(The Farm by Alexis Rockman)

• Mice
• Livestock: Cattle, Sheep, Goats and Pigs
• Birds and Fish
• Animal Cloning

Uses for Transgenic Animals

1. Improved production by farm animals

Ex.

• Milk -Higher yields
• Meat -Rapid weight gain and better efficiency of feed utilization
• Eggs -Increased laying frequency
• Wool -Quality

2. Production and secretion of human therapeutic proteins in milk

Ex.

• Interferons
• Blood clotting factors

Milk is easy to collect, and protein purification is simpler than from cell culture

Will likely have correct posttranslational modifications

3. Source of organs for transplantation to humans

Ex.

• Hearts from pigs genetically engineered to eliminate cell surface antigens

To prevent rejection by patient's immune system

4. Animal models for research on human diseases

Ex.

• Alzheimer disease
• Arthritis

To determine causes of a disease and for testing of potential therapies before use on humans

General Strategy for Producing Transgenic Animals

1. Obtain fertilized eggs or early stage of a developing embryo

2. Introduce cloned DNA into nucleus of egg or embryo cell

3. Implant egg or embryo into surrogate mother

4. Identify transgenic founder animals

Offspring carrying cloned DNA in germ line cells

Germ line cells produce gametes (egg or sperm)

Founder can pass transgene on to next generation

5. Breed transgenic founder strain to establish new genetic line carrying the foreign DNA

I. Infection of embryos with retroviral (RNA virus) vectors

Retro viral vectors

• High efficiency of gene transfer because transduction (infection) is highly efficient
• Genetically stable because of integration which is directed by long terminal repeats (LTRs) at the 5' and 3' ends
• Restricted to small DNA fragments (8 kb)

May lack regulatory sequences needed for expression

• Concerns about replication of retrovirus in animal and potential to cause cancer have limited the use of this method

Retroviruses integrate into host genome and may disrupt tumor supressor genes

(See Fig. 19.1)

1. Mate and harvest embryos

2. Transduce (infect) embryo with recombinant retrovirus (see below)

3. Implant embryo into surrogate mother

4. Test offspring to identify transgenic animals

5. Mate and test offspring to ensure that transgene is integrated into genome of germ line cells

The cells are diploid, but only one chromosome of a pair received the transgene, so the animals are heterozygous for the gene

6.. Mate positive offspring with each other to establish a homozygous transgenic line

Mate two heterozygous mice and 1 in 4 progeny would be expected to be homozygous, referred to as a Founder animal

Retroviruses are composed of RNA. How do you get your gene(s) into the viral genome and then into the animal cells?

• Retroviral vectors are designed to be replication incompetent for safety.

Packaging cells lines that supply missing viral genes are used to produce infectious viral particles

A packaging cell line contains the genes gag, pol and env

gag and pol are needed to produce infectious virus particles and are integrated into the packaging cell line genome

env determines range of cell types that can be infected (trophism). This gene may be integrated into the packaging cell genome as for gag and pol or may be present on a separate vector that is cotransfected with the recombinant vector into the packaging cell line. Different env genes can be used to infect various cell types depending on the cell's membrane receptor to which the virus binds.

Y⁺ is a packaging signal for recognition and packaging of RNA into viral particles.

Selectable marker Ex. Neomycin resistance for host cell and Amp^R for E. coli.

Note the resistance genes are under the control of a eukaryotic and prokaryotic promoters for selection of the cells being used.

• Use of the vector

1. The vector is a plasmid (DNA) that replicates in E. coli for cloning cDNA copies of the gene you want to introduce into the animal cells

2. After the recombinant vector is obtained, it is transfected into a packaging cell line for production of RNA and packaging into infectious viral particles

3. The viral particles are used to transduce host cells (Ex. mouse). The RNA will be reverse transcribed into DNA in the host cells which will integrate into the host cell genome

4. During expression, the integrated gene is transcribed and translated into a gene product

II. DNA microinjection

(Fig. 19.2)

1. Inject female mouse with pregnancy hormones to increase the numbr of eggs produced (superovulation)

-Pregnant mare's serum
-Human chorionic gonadotrophin

2. Mate to fertilize eggs

3. Harvest ~ 35 eggs

4. Inject transgene into male pronucleus

5. Implant 25-40 eggs into pseudopregnant surrogate mother

6. Identify transgenic founder using labeled transgene as probe or by PCR with primers specific for the gene

7. Mate to determine if transgene is in germ line cells

8. Breed transgenic progeny to establish a homozygous transgenic line

Problems Associated With Microinjection

(Fig. 19.3)

• Must be performed by highly skilled personnel
• Low overall efficiency for production of transgenic animals
• Foreign DNA is integrated at random sites of animal genome

o May not allow for very high expression levels

o May disrupt normal physiology of animal

III. Transfection of pleuripotent embryonic stem cells

(Fig. 19.4)

