Transcript Animal Biotechnology - Lectures For UG-5

Animal Biotechnology
Animal Biotech
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Animals provide a number of products we use in every day
life:
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Milk
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Leather
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Wool
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Eggs
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Meat
Animal Biotechnology
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Transgenics
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Characterizing genetic variability
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Reproductive technologies
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Artificial insemination (AI) and preservation of semen
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Improving health through developing vaccines
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Diagnostics and epidemiology
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Nutrition and feed utilization
Transgenics
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Transgenics are genetically modified organisms with DNA from
another source inserted into their genome
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A large number of transgenic animals have been created
Mice Cows Pigs Sheep Goats Fish Frogs Insects
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Currently, no transgenic animal or animal product is approved by
the FDA or USDA for human consumption
Goals of transgenic animal creation
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Improve livestock animals
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Genetic modification of animals may be aimed at improving their milk, meet,
wool etc production.
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Use of animals as bioreactors
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Genes are transferred into the animals to obtain a large scale production of
proteins encoded by these genes in milk, urine or blood of such animals; such
animals are called bioreactors and this method is called molecular farming or
gene farming.
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Research into animal and human disease
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A special case of gene transfer is alleviating or eliminating the symptoms
and consequent miseries of genetic diseases.
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Specific transgenic animals strains or lines are created to fulfill specialized
experimental and/or biomedical needs.
Eg. Konck out mice strains in which specific genes have been replaced
or knocked out by their disrupted counterparts through a process of
homologous recombination.
Transgenic Animal Creation
Making a transgene
1.
Structural gene which selected as per requirement
2.
Vectors; are carriers used to transfer the gene of interest from one
organism to another. Eg. SV40 vectors, bovine papilloma vectors,
retrovirus vectors, bacculovirus vectors
3.
Requirements of promoter/ enhancer seq in the transgene so that it can
be expressed in the host cells.
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A transgene must be integrated into the host genome for obtaining
transgenic cells/animals.
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Transgene must be present in proper orientation in relation to and in
association with various sequences required for its efficient transcription
and translation in host cell.
ts
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A transgene must be integrated into the host genome for obtaining transgenic
cells/animals.
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Transgene must be present in proper orientation in relation to and in association
with various sequences required for its efficient transcription ans translation in host
cell.
The sequences may be listed as follow;
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An efficient promotors/ enhancer
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The translation initiation codon (AUG in mRNA, and ATG in DNA)
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The chain termination codon
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Transcription termination sequence
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Polyadenylation cleavage/ addition site
Gene Construct
Schematic representation of a gene construct for expression in animal/ plant
cell.
P/E
ATG
Gene
TAG
Poly- (A)
Construction of Transgenic Animal
Isolation of transgene
Insertion into vector
Introduction into cell
Integration of gene into the genome
Development of organism
Fully transgenic
organisms
Mosaic organisms
Non transgenic
organisms
Creation of Transgenic Animals
by Nuclear Injection
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In vitro fertilization is used to start a
transgenic animal.
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Harvested eggs and sperm are
fertilized, and before the pronuclei
fuse, the transgene is injected into
the male pronucleus.
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The embryo continues to divide in
culture and is then implanted into a
mouse.
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The “foster mother” mouse has been
treated with hormones so that she
accepts the embryo and carries on
with the pregnancy.
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The offspring are screened for stable
integration of the transgene.
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Founder mice have one copy of the
transgene.
Gene injected into
the male pronuclei
Detection of Transgenics and Transgene Function
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Dot Blot technique
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PCR amplification using genomic DNAs extracted from mice
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Detection of mRNA Expression
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Assays for protein Expression
Large Transgenic Mice
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Express the rat somatotropin gene
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Under the control of the promoter from an unrelated mouse gene,
metallothionein , which is normally expressed in the liver
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Instead of being made in the pituitary gland, the normal site for growth
hormone, the rat somatotropin is manufactured in liver
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Human somatotropin has also been expressed in mice and also gives bigger
mice.
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Dwarfism
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Recombinant human somatotropin (rHST) is now used to treat the hormonedeficient type of dwarf.
Large Transgenic Mice
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Express the rat somatotropin gene
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Under the control of the promoter from
an unrelated mouse gene,
metallothionein , which is normally
expressed in the liver
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Instead of being made in the pituitary
gland, the normal site for growth
hormone, the rat somatotropin is
manufactured in liver
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Human somatotropin has also been
expressed in mice and also gives
bigger mice.
Trendy Transgenic Mice
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Marathon Mouse
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Can run about 1800 meters—more than a mile—before exhaustion. This is
twice as far as a normal mouse can last.
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Marathon mouse has enhanced PPAR-delta—a regulator of several genes
involved in burning fat and in muscle development.
Mighty Mouse
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Lack myostatin, a protein that slows muscle growth. The result is colossal
muscle development.
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There are one-two known cases of a human with a genetic defect leading to
lack of myostatin. A German boy, born in Berlin in 2000, has muscles twice
the size of other children his age.
Recombinant protein production
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The somatotropin gene from Cow
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Can be expressed in bacteria - the production of large amounts of the
hormone, rBST.
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Used in the dairy industry to increase milk production.
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Used to boost adult cow’s somatotropin, resulting in increased milk
production.
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If cloned in Cow?
Production of heterologous proteins
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Recombinant proteins can be produced in the milk from transgenic cows or
other farm animals.
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The cloned genes are placed under the control of a regulatory region that will
allow gene expression only in the mammary gland.
For small-scale production, transgenic goats are often used.
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E.g. transgenic goats producing rTPA
Knockout Mice
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The target gene is cloned and
disrupted by inserting a DNA
cassette.
This work is usually done in
bacteria.
Once the construct is made, it is put
back into a mouse by injection into
the male pronucleus during
fertilization.
After the transgenic offspring are
born, two heterozygotes are crossed
to create a homozygous knockout
mouse.
These are then screened for defects
due to inactivation of the target
gene.