Animal Biotechnolog

yA 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 – 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 Method1. 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 Method1. 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

• 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

• 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:– 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 & GoatsSuccessfully-transformed cells were then:• Fused with enucleated sheep eggs and implanted in the uterus of a ewe (female sheep).

• Several embryos survived until their birth, and two young lambs lived over a year.

• 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).

• 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.

• 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

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Transgenic Mice• 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:• 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

• a "target" gene, the one whose function is to be studied, flanked by loxP sequences

In the adult animal:• those cells that

– 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 cellwill 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 ChickensWhy 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 foreigngenes

1. Infecting embryos with a viral vector carrying:– 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 produceas much as 0.1 g of human protein in each egg that they lay

– 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 cancersAutoimmune 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