification of specific clones
Following are the screening techniques used for identification of specific clones.
Describing any 4 from the following:
• 1) Screening by DNA hybridization:
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• The target sequence in a DNA can be determined with a DNA probe.
• In this method, double-stranded DNA of interest is converted into single
strands by heat or alkali.
• The two DNA strands are kept apart by binding to solid matrix such as
nitrocellulose or nylon membrane. Now, the single strands of DNA probe
(10-1,000 bp) labeled with radioisotope are added.
• Hybridization takes place between the complementary nucleotide sequences
of the target DNA and the probe.
• The hybridized DNA can be detected by autoradiography.
• This can be achieved by DNA probes, random primer method and non-isotopic
DNA probes.
• 2) Screening by colony hybridization:
• Colony hybridization technique is also referred to as replica plating.
• In this technique, DNA sequence in the transformed colonies can be detected
by hybridization with radioactive DNA probes.
• In this method, samples of each colony are transferred to a solid matrix such
as nitrocellulose or nylon membrane.
• The colony cells are lysed and deproteinized. The DNA is denatured and
irreversibly bound to matrix.
• Then radiolabled DNA probe is added which hybridizes with the
complementary target DNA.
• The non-hybridized probe molecules are washed away.
• The colony with hybridized probe can be identified on autoradiograph. The
cells of this colony can be isolated and cultured.
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• 3) Screening by PCR:
• The Polymerase chain reaction (PCR) is a technique for screening DNA
libraries. But adequate information on the franking
• sequences of target DNA must be available to prepare primers for this method.
• The colonies are maintained in multiwell plates, each well is screened by PCR
and the positive wells are identified.
• 4) Screening by immunological assay:
• The immunological techniques can be used for the detection of protein or a
polypeptide, synthesized by a gene.
• In this method, the cells are grown as colonies on master plates which are
transferred to a nitrocellulose solid matrix.
• The colonies are then subjected to lysis and the released proteins bound to the
matrix.
• These proteins are then treated with a primary antibody which specifically
binds to the protein, encoded by the target DNA.
• After removing the unbound antibody by washings, binds to the first antibody.
• Again the unbound antibodies are removed by washings.
• The second antibody carries an enzyme label bound to it.
• The detection process is so devised that as a colourless substrate it is acted
upon by this enzyme, a coloured product is formed.
• The colonies which give positive result are identified. The cells of a specific
colony can be subcultured from the master plate.
• 5) Screening by protein function:
• This method of screening is used if the target DNA of the gene library is
capable of synthesizing a protein that is not normally produced by the host cell,
the protein activity can be used for screening.
• A specific substrate is used, and its utilization by a colony of cells indicates the
presence of an enzyme that acts on the substrate.
Q 4
10
Explain the production of insulin through recombinant DNA technology.
A 4
( Unit 6 ; Section 6.1 )
Production of insulin through recombinant DNA technology:
The insulin synthesis in E. coli has undergone several changes, for improving
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the yield, e.g. addition of signal peptide, synthesis of A and B chains separately
etc.
• The genes for insulin A chain and B chains are separately inserted to the
plasmids of two different E. coli cultures.
• The lac operon system (consisting of inducer gene, promoter gene, operator
gene and structural gene Z for
β-galactosidase) is used for expression of both the genes.
• The presence of lactose in the culture medium induces the synthesis of insulin
A and B chains in separate cultures.
• The so formed insulin chains can be isolated, purified and joined together to
give full-pledged human insulin.
Q 5
10
Discuss any 4 traditional methods of plant breeding.
( Unit 1 ; Section 1.5 )
A 5
Traditional methods of plant breeding:
1) Mass Selection
• In mass selection, a large number of plants of similar phenotype are selected
and their seeds are mixed together to constitute a new variety.
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• The plants are selected on the basis of their appearance or phenotype.
Therefore, selection is done for easily observable characters, like, plant height,
ear (inflorescence) type, grain colour, grain size, disease resistance, tillering
ability, lodging resistance, shattering resistance etc.
• If the plant population has variation for grain characteristics (like seed colour
and seed size), selection may be done for them before the seeds of selected
plants are mixed together.
• 2) Pureline selection
• A pureline is a progeny of a single homozygous self pollinated plant. As a
result individuals within a pureline have identical genotype and any variation in
pureline is solely due to the environment.
• In a self pollinated species, all the plants are expected be homozygous
because of continued self fertilization. Thus, progeny of single plant from a
population of a self pollinated crop would be a pure line.
• In pureline selection, a large number of plants are selected from a self
pollinated crop and are harvested individually; individual plant progenies from
them are evaluated ,and the best progeny is released as a pureline variety.
• A pureline variety is a variety obtained from a single homozygous plant of self
pollinated crop.
• 3) Pedigree Method
• In this method, individual plants are selected from F1 and the subsequent
generations, and their progenies are tested.
• During this operation, records of all the parent offspring relationships are
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kept and this record is called as pedigree record. Individual plant selection is
continued till the progenies show no segregation.
• 4) Bulk Method
• This method is called as population method or mass method.
• This method was first used by Nilsson-Ehlein (1908). In this method F2 and
subsequent generations are harvested in mass or as bulk to raise the next
generation.
• At the end of bulking period, individual plants are selected and evaluated in a
similar manner as a pedigree method of breeding. Duration of bulking ranges
from 6-30 generation.
• 5) Back Cross Method
• A cross between hybrid and one of its parents is called back cross. In this
method, hybrid and the progenies are repeatedly back crossed to one of their
parents.
• At the end of 6-8 backcrosses the progeny would be identical with parent used
for back crossing.
• The objective is to improve one or two specific defects of high yielding variety.
Q 6
10
Discuss the advantages of edible vaccines.
( Unit 2 ; Section 2.5 )
A 6
Advantages of edible vaccines:
Edible plants are very effective as a delivery vehicle for inducing oral
immunization.
• In edible plants, Adjuvant for immune response is not necessary.
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• Edible plants feasibility of oral administration is excellent compared to injection.
• It is easy for separation and purification of vaccines from plant materials.
• They have effective prevention of pathogenic contamination from animal cells.
• They are convenient and safety in storing and transporting vaccines.
• They have effective maintenance of vaccine activity by controlling the
temperature in plant cultivation.
• They are easy for mass production system by breeding compared to an animal
system.
• Its possible for production of vaccines with low costs.
• Reduced need for medical personnel and sterile injection conditions.
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• They are economical to mass produce and transport.
• They are heat stable, eliminating the need for refrigeration.
• The antigen protection happens through bioencapsulation.
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