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Transgenic Animals

A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to the gene itself, the DNA usually includes other sequences to enable it These animals should eventually prove to be valuable sources of proteins for human therapy.
In July 2000, researchers from the team that produced Dolly reported success in producing transgenic lambs in which the transgene had been inserted at a specific site in the genome and functioned well. [More]

Transgenic mice have provided the tools for exploring many biological questions.

An example:

Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-manipulated model for studying the disease. However, transgenic mice expressing the human gene for the polio virus receptor

Two methods of producing transgenic mice are widely used:

The Embryonic Stem Cell Method (Method "1")

Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.
Link to discussion of embryonic stem cells.

1. Make your DNA

Using recombinant DNA methods, build molecules of DNA containing

2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.

3. Select for successfully transformed cells. [Method]

4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.

5. Embryo transfer

6. Test her offspring

7. Establish a transgenic strain

The Pronucleus Method (Method "2")

1. Prepare your DNA as in Method 1

2. Transform fertilized eggs

3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.

An Example

This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right) with a normal littermate (left). The giant mouse developed from a fertilized egg transformed with a recombinant DNA molecule containing: The levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice.

Random vs. Targeted Gene Insertion

The early vectors used for gene insertion could, and did, place the gene (from one to 200 copies of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a particular gene, it is possible to design vectors that replace that gene. The replacement gene can be one that In either case, targeted gene insertion requires

Step 1

Treat culture of ES cells with preparation of vector DNA.

Results:

Step 2

Culture the mixture of cells in medium containing both G418 and ganciclovir.

Step 3

Inject these into the inner cell mass of mouse blastocysts.

Knockout Mice: What do they teach us?

If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the nonfunctional gene (both copies of the gene at that locus have been "knocked out").

Knockout mice are valuable tools for discovering the function(s) of genes for which mutant strains were not previously available. Two generalizations have emerged from examining knockout mice:

Tissue-Specific Knockout Mice

While "housekeeping" genes 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).

Link to a discussion of cell-specific gene expression.

To study such genes, one might expect that the methods described above would work. However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied.

Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell.

The Cre/loxP System

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).

Using "Method 1" (above), mice can be made transgenic for In the adult animal,

The result: a mouse with a particular gene knocked out in only certain cells.

Knock-in Mice

The Cre/loxP system can also be used to

Such transgenic mice are called "knock-in" mice.

Transgenic Sheep and 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, and two of the animals expressed large quantities of the human protein in their milk.

This is how it was done.

Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that contained these segments of DNA:

  1. 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.

  2. A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector. [Link to technique]
  3. 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).

  4. Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in milk-producing cells.
  5. Binding sites for ribosomes for efficient translation of the beta-lactoglobulin mRNAs.
Successfully-transformed cells were then

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 Chickens

Chickens Two methods have succeeded in producing chickens carrying and expressing foreign genes.

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.

Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens will probably add the correct sugars to glycosylated proteins — something that E. coli cannot do.

Transgenic Pigs

Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may someday be able to produce transgenic pigs that can serve as a source of transplanted organs for humans. [More]

Transgenic Primates

In the 28 May 2009 issue of Nature, Japanese scientists reported success in creating transgenic marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into the germline and passed on to the animal's offspring. The hope is that these transgenic animals will provide the best model yet for studying human disease and possible therapies.

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13 June 2011