Stem cells are undifferentiated cells

Embryonic stem cells are pleuripotent

Can differentiate and become any cell type

Ex. neural, bone marrow (blood), organ tissue, germline

1. Obtain stem cells from blastocyst stage of a developing embryo

Grown in cell culture

2. Transfect cells with plasmid vector containing cloned DNA

Target a nonessential gene for integration of foreign DNA into mouse genome

Use a targeting vector and select for cells with transgene integrated at target site (see below)

Embryonic stem cells have not been discovered for most animals

**Human stem cells discovered in 1998

3. Enrich for transfected cells

4. Microinject cells into a recipient blastocyst

5. Implant blastocyst into surrogate mother

6. Identify chimeric offspring that have the transgene

Chimeras are animals inwhich some but not all cells have the transgene

7. Mate chimeras to identify those with transgene in germline to estabilish a founder

Use of a Targeting Vector

(Fig. 19.5)

• Integrates DNA at a specific site within an animal's genome

Genetic elements present on the vector

1. HB1 and HB2

Blocks of DNA with nucleotide sequences that are homologous to DNA of target site of animal genome

Target site is within a gene that is not essential to the animal

2. Transgene (located between HB1 and HB2)

3. Antibiotic resistance gene (also between HB1 and HB2)

Ex. npt, gene that provides resistance to neomycin and G-418 (related to neomycin)

Cells with integrated DNA are resistant to G-418 (positive selection)

Cells without integrated DNA are killed by G-418

4. Thymidine kinase (tk) genes (from herpes simplex virus)

Thymidine kinase transforms ganciclovir to a toxic compound that kills cells

tk genes are located outsie of HB1 and HB2 and their expression kills cells containing nonspecifically integrated DNA (negative selection)

After transfection of mouse embryonic stem cells, enrich for transfected cells by applying positive-negative selection by treating cells with G-418 and ganciclovir

Cells with integrated DNA survive because of neomycin resistance gene

The antibiotic G-418 kills cells without integrated DNA

Cells with DNA integrated at a nonspecific site are killed because the thymidine kinase gene is present

Thymidine kinase monophosphorylates ganciclovir (a prodrug) and cellular kinase add 2 additional phosphates resulting in a triphosphorylated product that is toxic and kills the cells

Cells with DNA integrated at the target site survive exposure to ganciclovir

tk genes are not present, so ganciclovir isn't converted to toxic compound

Expression of Transgenes in the Mammary Gland

(Tables 19.2 and 19.3)

• For production of authentic human therapeutic proteins secreted in milk by cows and goats

Ex. glycosylated proteins

• High expression levels and large volumes can be achieved compared to cultured mammalian cells

o Dairy cow can produce ~10,000 liters/year, goat ~900 liters/year

• Want high-level expression by mammary gland cells only

o Control expression with promoters of genes for milk proteins

Ex. Caseins and lactalbumins

• Transgenic cows take longer to develop than goats (3 years vs. 1.5 years)

Transgenic Chickens and quail

(Fig. 19.20)

• Relatively easy to raise
• Foreign proteins may be secreted in eggs and then purified

1. Blastoderm cells are isolated from a donor egg

2. Transgene is introduced on a vector via lipofection

Lipofection: DNA is encapsulated into lipid vesicles (liposomes)

DNA enters cells by fusion with cell membrane

3. A recipient egg is irridated to destroy most of the blastoderm cells and the egg is injected with with the transfected donor cells

4. Egg is incubated and chicks are screened for a transgene

These animals will be chimeric, an individual will have some cells with and some without the transgene

5. Mate to identify those with transgene in germline and to produce a founder

• Can be raised for food using aquaculture

Ex. carp, catfish, trout, salmon, tialpia

• Transgenes can be introduced by microinjection or electroporation of fertiltized eggs with high efficiency
• Eggs develop externally for many fish, no need for implantation into surrogate mother
• Desirable transgenic fish traits:

-Resistance to disease and environmental stress

-Increased growth rate in cold waters

Ex. Insert transgene for growth hormone, under control of an antifreeze protein promoter, into salmon genome

Transgenic Atlantic salmon grow to market weight in about 1/2 the time and use less feed

Probably will be the first transgenic animal marketed for human consumption approved by the FDA

• Need for biocontainment to prevent escape into the wild

Might compete with wild populations or spread transgene to wild gene pool

May affect ecological balance between size of predator and prey

(Fig. 19.17)

• Creates an animal that is genetical identical to an existing animal

May be used to create a herd of genetically identical:

Transgenic animals without breeding

Nontransgenic animals with known properties, such as rate of growth or milk production

• Donor nucleus comes from differentiated cell of animal to be cloned (e.g from mammaary gland)
• After starvation, donor cell is fused to an egg that has had its nucleus removed
• The egg-cell fusion is cultured in vitro
• After several rounds of cell division the embryo is implanted into a surrogate mother for development and birth
• The off spring should have the same genetic makeup as the donor animal

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Last updated: November December13, 2004 /jdh

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