Download Animal Biotechnology - Killingly Public Schools

Animal
Biotechnology
A New Kind of
Pharming
Transgenic Animals
• Animal that carries a foreign
gene that has been deliberately
inserted into its genome
• The foreign gene is constructed
using recombinant DNA
methodology
• Can be used for
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Hormone production
Antibody production
Protein production
Diagnostics
Gene Function Research
Advantages
• Increased growth
• Disease resistance
• Improve nutritional
quality of food
• Increase muscle mass
• Better wool production
• Study disease better
Disadvantages
• Inserted gene has
multiple functions
• Breeding problems
• Sometimes leads to
mutagenesis and
functional disorders
• Low survival rate of
transgenic animals
• Ethical dilemmas
Methods to Produce Transgenic
Animals
• Embryonic Stem Cell – transforming ES cells
growing in tissue culture with the desired DNA
• Pronucleus – injecting the desired gene into
th pronuleus of a fertilized egg
• Retrovirus mediated gene transfer
Embryonic Stem Cell Method
1. Embryonic Stem Cells with the
desired DNA
2. Injection into blastocysts
3. Injection into foster mother
4. Formation of new individual
5. Test offspring for presence of gene
Pronucleus Method
1. Formation of DNA as in first method
2. Fusion of male pronucleus (nucleus of
sperm) with desired DNA
3. Formation of diploid zygote
4. Mitotic division to form a two celled
embryo
5. Injection into foster mother
6. Formation of new individual
7. Test offspring for presence of gene
Example
• Comparison between a
transgenic mouse and a
normal mouse
• The giant mouse developed
from a fertilized egg
transformed with a
recombinant DNA molecule
containing the Human
Growth Hormone
Retro Virus Mediated Gene Transfer
• Retroviruses (viruses that
have RNA only. Use
reverse transcriptase to
make DNA from RNA) are
used as vectors to transfer
genetic material into the
host cell, resulting in a
chimera, an organism
consisting of tissues or
parts of diverse genetic
constitution
Examples of Transgenic Animals
Transgenic Cow
• Transgenic cows carrying extra copies of two
types of casein genes produce 13% more milk
protein
Transgenic Pigs
• Produced by fertilizing normal eggs with
sperm cells that have incorporated foreign
DNA (sperm mediated gene transfer)
– Possible source of organs for humans
Transgenic Fish
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Tilapia
Salmon
Trout
Catfish
Can grow up to 6 times faster than wild fish
Most have extra copies of a growth hormone
gene
Transgenic Sheep & Goats
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Until recently, the transgenes introduced into sheep inserted randomly in the genome and
often worked poorly. However, in July 2000 success at inserting a transgene into a specific
gene locus was reported. The gene was the human gene for alpha1-antitrypsin (without it,
causes people to be prone to emphysema/lung inflamation), and two of the animals
expressed large quantities of the human protein in their milk.
How it was done:
• Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a
vector that contained these segments of DNA:
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2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen. (Its absence in
humans causes the inherited disease osteogenesis imperfecta) This locus was chosen because
fibroblasts secrete large amounts of collagen and thus one would expect the gene to be easily
accessible in the chromatin.
A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector.
The human gene encoding alpha1-antitrypsin. Some people inherit two non- or poorly-functioning
genes for this protein. Its resulting low level or absence produces the disease Alpha1-Antitrypsin
Deficiency (A1AD or Alpha1). The main symptoms are damage to the lungs (and sometimes to the
liver).
Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in
milk-producing cells.
Binding sites for ribosomes for efficient translation of the beta-lactoglobulin mRNAs.
Transgenic Sheep & Goats
Successfully-transformed cells were then:
• Fused with enucleated sheep eggs and implanted in the uterus of a ewe (female sheep).
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Several embryos survived until their birth, and two young lambs lived over a year.
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When treated with hormones, these two lambs secreted milk containing large amounts of
alpha1-antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion
of the transgene).
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On June 18, 2003, the company doing this work abandoned it because of the great expense
of building a facility for purifying the protein from sheep's milk. Purification is important
because even when 99.9% pure, human patients can develop antibodies against the tiny
amounts of sheep proteins that remain.
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However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won
preliminary approval to market a human protein, antithrombin, in Europe. Their protein —
the first made in a transgenic animal to receive regulatory approval for human therapy —
was secreted in the milk of transgenic goats.
Transgenic Sheep & Goats
• Spider silk is regarded as the
strongest material known
• Genes from the dragline silk were
isolated and put into a goat in
order to be expressed in the
mammary gland in the form of
milk
• The silk proteins found in the milk
can be used make lighter and
stronger bullet proof vests,
thinner tread for surgery,
clothing, etc.
Transgenic Mice
• While "housekeeping" genes (responsible for routine
metabolic functions) are expressed in all types of cells at all
stages of development, other genes are normally expressed in
only certain types of cells when turned on by the appropriate
signals (e.g. the arrival of a hormone). But how do we study
these genes?
• Gene knockout is a genetic technique in which one (or more)
of an organism's genes is made inoperative
– knockout organisms are used in learning about a gene that has been
sequenced, but which has an unknown or incompletely known
function
– can draw inferences from the difference between the knockout
organism and normal individuals
– Can also have knock in organisms where researchers insert a gene in
mice they wish to study
Transgenic Mice
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One of the bacteriophages that infects E. coli, called P1,
produces an enzyme — designated Cre — that cuts its
DNA into lengths suitable for packaging into fresh virus
particles. Cre cuts the viral DNA wherever it encounters
a pair of sequences designated loxP. All the DNA
between the two loxP sites is removed, and the
remaining DNA ligated together again (so the enzyme is
a recombinase.
Mice can be made transgenic for:
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the gene encoding Cre attached to a promoter that will
be activated only when it is bound by the same
transcription factors that turn on the other genes
required for the unique function(s) of that type of cell
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a "target" gene, the one whose function is to be studied,
flanked by loxP sequences
In the adult animal:
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those cells that
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receive signals (e.g., the arrival of a hormone or cytokine)
to turn on production of the transcription factors needed
to activate the promoters of the genes whose products are
needed by that particular kind of cell
will also turn on transcription of the Cre gene. Its protein
will then remove the "target" gene under study. All
other cells will lack the transcription factors needed to
bind to the Cre promoter (and/or any enhancers) so the
target gene remains intact.
The result: a mouse with a particular gene knocked out
in only certain cells.
Transgenic Primates
• Marmosets
• In some cases, the
transgene (for green
fluorescent protein) was
incorporated into the
germline and passed on to
the animal's offspring
• Possible best model yet for
studying human disease and
possible therapies
Transgenic Chickens
Why Chickens?
• Grow faster than sheep and goats and large numbers can be grown in close quarters
• Synthesize several grams of protein in the "white" of their eggs
Two methods have succeeded in producing chickens carrying and expressing foreign
genes
1. Infecting embryos with a viral vector carrying:
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the human gene for a therapeutic protein
promoter sequences that will respond to the signals for making proteins in egg white
2. Transforming rooster sperm with a human gene and the appropriate promoters and
checking for any transgenic offspring
Preliminary results from both methods indicate that it may be possible for chickens to produce
as much as 0.1 g of human protein in each egg that they lay
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Should this cost less than producing therapeutic proteins in culture vessels
Chickens will probably add the correct sugars to glycosylated proteins — something that E. coli
cannot do
Antibodies
• Proteins developed by
the immune system
that recognize specific
molecules (antigens)
• Antigens = A substance
that reacts with the
products of a specific
immune response
Monoclonal Antibodies
• Usually made by fusing
myeloma cells with the
spleen cells from a mouse
that has been immunized
with the desired antigen
• Fusing antibody-producing
spleen cells, which have a
limited life span with cells
derived from an immortal
tumor of lymphocytes
(myeloma) results in a
hybridoma that is capable
of unlimited growth
Applications of
Monoclonal Antibodies
Diagnostic tests
• Once monoclonal antibodies for a given substance have been produced, they can
be used to detect the presence of this substance. The Western blot test and
immuno dot blot tests detect the protein on a membrane.
Monoclonal antibody therapy
• Use of monoclonal antibodies to specifically bind to target cells or proteins;
possibly stimulating the patient's immune system to attack those cells
• Possible to create a mAb specific to almost any extracellular/cell surface target
such as: rheumatoid arthritis, multiple sclerosis, and different types of cancers
Autoimmune diseases
• Monoclonal antibodies used for autoimmune diseases include infliximab and
adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and
ulcerative Colitis (form of IBD) by their ability to bind to and inhibit TNF-α(tumor
necrosis factor – regulates immune cells)
• Basiliximab and daclizumab inhibit Iinterleukin-2 (cytokine that stimulates the
growth of T cells) on activated T cells preventing rejection of kidney transplants
• Omalizumab inhibits human immunoglobulin E (key component in allergic
reactions) and is useful in moderate-to-severe allergic asthma